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Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Teerlink JR, Cotter G, Davison BA, et al, for the RELAXin in Acute Heart Failure (RELAX-AHF) Investigators. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial. Lancet 2012; published online Nov 7. http://dx.doi.org/10.1016/S0140-6736(12)61855-8.

Supplemental Appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Teerlink JR, Cotter G, Davison BA, et al, for the RELAXin in Acute Heart Failure (RELAX-AHF) Investigators. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial. Lancet 2012; published online Nov 7. http://dx.doi.org/10.1016/S0140-6736(12)61855-8. Table of Contents: Section 1: Supplemental Methods 2

A. Study Personnel 2

B. Study Conduct and Oversight 4

C. Inclusion and Exclusion Criteria 4

D. Additional Study Procedures 5

E. Additional Efficacy Analyses 5

F. Method for calculating the Dyspnoea VAS AUC 6

G. Adjudication of Endpoints 6

Section 2: Supplemental Results 9

Appendix Figure A1: Change in systolic blood pressure 9

Appendix Figure A2: All-cause death through 180 days (Safety) 10

Appendix Table A1: Physician assessment of HF signs and symptoms 11

Section 3: Adverse Events and Safety 13

Protocol 18

Statistical Analysis Plan 117

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Section 1: Supplemental Methods A. Study Personnel Investigative sites by Country (patients enrolled): Investigators in descending order of patients enrolled (affiliation, city): Argentina (71): Gustavo Maria Ferrari, M.D. (Hospital Britanico de Buenos Aires, Buenos Aires); Ariel Quiroga, M.D. (Hospital Español, Rosario); Alberto Fernandez, M.D. (Sanatorio Modelo de Quilmes, Quilmes); Eduardo Perna, M.D. (Instituto de Cardiologia de Corrientes "J. F. Cabral", Corrientes); María Silvina Ramos, M.D. (Clinica Constituyentes, Moron); Luis Guzman, M.D. (Sanatorio Allende, Cordoba); Guillermo Cursack, M.D. (Instituto Cardiovascular de Rosario, Rosario - Santa Fe); Oscar Allall, M.D. (Hospital Cordoba, Cordoba); Marcelo Gustavo Masuelli, M.D. (Complejo Medico de la Policia Dederal Argentina Churruca Visca, Buenos Aires); Carlos Rapallo, M.D. (Sanatorio Itoiz S.A., Avellaneda). France (21): Alain Cohen-Solal, M.D., Ph.D. (Hopital Lariboisiere, Paris); Michel Galinier, M.D. (CHU de Rangueil, Toulouse); Guillaume Jondeau, M.D., Ph.D. (Hôpital Bichat, Paris); Richard Isnard, M.D., Ph.D. (Hopital Pitie-Salpêtrière, Paris). Germany (78): Hans-Georg Olbrich, M.D. (Asklepios Klinik Langen, Langen); Veselin Mitrovic, M.D. (Kerckhoff-Klinik GmbH, Bad Nauheim); Karl Werdan, M.D. (Martin-Luther-Universität Halle-Wittenberg, Halle); Stephan Felix, M.D. (Universitätsklinikum der Ernst-Moritz-Arndt-Universität Greifswald, Greifswald); Thomas Heitzer, M.D. (Klinikum Dortmund); Gerhard Cieslinski, M.D. (Krankenhaus Nordwest GmbH, Frankfurt am Main); Karl Stangl, M.D. (Charité - Universitätsmedizin Berlin, Berlin). Hungary (151): János Tomcsányi, M.D., Ph.D. (Budai Irgalmasrendi Korhaz, Budapest); Dezso Apró, M.D. (Allami Szivkorhaz, Balatonfured); Kálmán Tóth, M.D., Ph.D., Sc.D., F.E.S.C. (Pecsi Tudomanyegyetem Klinikai Kozpont, Pecs); András Vértes, M.D. (Egyesített Szent István és Szent László Kórház-Rendelőintézet, Budapest); Géza Lupkovics, M.D., Ph.D. (Zala Megyei Korhaz, Zalaegerszeg); Zoltán László, M.D., Ph.D. (Fővárosi Önkormányzat Szent János Kórháza, Budapest); Attila Cziraki, M.D., Ph.D. (Pécsi Tudományegyetem, Szívgyógyászati Klinika, Pécs). Israel (210): Alon Marmor, M.D. (Ziv Medical Center, Safed); Sorel Goland, M.D. (Kaplan Medical Center, Rehovot); Amos Katz, M.D. (Barzilai Medical Center, Ashkelon); Reuven Zimlichman, M.D. (Wolfson Medical Center, Holon); Doron Aronson, M.D. (Rambam Health Care Campus, Haifa); Adi Butnaru, M.D. (Shaare-Zedek Medical Center, Jerusalem); Muhamad Omary, M.D. (Nazareth Hospital EMMS, Nazareth); Xavier Alejandro Piltz, M.D. (Bikur Cholim Hospital, Jerusalem); Doron Zahger, M.D. (Soroka University Medical Center, Beer-Sheva). Italy (77): Marco Metra, M.D. (Azienda Ospedaliera Spedali Civili di Brescia, Brescia); Andrea Mortara, M.D. (Policlinico di Monza, Monza); Manrico Balbi, M.D. (Azienda Ospedaliera Universitaria "San Martino", Genova); Franco Cosmi, M.D. (Ospedale Valdichiana Santa Margherita, Cortona); Salvatore DiSomma, M.D., (Azienda Ospedaliera S. Andrea, Roma); Maria Cristiana Brunazzi, M.D. (Ospedale Civile “Destra Secchia”, Pieve di Coriano Mantova). Netherlands (10): Adriaan A. Voors, M.D., Ph.D. (University Medical Center Groningen, Groningen); Peter E.F. Bendermacher, M.D. (Elkerliek, Helmond); Gert-Jan Milhous, M.D. (Beatrix Ziekenhuis, Banneweg); Paul L. van Haelst, M.D., Ph.D. (Antonius Ziekenhuis, Hanzeplein); Peter Dunselman, M.D. (Amphia Ziekenhuis, Breda). Poland (258): Piotr Ponikowski, M.D., Ph.D. (Osrodek Chrob Serca, 4 Wojskowy Szpital Kliniczny, Wroclaw); Piotr Jankowski, M.D. (Sadmodzielny Publiczny Zaklad Opieki Zdrowotnej Szpital Uniwersytecki w Krakowie, Krakow); Andrzej Wysokinski, M.D., Ph.D. (Samodzielny Publiczny Szpital Kliniczny Nr 4 w Lublinie, Lublin); Miroslaw Dluzniewski, M.D., Ph.D. (Wojewodzki Szpital Brodnowski, Warszawa); Janina Stepinska, M.D., Ph.D. (Instytut Kardiologii im. Prymasa Tysiaclecia Stefana Kardynala Wyszynskiego, Warszawa); Wieslawa Tracz, M.D., Ph.D. (Krakowski Szpital Specjalistyczny im. Jana Pawla II, Krakow); Maria Krzeminska-Pakula, M.D., Ph.D. (Wojewodzki Specjalistyczny Szpital im. WI. Bieganskiego, Lodz); Jacek Grzybowski, M.D., Ph.D. (Instytut

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Kardiologii im. Prymasa Tysiaclecia Stefana Kardynala Wyszynskiego, Warszawa); Krystyna Loboz-Grudzien, M.D., Ph.D. (Dolnoslaski Szpital Specjalistyczny im. T. Marciniaka, Wroclaw). Romania (153): Dan-Domenic Ionescu, M.D., Ph.D. (Craiova Cardiology Centre, Craiova); Costel Sorin Stamate, M.D., Ph.D. ("Sf. Pantelimon" Emergency Clinical Hospital, Bucharest); Maria Dorobantu, M.D., Ph.D. ("Floreasca" Emergency Clinical Hospital, Bucharest); Calin Pop, M.D., Ph.D. ("Dr. Constantin Opris" Emergency Clinical County Hospital, Baia Mare); Adrian Matei, M.D., Ph.D. (Cardiovascular Diseases and Transplant Institute, Targu Mures); Tiberiu Nanea, M.D., Ph.D. (Caritas Clinical Hospital, Bucharest); Mariana Radoi, M.D., Ph.D. (Brasov Emergency Clinical County Hospital, Brasov); Adriana Salajan, M.D., Ph.D. (Oradea Emergency Clinical County Hospital, Oradea). Spain (18): Josep Masip, M.D., Ph.D. (Hospital Sant Joan Despí Moises Broggi, Barcelona); Domingo Pascual, M.D., Ph.D. (Hospital Universitario Virgen de la Arrixaca, El Palmar – Murcia); Manuel Gomez Bueno, M.D., Ph.D. (H. Puerta de Hierrro de Majadahonda, Majadahonda); Roberto Muñoz, M.D., Ph.D. (Hospital Infanta Leonor, Madrid). United States of America (114): Sheba Meymandi, M.D. (Olive View-UCLA Medical Center, Sylmar, CA); Philip Levy, M.D., M.P.H. (Wayne State University/ Detroit Receiving Hospital, Detroit, MI); Peter Pang, M.D. (Northwestern University, Chicago, IL); Craig Clark, D.O. (Iowa Health Cardiology, Des Moines, IA); Gregory Fermann, M.D. (University of Cincinnati University Hospital, Cincinnati, OH); Kirkwood Adams, Jr., M.D. (University of North Carolina, Chapel Hill, NC); Biykem Bozkurt, M.D., Ph.D. (Baylor College of Medicine/ Michael E DeBakey VA Medical Center, Houston, TX); James Fulmer, M.D. (River City Clinical Research, Jacksonville, FL); Donna Mancini, M.D. (New York Presbyterian Hospital - Columbia University Medical Center, New York, NY); Timothy Vittorio, M.S., M.D. (Mount Sinai Medical Center, New York, NY); Ronald Zolty, M.D., Ph.D. (Albert Einstein College of Medicine, Bronx, NY); Barry Greenberg, M.D., (University of California San Diego Medical Center, La Jolla, CA); Eugene Chung, M.D. (The Lindner Research Center, Cincinnati, OH); Viorel Florea, M.D., Ph.D., Sc.D. (Minneapolis VA Medical Center, Minneapolis, MN); John Heilman, III, M.D. (Black Hills Cardiovascular Research, Rapid City, SD); Alan Storrow, M.D. (Vanderbilt University Medical Center, Nashville, TN); Maria Rosa Costanzo, M.D. (Midwest Heart Foundation, Oakbrook Terrace, IL); Gervasio Lamas, M.D. (Mount Sinai Medical Center, Miami Beach, FL); Mitchell Greenspan, M.D. (Grand View - Lehigh Valley Health Services, Sellersville, PA); Marc Klapholz, M.D. (University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ); Joaquin Martinez-Arraras, M.D. (Amarillo Heart Clinical Research Institute Inc., Amarillo, TX); Wm. Frank Peacock, M.D. (The Cleveland Clinic, Cleveland, OH); Nizar Saleh, M.D. (St. Mary's Duluth Clinic Health System, Duluth, MN); Roy Small, M.D. (Lancaster Heart & Stroke Foundation, Lancaster, PA); John R. Teerlink, M.D. (San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, CA); Benjamin Trichon, M.D. (Asheville Cardiology Associates, Asheville, NC); Detlef Wencker, M.D. (Hartford Hospital University of Connecticut, Hartford, CT). Executive Committee: Marco Metra, M.D. (Co-Chairperson; University of Brescia, Brescia, Italy); John R. Teerlink, M.D. (Co-Chairperson; University of California at San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco, CA, USA); Gad Cotter, M.D. (Momentum Research Inc., Durham, NC, USA); Beth A. Davison, Ph.D. (Momentum Research Inc., Durham, NC, USA); G. Michael Felker, M.D., M.H.S. (Duke University School of Medicine and Duke Heart Center, Durham, NC, USA); Gerasimos Filippatos, M.D. (Athens University Hospital, Attikon, Athens, Greece); Barry H. Greenberg, M.D. (University of California at San Diego, San Diego, CA, USA); Piotr Ponikowski, M.D., Ph.D. (Medical University, Clinical Military Hospital, Wroclaw, Poland); Thomas Severin, M.D. (Novartis Pharma AG, Basel, Switzerland); Sam L. Teichman, M.D. (Corthera, Inc., San Carlos, CA, USA); Elaine Unemori, Ph.D. (Corthera, Inc., San Carlos, CA, USA); Adriaan A. Voors, M.D., Ph.D. (University of Groningen, Groningen, the Netherlands). Steering Committee: Kirkwood F. Adams, Jr., M.D. (University of North Carolina, Chapel Hill, NC, USA); Maria Dorobantu, M.D. ("Floreasca" Emergency Clinical Hospital, Bucharest, Romania); Liliana Grinfeld, M.D. (Hospital Italiano, Buenos Aires, Argentina); Guillaume Jondeau, M.D. (Hôpital Bichat, Paris, France); Alon Marmor, M.D. (Ziv Medical Center, Safed, Israel); Josep Masip, M.D. (Consorci Sanitari Integral, University of Barcelona, Barcelona, Spain); Peter S. Pang, M.D. (Northwestern University, Chicago, IL, USA); Karl Werdan, M.D. (Martin-Luther-Universität Halle-Wittenberg, Halle, Germany).

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Data and Safety Monitoring Board: Barry M. Massie (Chairperson; University of California at San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco, CA, USA); Michael Böhm, M.D. (Klinik fur Innere Medizin III Kardiologie, Hamburg, Germany); Sidney Goldstein, M.D. (Henry Ford Hospital, Detroit, MI, USA); Gary Francis, M.D. (University of Minnesota, Minneapolis, MN, USA). Clinical Events Committee: G. Michael Felker, M.D., M.H.S. (Chairperson; Duke Clinical Research Institute, Durham, NC, USA); Javed Butler, M.D. (Emory University Hospital Center for Heart, Atlanta, GA, USA); Adrian F. Hernandez, M.D. (Duke Clinical Research Institute, Durham, NC, USA); Alan B. Miller, M.D. (University of Florida Health Sciences Center, Jacksonville, FL, USA). B. Study Conduct and Oversight: RELAX-AHF was a randomised, double-blind, placebo-controlled, international trial comparing serelaxin with placebo in patients hospitalised for acute heart failure at 96 sites in 11 countries. The study, sponsored by Corthera (a Novartis company), was part of a Phase II/III trial designed by an executive committee consisting of 5 investigators, two Momentum clinical scientists, and two Corthera clinical scientists.18 The background and design of this study have been published,19 and the protocol is included in the Supplementary Appendix. The protocol was approved by the ethics committee at each participating centre, and patients provided written informed consent. The trial was conducted in accordance with current Good Clinical Practice. An independent data and safety monitoring committee, supported by an independent statistical centre (Statistics Collaborative, Inc.), reviewed accumulated safety data after 222, 419, 574 and 759 patients had Day 14 data. During enrolment, the DSMC Chair reviewed monthly summaries of SAEs, demographics and protocol compliance. There were no pre-specified stopping rules for efficacy. Hospitalisations and deaths within 60 days were adjudicated by an independent clinical events committee that was unaware of treatment assignments, and deaths from day 61 to 180 days were investigator reported. Post-database lock and still blinded to treatment assignment, the clinical events committee also adjudicated all of the deaths within 180 days after randomisation. Three independent contract research organisations (Covance, PPD, Tango) monitored sites. Statistical analyses were performed by Accovion GmbH with the use of SAS software, version 9·2 (SAS Institute; Cary, NC), and these analyses were subsequently confirmed by the sponsor. The executive committee had full access to the final data set. The co-chairpersons (J.R.T.; M.M.) prepared the initial draft of the manuscript, which was revised on the basis of the comments of the other authors, each of whom approved the final version. The decision to submit the manuscript for publication was made by the members of the Executive Committee, who vouch for the data and the analysis and for the fidelity of the study to the protocol. (ClinicalTrials.gov Identifier NCT00520806) C. Inclusion and Exclusion Criteria: Inclusion Criteria: Subjects must fulfill all of the following criteria at screening to be eligible for the study. The screening period is defined as that interval that begins at the time the informed consent is signed and ends with the qualification of the subject for entry into the study (i.e. when subject is determined to meet all eligibility criteria). 1. Able to provide written informed consent 2. Male or female ≥ 18 years of age, with body weight < 160 kg 3. Systolic blood pressure > 125 mmHg at the start of screening and at the end of screening 4. Hospitalised for AHF. AHF is defined as including all of the following measured at any time between

presentation (including the emergency department [ED]) and the end of screening: a. Dyspnoea at rest or with minimal exertion b. Pulmonary congestion on chest radiograph c. BNP ≥ 350 pg/mL or NT-pro-BNP ≥ 1400 pg/mL 5. Able to be randomised within 16 hours from presentation to the hospital, including the ED 6. Received IV furosemide of at least 40 mg (or equivalent) at any time between admission to emergency services

(either ambulance or hospital, including the ED) and the start of screening for the study 7. Impaired renal function defined as an estimated glomerular filtration rate (eGFR) on admission between 30-75

mL/min/1·73 m2, calculated using the simplified Modification of Diet in Renal Disease (sMDRD) equation

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Exclusion Criteria: 1. Pregnant or breast-feeding women (women of child bearing potential must have the results of a negative

pregnancy test recorded prior to study drug administration) 2. Administration of intravenous radiographic contrast agent within 72 hours prior to screening or acute contrast-

induced nephropathy at the time of screening 3. Temperature >38°C (oral or equivalent) or sepsis or active infection requiring IV anti-microbial treatment 4. Current (within 2 hours prior to screening) or planned (through the completion of study drug infusion) treatment

with any IV therapies, including vasodilators (including nesiritide), positive inotropic agents and vasopressors, or mechanical support (intra-aortic balloon pump, endotracheal intubation, mechanical ventilation, or any ventricular assist device), with the exception of IV furosemide (or equivalent), or of IV nitrates at a dose of < 0·1 mg/kg/hr if the patient has a systolic BP > 150 mmHg at screening

5. Current or planned ultrafiltration, hemofiltration, or dialysis 6. Known significant pulmonary disease 7. Known significant valvular disease (including any of the following: severe aortic stenosis [AVA < 1·0 or mean

gradient > 50 on prior or current echocardiogram], severe aortic regurgitation, or severe mitral stenosis) 8. Any organ transplant recipient, or patient currently listed for imminent transplant (i.e., does not exclude patients

on an administrative transplant waiting list), or admitted for any transplantation 9. Major surgery within 30 days 10. Hematocrit < 25% or blood transfusion in the prior 14 days or active, life-threatening GI bleeding; or active

menorrhagia or metrorrhagia 11. Major neurologic event, including cerebrovascular events, in the prior 60 days 12. Clinical diagnosis of acute coronary syndrome within 45 days prior to screening (including the present

admission) as determined by both clinical and enzymatic criteria 13. Troponin > 3 times the upper limit of normal (including "borderline/intermediate") between presentation and the

end of screening 14. AHF due to significant arrhythmias (including any of the following: ventricular tachycardia, bradyarrhythmias

with ventricular rate <45 beats per minute or any second or third degree AV block or atrial fibrillation/flutter with ventricular response of >120 beats per minute)

15. Acute myocarditis or hypertrophic obstructive, restrictive, or constrictive cardiomyopathy (does not include restrictive mitral filling patterns seen on Doppler echocardiographic assessments of diastolic function)

16. Known hepatic impairment 17. Non-cardiac pulmonary oedema, including suspected sepsis 18. Administration of an investigational drug or implantation of investigational device, or participation in another

trial, within 30 days before screening or previous treatment with relaxin 19. Inability to follow instructions or comply with follow-up procedures 20. Known hypersensitivity to relaxin or similar substances or to any of the excipients D. Additional Study Procedures: Physical examinations were performed at all scheduled time points. Non-serious adverse events were reported through day 5, and serious adverse events through day 14. Blood pressure and heart rate were monitored frequently during study drug infusion and through 48 hours after study drug discontinuation. More frequent monitoring was required for systolic blood pressure decreases >40 mmHg from baseline or to <100 mmHg. Central laboratories analysed standard haematology, chemistry and urinalysis, as well as neurohormonal assays. All dyspnoea assessments were performed with the patient resting supine, with the bed's head elevated to 30° with no supplemental oxygen for 3 to 5 minutes. Trained site staff members administered the assessment by reading verbatim from a linguistically validated translation of the scales. E. Additional Efficacy Analyses The following analyses were pre-specified, but there were no adjustments for multiple comparisons. • Change from baseline in dyspnea score by VAS at 6, 12, 24 and 48 hours and at Days 5 and 14; • Area under the curve representing the change in dyspnea VAS from baseline for baseline to Day 14, Day 1 to Day

5 and Day 1 to Day 14; • Markedly or moderately better dyspnea compared to baseline by Likert scale assessed separately at 6, 12, and 24

hours and then daily to Day 5 and at Day 14;

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• Time to moderately or markedly better self-assessed dyspnea on the 7-point Likert scale up to Day 5; • Time to worsening heart failure (WHF) through Day 5 and through Day 14; • Total doses of IV loop diuretics and oral loop diuretics through Day 5 or discharge if earlier; • Change in body weight from randomization through Day 5 and Day 14; • Length of hospital stay; • All cause death or rehospitalization due to heart failure or renal failure through Day 60; • Days alive and out of hospital through Day 30; • Cardiovascular death through Day 180; • Change from baseline in heart failure signs and symptoms (subject-reported dyspnea and general well-being using

Likert scale and VAS, and physician assessments). • Days of the initial hospitalization from randomization spent in the intensive care unit/coronary care unit

(ICU/CCU) • All cause death through Day 30; and • All cause death or worsening heart failure or rehospitalization due to heart failure through Day 30. • Cardiovascular (CV) death or rehospitalization due to heart failure (HF) or renal failure (RF) through Day 30 • Cardiovascular death or rehospitalization due to heart failure or renal failure through 30 days following discharge

from the index hospitalization F. Method for calculating the Dyspnoea Visual Analogue Scale Area Under the Curve The area under the curve representing the change from baseline in dyspnea VAS score from baseline through Day 5 (VAS AUC) will be computed after applying the data handling conventions described in Section . For each subject, the change in dyspnea VAS scores from baseline (Day 0) will be computed by subtracting the baseline score from each post-baseline dyspnea VAS score. The area under the curve for the change from baseline values (mm-hour) will be computed using the trapezoidal rule. Subjects who die or have a worsening heart failure event (either during the index hospital or rehospitalization due to heart failure) will have their score imputed to be the worst observed score in any subject at any time point in the respective analysis population carried forward from the day of death or WHF regardless of whether their original data are missing. For post-baseline visual analog scale (VAS) values otherwise missing, a missing score will be imputed using linear interpolation between the last preceding and first following non-missing values. If no following non-missing value is available, the last available preceding value will be carried forward. A missing baseline score will be imputed as the earliest, non-missing score within 24 hours for the subject minus the average change from baseline in the study population to that time point; post-baseline scores for subjects for whom a missing baseline cannot be thus imputed will be included in the analysis as no change from baseline. Except for subjects who die or who experience WHF, subjects who are missing all post-baseline dyspnea VAS scores will be included in the analysis as having no change from baseline at any time point. G. Adjudication of Endpoints Day 60 EndpointAdjudications. Momentum Research, Inc. (MRI) will coordinate with Medpace to identify all events that may be potential endpoints that have occurred in the trial. Medpace will work with the DCC and the site, and as appropriate, the contract research organization (CRO), in the collection of CRFs and medical records for the event. Medpace will review all available CRF and source documentation for the event and determine whether additional medical records will be required to support adjudication of the event. Medpace will query the site for available supporting medical records prior to sending the packet to the CEC for review. All queries and responses will be recorded and tracked. Should the requested information not be available, this will be noted on the Adjudication Worksheet. Medpace will proceed with a thorough evaluation of the suspected event using appropriate clinical judgment. The Adjudication Worksheet will be used as a guide in forwarding supporting case materials (e.g., catheterization reports, hospital records, autopsy reports, etc.). The supporting medical records will have all patient identifiers (e.g., name, social security number, etc.) removed or completely obscured prior to submission to Medpace. Medpace will ensure that all relevant source documentation has been redacted and translated into English prior to declaring the packet complete and ready for adjudication. Once the event(s) has been reviewed by Medpace, the Adjudication Worksheet, copies of the pertinent source documentation, and relevant CRF information will be organized by Medpace into a complete endpoint package. The package will be reviewed for completeness by Medpace prior to posting for CEC for adjudication. Rehospitalization cases will be assigned to reviewers using a randomized sequential listing. For efficiency, 3 or more cases will

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typically be assigned at a given time in a batch. Each batch will be assigned to two committee members. The individual cases will be assigned a batch number and then scanned and posted to a secure website for CEC review. Because all death cases require adjudication by all four committee members, these will be posted on an ongoing basis for committee review. Day 180 Endpoint Adjudications: Deaths occurring from Day 61 through Day 180 will be adjudicated retrospectively following the Day 180 clinical database lock. Data on these deaths have been collected on an ongoing basis by Medpace Safety and narrative written in order to supplement the safety database. No new information will be gathered specifically for the adjudications. The available information will be assembled by Medpace into a package for posting, as described above. Definition of Events for CEC Adjudication: Events requiring adjudication include: � Death within 180 days � Rehospitalization within 60 days All deaths that occur within 180 days from the start of study drug infusion or date of randomization if the subject was randomized but not treated with study drug will be adjudicated. All hospitalizations that occur within 60 days from the start of study drug infusion and after discharge from the index hospitalization will be adjudicated. If the subject dies within 24 hours of hospital presentation, this event will be adjudicated as a death. If the subject was randomized but not treated with study drug, the date of randomization will be used in place of start of study drug infusion. In addition, a rehospitalization for the purposes of this study must be for an urgent, not elective, reason and must be of duration of 24 hours or greater from presentation to discharge, including any time in the emergency room or observation unit. If the time of either presentation or discharge is unknown, then a calendar date change should be considered when assessing the latter requirement. All unplanned rehospitalizations will be reviewed and the primary reason for hospitalization adjudicated. Source documentation will be reviewed for determination as to whether the event meets the classification criteria established in this charter or based upon subsequent adjudication precedents and described in the meeting minutes to ensure consistency. Decisions by the committee will be based on the documentation provided and good clinical judgment. If the event does not fulfill all of the criteria, but the clinical picture is strongly suggestive of an event of interest, good clinical judgment will be used to classify the event as meeting the criteria. These will be discussed at meetings and described in the meeting minutes to ensure committee consensus and subsequent consistency. Individual decisions will be justified on the individual committee member Adjudication Forms. Selected Endpoint Definitions: All unplanned rehospitalizations will be reviewed and the primary reason for the rehospitalization adjudicated and classified into one of the following categories: � Rehospitalization due to heart failure � Rehospitalization due to renal failure � Rehospitalization due to causes other than heart failure or renal failure Rehospitalization is defined as an urgent and not previously scheduled admission to an inpatient unit or a visit to an emergency department that results in at least a 24 hour stay (or a date change if the time of admission/ discharge is not available). Elective, scheduled admissions for diagnostic (eg, coronary angiography) or therapeutic (eg, pacemaker revision) reasons that occur as a follow-up to the index hospitalization or other rehospitalization while on-study will not be classified as a study endpoint. The CEC will adjudicate whether a rehospitalization was urgent and not previously scheduled or was elective. The CEC will adjudicate whether an emergency department visit or other visit to an inpatient unit met study criteria for rehospitalization, ie, lasted >24 hours or in the absence of clock times, whether a calendar date change occurred between admission and discharge. Rehospitalization due to heart failure will be defined as an event that occurred primarily because of the documented presence of at least 2 of the following 3 criteria: � Clinical manifestations of heart failure � Biomarker or radiographic evidence consistent with heart failure � Use of additional or increased pharmacologic or mechanical interventions directed at the treatment of heart failure

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Clinical manifestations of heart failure including the following signs and symptoms: New or worsening � dyspnea � orthopnea � paroxysmal nocturnal dyspnea � edema � pulmonary rales � jugular venous distension � new or worsening third heart sound or gallop rhythm � hypotension or cardiogenic shock not occurring in the context of an acute myocardial infarction or as the consequence of an arrhythmia, or

� other clinical evidence of new or worsening heart failure, eg, weight gain, or confinement to bed predominantly due to heart failure symptoms

Biomarker results consistent with heart failure include documented increased or increasing levels of a natriuretic peptide (BNP or NTproBNP) Radiographic evidence consistent with heart failure includes documented worsening pulmonary congestion or pulmonary edema on chest X-ray or other generally recognized imaging pattern. The use of additional or increased pharmacologic or mechanical interventions directed at the treatment of heart failure includes: � Initiation of intravenous diuretic, inotropic, or vasodilator therapy � Significant addition or increase in oral heart failure therapy � Up-titration of intravenous therapy, if already on therapy � Initiation of mechanical or surgical intervention (mechanical circulatory or ventilatory support, heart transplantation or ventricular pacing to improve cardiac function), or the use of ultrafiltration, hemofiltration, or dialysis that is specifically directed at treatment of heart failure.

If the reviewer judges that heart failure was not the primary reason for the rehospitalization, but that heart failure contributed to the rehospitalization, this should be indicated on the adjudication form. Rehospitalization due to renal failure is defined as when the reason for hospitalization is primarily due to signs, symptoms or laboratory abnormalities directly related to renal dysfunction; no other acute perturbation is responsible for the admission; and therapeutic interventions are directed toward renal failure. Renal failure requiring rehospitalization is classified as one of the following: � Acute renal failure � Acute on chronic renal failure / Worsening chronic renal failure Acute renal failure will be determined to be the cause of rehospitalization when a patient with a recently documented normal creatinine (eg, less than 1.5 mg/dl) meets the above criteria for a hospital admission and the primary reason is a documented greater than 50% increase in creatinine compared to the most recently available measurement, and/or oliguria or anuria upon presentation. Acute on chronic renal failure / Worsening chronic renal failure will be determined to be the cause of rehospitalization when a patient with a recently documented abnormal creatinine (eg, greater than 1.5 mg/dl creatinine) meets the above criteria for a hospital admission and the primary reason is a documented greater than 0.5 mg/dl increase in creatinine compared to the most recent available measurement, or oliguria or anuria is present upon presentation, and/or is admitted for dialysis. For additional specific criteria, the CEC referred to the US FDA draft guidance entitled, Standardized Definitions for Cardiovascular Outcomes Trials, dated 22 July 2009.

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Section 2: Supplemental Results Appendix Figure A1: Change in systolic blood pressure during study drug infusion

Mean (SEM)

9

Appendix Figure A2: All cause death through day 180 by treatment group (Safety population)

Placebo: 64 deaths (11.3%) Serelaxin: 41 deaths (7.3%) HR 0.63 (0.42, 0.93) p=0.02

10

Appendix Table A1: Physician assessment of heart failure signs and symptoms Placebo Serelaxin P-value† Change from baseline in dyspnoea on exertion

scoreA

Hour 6 -0·4 (0·61) -0·4 (0·62) 0·505 Hour 12 -0·6 (0·76) -0·7 (0·74) 0·014 Day 1 -0·9 (0·88) -1·0 (0·78) 0·061 Day 2 -1·1 (0·92) -1·3 (0·88) 0·017 Day 5 -1·5 (1·07) -1·5 (0·97) 0·285 Day 14 -1·4 (1·14) -1·5 (1·08) 0·461 Change from baseline in orthopnoea scoreB Hour 6 -0·4 (0·69) -0·4 (0·70) 0·585 Hour 12 -0·6 (0·83) -0·8 (0·85) 0·004 Day 1 -1·0 (0·97) -1·1 (0·93) 0·005 Day 2 -1·2 (1·05) -1·4 (1·00) 0·002 Day 5 -1·3 (1·18) -1·4 (1·09) 0·075 Day 14 -1·2 (1·27) -1·3 (1·22) 0·444 Change from baseline in oedema scoreC Hour 6 -0·1 (0·51) -0·2 (0·48) 0·125 Hour 12 -0·3 (0·65) -0·4 (0·65) 0·041 Day 1 -0·6 (0·84) -0·7 (0·82) 0·116 Day 2 -0·7 (1·02) -0·9 (0·95) 0·010 Day 5 -0·8 (1·21) -1·0 (1·13) 0·031 Day 14 -0·7 (1·32) -0·9 (1·30) 0·070 Change from baseline in rales scoreD Hour 6 -0·4 (0·62) -0·4 (0·61) 0·109 Hour 12 -0·6 (0·77) -0·7 (0·72) 0·024 Day 1 -0·8 (0·86) -1·0 (0·81) 0·002 Day 2 -1·0 (0·98) -1·2 (0·86) 0·008 Day 5 -1·1 (1·15) -1·2 (0·99) 0·020 Day 14 -1·0 (1·28) -1·1 (1·15) 0·052 Change from baseline in JVP scoreE Hour 6 -0·2 (0·49) -0·2 (0·46) 0·130 Hour 12 -0·3 (0·60) -0·4 (0·56) 0·239 Day 1 -0·5 (0·76) -0·6 (0·68) 0·026 Day 2 -0·6 (0·85) -0·7 (0·75) 0·058 Day 5 -0·6 (0·94) -0·7 (0·82) 0·202 Day 14 -0·6 (1·01) -0·6 (0·92) 0·499

11

†Wilcoxon rank sum test

ANYHA=New York Heart Association class; 0=none (NYHA I), 1=mild (NYHA II), 2=moderate (NYHA III), 3=severe (NYHA IV) B0=none, 1=1 pillow (10 cm), 2=2 pillows (20 cm), 3=>30 degrees C0=0 (Complete absence of skin indentation with mild digital pressure in all dependent areas), 1=1+ (Indentation of skin that resolves over 10–15 seconds), 2=2+(Indentation of skin is easily created with limited pressure and disappears slowly (15–30 seconds or more)), 3=3+(Large areas of indentation easily produced and slow to resolve (> 30 seconds)) D0=no rales, 1=rales <1/3, 2=rales 1/3-2/3, 3=rales>2/3 EJVP=Jugular venous pressure; 0=<6 cm, 1=6-10 cm, 2=>10 cm

12

Section 3: Adverse Events and Safety

Overview of all-treatment emergent AEs

All treatment-emergent adverse events (AEs), i.e., non-serious AEs through Day 5, serious adverse events (SAEs) through Day 14, were reported for a total of 56·1% of patients on placebo and 53·7% of patients treated with serelaxin. A total of 3·9% on placebo and 4·6% of patients on serelaxin discontinued due to an AE. SAEs and SAEs leading to discontinuation through Day 14 (Appendix Table A2) occurred in 13·7% and 0·5% of patients receiving placebo and in 15·1% and 0·9% of those receiving serelaxin, respectively.

Appendix Table A2 RELAX-AHF: Overview of incident adverse events (AEs) – (safety analysis set)

Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Subjects With any AE 320 (56·1) 305 (53·7)

Subjects With Any Drug-Related AE[1] 46 ( 8·1) 47 ( 8·3)

Subjects with Any AE Leading to Study Drug Discontinuation 22 ( 3·9) 26 ( 4·6)

Subjects with any SAE 78 (13·7) 86 (15·1)

Subjects with any Drug-Related SAEs[1] 2 ( 0·4) 3 ( 0·5)

Subjects with Any SAE Leading to Drug Discontinuation 3 ( 0·5) 5 ( 0·9)

Serious AE With an Outcome of Death 15 ( 2·6) 10 ( 1·8)

[1] AEs assessed as definite, probable, or possible are considered drug-related. Note: Incident AEs are considered those AEs with an onset date and time after the initiation of study drug.

All-treatment emergent AEs through Day 5

Due to the different collection periods of non-serious and serious AEs, an analysis of all AEs by system organ class (SOC) through Day 5 has been performed (Appendix Table A3). As expected for this study population with acute heart failure (AHF), the majority of AEs were observed in the SOCs of Cardiac disorders (placebo: 15·8%; serelaxin: 12·3%) with Cardiac failure congestive (placebo: 5·6%; serelaxin: 3·3%), Cardiac failure (placebo: 1·6%; serelaxin 0·7%) and Ventricular tachycardia (placebo: 1·8%; serelaxin 0·7%) as the most frequently reported AEs in this SOC.

Broadly, the AE profile by SOC and preferred term (PT) was comparable between serelaxin and placebo, and in particular did not suggest a deleterious effect of serelaxin. The majority of observed PTs reflected the underlying conditions of AHF and concomitant diseases. Notably, there was a trend towards more frequently reported AEs in the placebo arm compared to serelaxin, which was more prominent for the SOCs (>1·5% difference) of Cardiac disorders, Gastrointestinal disorders, Hepatobiliary disorders, Investigations, Nervous system disorders, Psychiatric disorders, Respiratory, thoracic and mediastinal disorders and Vascular disorders. Although specific PT incidence rates were small and do not allow for generalisation, PT analysis per SOC may indicate that serelaxin treatment resulted in preserved organ function, including less congestion, compared to placebo. For AEs of interest see below.

Appendix Table A3 RELAX-AHF: Incidence of adverse events by System Organ Class (SOC) from study drug initiation to Day 5 (safety analysis set)

System Organ Class

Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Subjects with at least one AE 305 (53·5) 280 (49·3)

Total number of AEs 703 539

13

System Organ Class

Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Blood and lymphatic system disorders 6 ( 1·1) 10 ( 1·8)

Cardiac disorders 90 (15·8) 70 (12·3)

Ear and labyrinth disorders 3 ( 0·5) 2 ( 0·4)

Endocrine disorders 3 ( 0·5) 1 ( 0·2)

Eye disorders 1 ( 0·2) 3 ( 0·5)

Gastrointestinal disorders 59 (10·4) 45 ( 7·9)

General disorders and administration site conditions 39 ( 6·8) 32 ( 5·6)

Hepatobiliary disorders 10 ( 1·8) 1 ( 0·2)

Infections and infestations 27 ( 4·7) 24 ( 4·2)

Injury, poisoning and procedural complications 0 3 ( 0·5)

Investigations 40 ( 7·0) 31 ( 5·5)

Metabolism and nutrition disorders 68 (11·9) 71 (12·5)

Musculoskeletal and connective tissue disorders 21 ( 3·7) 27 ( 4·8)

Neoplasms benign, malignant and unspecified (incl cysts and polyps)

2 ( 0·4) 4 ( 0·7)

Nervous system disorders 45 ( 7·9) 30 ( 5·3)

Psychiatric disorders 31 ( 5·4) 22 ( 3·9)

Renal and urinary disorders 32 ( 5·6) 26 ( 4·6)

Reproductive system and breast disorders 0 2 ( 0·4)

Respiratory, thoracic and mediastinal disorders 59 (10·4) 31 ( 5·5)

Skin and subcutaneous tissue disorders 2 ( 0·4) 7 ( 1·2)

Vascular disorders 41 ( 7·2) 29 ( 5·1)

Serious adverse events (SAEs) through Day 14

Overall, no clinically significant differences were observed in the SAE incidence rates between treatment groups. SAEs with an incidence rated of >0·5% by treatment group through Day 14 are presented in Appendix Table A4.

Slightly more patients on serelaxin experienced an SAE of heart failure including the PTs Cardiac failure, Cardiac failure acute, Cardiac failure congestive (placebo: 17 SAEs, serelaxin: 21) or pulmonary oedema (placebo: 0 SAEs, serelaxin: 5; PTs - Pulmonary oedema, Pulmonary oedema acute). Relative to the length of the time interval (Day 6-14 versus Day 0-5), the majority of SAEs were observed within the first five days; there was no notable difference in the safety profile of SAEs reported through Day 5 and Day 14, respectively.

There were 15 (2·6%) patients on placebo and 10 (1·8%) patients on serelaxin who experienced a fatal SAE (Appendix Table A5), however it is of note that the majority of deaths in this trial were observed after Day 14 and either collected as efficacy endpoint (Cardiovascular death through Day 60) or as safety endpoint (all-cause mortality through Day 180). None of the SAEs were assessed as suspected and unexpected (SUSAR) and there was no finding suggesting a significant safety risk to patients.

Appendix Table A4 RELAX-AHF: Incidence of serious adverse events at a rate >0·5% from study drug initiation to Day 14 (safety analysis set)

Preferred Term Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Subject with any SAE 78 (13·7) 86 (15·1)

Total Number of SAEs 107 105

14

Anaemia 3 ( 0·5) 0

Acute myocardial infarction 3 ( 0·5) 3 ( 0·5)

Bradyarrhythmia 4 ( 0·7) 2 ( 0·4)

Cardiac arrest 3 ( 0·5) 0

Cardiac failure acute 2 ( 0·4) 4 ( 0·7)

Cardiac failure congestive 12 ( 2·1) 10 (1·8)

Cardiac failure 3 ( 0·5) 7 ( 1·2)

Ventricular fibrillation 1 ( 0·2) 3 ( 0·5)

Pneumonia 5 ( 0·9) 1 ( 0·2)

Cerebrovascular accident 3 ( 0·5) 2 ( 0·4)

Renal failure 6 ( 1·1) 6 ( 1·1)

Acute pulmonary oedema 0 4 ( 0·7)

At each level of summation (System Organ Class (SOC) and Preferred Term), subjects are counted once per SOC and preferred term. Incident AEs are considered those AEs with an onset date and time after the initiation of study drug.

Appendix Table A5 Incidence of Serious Adverse Events with an outcome of death through Day 14 (safety analysis set)

System Organ Class Preferred Term

Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Subject with SAEs with an outcome of death 15 ( 2·6) 10 ( 1·8)

Total number of SAEs with an outcome of death 17 10

Cardiac disorders 8 ( 1·4) 4 ( 0·7)

Acute myocardial infarction 1 (0·2) 0

Cardiac arrest 2 (0·4) 0

Cardiac failure acute 0 1 (0·2)

Cardiac failure congestive 1 (0·2) 0

Cardiac failure 2 (0·4) 1 (0·2)

Cardiac tamponade 1 (0·2) 0

Cardiogenic shock 1 (0·2) 1 (0·2)

Dissecting coronary artery aneurysm 1 (0·2) 0

Pulseless electrical activity 0 1 (0·2)

Infections and infestations 1 (0·2) 1 (0·2)

Pneumonia 1 (0·2) 0

Sepsis 0 1 (0·2)

Neoplasms benign, malignant and unspecified (incl. cysts and polyps)

1 (0·2) 1 (0·2)

Lung cancer metastatic 1 (0·2) 0

Lung neoplasm 0 1 (0·2)

Nervous system disorders 3 (0·5) 2 (0·4)

Cerebrovascular accident 2 (0·4) 2 (0·4)

Embolic stroke 1 (0·2) 0

Renal and urinary disorders 1 (0·2) 1 (0·2)

Renal failure 1 (0·2) 1 (0·2)

Respiratory, thoracic and mediastinal disorders 2 (0·4) 1 (0·2)

Acute pulmonary oedema 0 1 (0·2)

Pulmonary haemorrhage 1 (0·2) 0

15

System Organ Class Preferred Term

Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Respiratory failure 1 (0·2) 0

Note: At each level of summation (System Organ Class (SOC) and Preferred Term), subjects are counted once per SOC and Preferred Term. Incident AEs are considered those AEs with an onset date and time after the initiation of study drug.

Adverse events of interest

AEs of special interest included events related to hypotension, renal impairment, hypersensitivity reactions and changes in RBC parameters including anaemia, which were reported through day 14. While slightly more patients with AEs indicative of hypotension were reported for patients treated with serelaxin, there were more patients in the placebo group who experienced an AE of renal and hepatic dysfunction. Incidence rates of these events through Day 5 (Appendix Table A6) and Day 14 (Appendix Table A7) are presented below. Events suggesting changes in RBC parameters/Anaemia, Hypersensitivity reactions were occasionally reported in both treatment groups and comparable; events indicative of QT prolongation comprised a variety of different PTs (SMQ) and their incidence rate was slightly higher in the placebo arm versus serelaxin (8·4% versus 6·2%).

Appendix Table A6 Incidence of adverse events of interest through Day 5 (safety analysis set)

AEs of interest Placebo (N=570)

n (%)

Serelaxin (N=568)

n (%)

Total patients, who experienced an AE indicative of hypotension

25 (4·4) 28 (4·9)

Blood pressure decreased 2 (0·4) 3 (0·5)

Dizziness 11 (1·9) 7 (1·2)

Loss of consciousness 0 1 (0·2)

Hypotension 9 (1·6) 15 (2·6)

Orthostatic hypotension 0 1 (0·2)

Presyncope 1 (0·2) 0

Somnolence 0 1 (0·2)

Syncope 2 (0·4) 1 (0·2) Total % of patients, who experienced an AE indicative of renal impairment 49 (8·6) 26 (4·6)

Azotaemia 1 (0·2) 1 (0·2)

Blood creatinine increased 22 (3·9) 14 (2·5)

Oliguria 1 (0·2) 0

Proteinuria 2 (0·4) 0

Renal failure 23 (4·0) 9 (1·6)

Renal failure acute 0 1 (0·2)

Renal impairment 1 (0·2) 1 (0·2)

Total % of patients, who experienced an AE indicative of hepatic impairment

16 (2·8) 4 (0·7)

Blood bilirubin increased 2 (0·4) 0

Cholestasis 1 (0·2) 0

Hepatic congestion, 3 (0·5) 0 Hepatic cyst, 1 (0·2) 0

Hepatic steatosis 1 (0·2) 0

16


n (%)

Serelaxin (N=568)

n (%)

Hyperbilirubinaemia 2 (0·4) 0 Hypoalbuminaemia 1 (0·2) 2 (0·4)

INR increased 4 (0·7) 2 (0·4)

Liver disorder 1 (0·2) 0

Appendix Table A7 Incidence of adverse events of interest through Day 14 (safety analysis set)


n (%)

Serelaxin (N=568)

n (%)

Total patients, who experienced an AE indicative of hypotension

27 (4·7) 28 (4·9)

Blood pressure decreased 2 (0·4) 3 (0·5)

Dizziness 11 (1·9) 7 (1·2)

Loss of consciousness 0 1 (0·2)

Hypotension 9 (1·6) 15 (2·6)

Orthostatic hypotension 0 1 (0·2)

Presyncope 1 (0·2) 0

Somnolence 0 1 (0·2)

Syncope 4 (0·7) 1 (0·2) Total % of patients, who experienced an AE indicative of renal impairment 51 (8·9) 32 (5·6)

Azotaemia 1 (0·2) 1 (0·2)

Blood creatinine increased 23 (4·0) 14 (2·5)

Oliguria 1 (0·2) 0

Proteinuria 2 (0·4) 0

Renal failure 25 (4·4) 14 (2·5)

Renal failure acute 0 2 (0·4)

Renal impairment 1 (0·2) 1 (0·2)

Total % of patients, who experienced an AE indicative of hepatic impairment

16 (2·8) 5 (0·9)

Blood bilirubin increased 2 (0·4) 0

Cholestasis 1 (0·2) 0

Hepatic congestion, 3 (0·5) 0 Hepatic cyst, 1 (0·2) 0

Hepatic steatosis 1 (0·2) 0

Hyperbilirubinaemia 2 (0·4) 0 Hypoalbuminaemia 1 (0·2) 2 (0·4)

INR increased 4 (0·7) 3 (0·5)

Liver disorder 1 (0·2) 0

17

RLX.CHF.003 (PRE-RELAX-AHF / RELAX-AHF) PAGE 1 OF 99 AMENDED PROTOCOL VERSION 05 CLEAN

Corthera, Inc. January 12, 2012 CONFIDENTIAL

1 TITLE PAGE

CORTHERA, INC.

PROTOCOL

Study Title: A Phase II/III, Multicenter, Randomized, Double-blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety of Relaxin in Subjects With Acute Heart Failure

Protocol Number: RLX.CHF.003 (Pre-RELAX-AHF/ RELAX–AHF) Investigational Product:

Human Relaxin (RLX030)

Indication: Acute Heart Failure Sponsor: Corthera, Inc.

150 Industrial Road San Carlos, CA 94070

Phone: (650) 235-3555 Fax: (650) 235-3579

Legal Representative Europe

Corthera (Europe) Ltd Frimley Business Park Frimley, Camberley, Surrey. GU16 7SR United Kingdom

EUDRACT Number 2007-004271-19 Development Phase: II/III Responsible Medical Officer:

Thomas Severin, MD, Global Program Medical Director

Phone: +41 61 324 5424 Fax: + 41 61 324 2130

Sponsor Signatory: Elaine Unemori, Ph.D. Medical Monitors: Gad Cotter, MD

3100 Tower Blvd. Suite 802 Durham, NC 27707 Office: 919-287-1824 Mobile: 919-599-0949 Fax: 919-287-1825 E mail: [email protected]

Thomas Severin, MD Global Program Medical DirectorCardiovascular and Metabolism Novartis Pharma AG CH-4002 Basel, Switzerland Phone: + 41 61 324 5424 Mobile: + 41 79 591 0948 E mail: [email protected]

Date: Amended Protocol Version 0512 Jan 2012

Confidential----May not be divulged, published, or otherwise disclosed to others without written approval from Corthera, Inc. This study will be conducted according to the principles of Good Clinical Practice as described in International Conference on Harmonization guidelines, including the archiving of essential documents

This document (090095af8323e33d in docbase CREDI_EH) has been digitally signed with external signatures using Entrust PKI.Signatures manifested as of 2/5/2012 3:43:10 PM, signing status at this time: Completed (1 of 1 signatures) Approved for report publication by Trapani Angelo in East Hanover at Sun, 05 Feb 2012 10:40:35 AM EST

18



1.1 Signature Page

RLX.CHF.003 A Phase II/III, Multicenter, Randomized, Double-blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety of Relaxin in Subjects With Acute Heart Failure

I agree to the procedures relating to this study as set out in this Protocol and Case Report Form. I fully understand that any changes instituted by the Investigator(s) without previous discussion with the Sponsor would constitute a violation of the protocol, including any ancillary studies or procedures performed on study subjects (other than those procedures necessary for the well being of the subjects).

I agree to follow ICH guidelines for good clinical practice and will obtain approval from the IRB/IEC prior to study start, allow direct access to source documents and agree to inspection by auditors from the Sponsor and regulatory authorities, as required by ICH GCP. I will assure that the investigational product(s) supplied by the Sponsor will be used only as described in this protocol.

I have read the protocol and agree to conduct the study in compliance with all applicable regulations and guidelines as stated in the protocol and other information supplied to me, including ICH Topic E6.

Investigator Signature Date

Print name:

On behalf of Corthera, Inc., I confirm that the Sponsor will comply with obligations as detailed in all applicable regulations and guidelines. I will ensure that the Investigator is informed of all relevant information that becomes available during the conduct of this study.

VP Clinical Development Signature Date

Print name:


19



2 CONTACT INFORMATION

Sponsor: Corthera, Inc.

150 Industrial Road San Carlos, CA 94070

Phone: +1 650 235 3555 Fax: +1 650 235 3579 Legal Representative Europe Corthera (Europe) Ltd

Frimley Business Park Frimley, Camberley, Surrey. GU16 7SR United Kingdom

Coordinating Center MOMENTUM Research, Inc 3100 Tower Boulevard, Suite 802 Durham, NC 27707 Phone: + 1 919 287 1824 Fax: +1 919 287 1825


20



AMENDMENT 5 TO PROTOCOL RLX.CHF.003

Amendment rationale The principal purpose of Amendment 5 is to: 1) clarify three eligibility criteria (two of which were previously provided to clinical sites in protocol clarification memos), 2) provide a study-specific definition of Worsening Heart Failure (WHF), 3) align endpoints with the Statistical Analysis Plan and use wording consistent with the Statistical Analysis Plan when referencing analysis populations, 4) clarify information related to the Day 60 and Day 180 database locks, and 5) specify anti-relaxin antibody measurements as a part of PK-related assessments.

These changes are described in detail below.

Changes to the protocol • Exclusion criterion #4 (excluding patients with current or planned treatment with any

IV therapies) has been updated to clarify that administration of IV furosemide (or equivalent) is not part of the exclusion. Administration of IV furosemide (or equivalent) is required per inclusion criterion #6; this exception is now explicitly stated in exclusion criterion #4. This clarification was provided to sites previously in a Clarification Memo dated 18Jan2010.

• Exclusion criterion #8 (excluding organ transplant recipients or patients currently listed for transplant) has been updated to clarify that patients on an administrative transplant list are not excluded from the study. The intent of this criterion is to exclude patients with end-stage cardiac failure who are awaiting urgent cardiac transplant; it is not the intent to exclude patients who are on a non-urgent administrative transplant list (i.e., patients that may require transplantation in the future if their disease progresses). This clarification was provided to sites previously in a Clarification Memo dated 27Oct2010.

• Exclusion criterion #13 (excluding patients with troponin > 3 times the upper limit of normal (including "borderline/intermediate") between presentation and screening) has been revised to make it clear that troponin can be measured until the end of screening (consistent with Section 7.6.1).

• Section 7.5 has been updated to specify that blood samples will be used for measurement of anti-relaxin antibodies as part of relaxin PK-related assessments.

• The study-specific definition for worsening heart failure (WHF) is described in other study documents (i.e., in the User Guide for Subject Self Report of Symptoms and Physician Daily Subject Assessment Worksheets), but was not specifically stated in the protocol; the definition for WHF has been added to Section 7.4.1.2. WHF is defined for this study as worsening signs and/or symptoms of heart failure that require an intensification of intravenous therapy for heart failure or mechanical ventilatory or circulatory support. Such treatment can include the institution or up-titration of IV furosemide, IV nitrates or any other IV medication for heart failure, or institution of mechanical support such as mechanical ventilation, IABP, etc. It is important to note that medications for heart failure (such as IV treatment for hypertension control) can be added for reasons other than WHF.


21



• Definitions of analysis populations in section 9.4 have been modified to provide consistent wording.

• In sections 9.5, 9.6, 9.9, 9.12 and 9.14, the analysis population names have been changed to be consistent with changes made in section 9.4.

• Section 19 of the protocol has been amended to specify that there will be a database lock when all subjects have completed through Day 60, after which efficacy and safety data will be unblinded. Individual treatment assignments will remain blinded until a second database lock after all subjects have completed through Day 180, which includes only vital status information, occurs. The Day 180 database lock signals completion of the study. Additional text is included to describe data publication or reporting outside of the study from the Day 60 lock.

Changes to specific sections of the protocol are shown in the track changes version of the protocol using strike through red font for deletions and red underlined for insertions. Sections of existing text pertaining to the completed, Phase II portion of the protocol are retained for reference, but are displayed in italicized type.

A copy of this amended protocol will be sent to the Institutional Review Board (IRBs)/Independent Ethics Committee (IECs) and Health Authorities.

The changes described in this amended protocol require IRB/IEC approval prior to implementation. The changes herein do not affect key elements of the Informed Consent, however, because the Informed Consent for some sites specifically references the previous version of the protocol (Amendment 4), these sites will be required to update and submit for approval a revised Informed Consent which references the new version of the protocol (Amendment 5).

Summary of amendments issued before January 12, 2012 Amendment 4

The following summarizes major changes made to the protocol in an amendment dated March 22, 2011.

• The main purpose of Amendment 4 was to incorporate an ECG sub-study into the RELAX-AHF trial. The sub-study is expected to provide information that will contribute to the cardiac safety profile of relaxin in the target population. Details of the procedures for the ECG sub-study were described in Section 21.5 and added to the Schedule of Events in Section 21.1.

• The address of Corthera (Europe) Ltd was changed.

• The Responsible Medical Officer and one of the Medical Monitors for the study were changed on the title page.

• Three terms were added to the list of abbreviations and typographical errors were corrected throughout the protocol.

• The image of the visual analog scale (VAS) for General Well Being in Section 21.3.2 was inadvertently omitted in Amendment 3 and was added back in Amendment 4.

Amendment 3


22



The following summarizes major changes made to the protocol in an amendment dated December 1, 2010.

• Change in sample size from 880 to 1160 patients.

• The assumptions and calculations for estimates of power for assessing the primary endpoints of dyspnea using the Likert scale and visual analog scale (VAS), as well as the secondary endpoints of cardiovascular death or hospitalization for heart failure or renal failure, and days alive and out of hospital through day 60 were summarized.

• The intent-to-treat (ITT) set replaces the full analysis set (FAS) as the primary analysis population for the primary and secondary efficacy endpoints. The FAS will be used in sensitivity analyses.

• Additional exploratory efficacy endpoints were described.

• Safety analyses were described in further detail.

• Rules for imputing missing VAS scores were further detailed.

• Exclusion criteria 7 and 10 were modified and a new exclusion criterion (#20) was added.

• The Corthera, Inc. address was changed.

• The Responsible Medical Officer and one of the Medical Monitors were changed.

Amendment 2

The following summarizes major changes made to the protocol in an amendment dated January 14, 2009.

• The sponsors name was changed from BAS Medical to Corthera, Inc.

• The primary and secondary endpoints were revised for Phase III.

• The dose of 30 µg/kg/day was selected for evaluation.

• Enrolment was set at 880 patients.

• Inclusion criteria 2, 3, 4, 4b, 6, and 7, and exclusion criteria 6, 7, 13, 14, and 16 were clarified or modified.

• Study drug down-titration and discontinuation rules were modified.

• Various assessments related to labs, visits, and the collection/reporting of adverse events and SAEs were clarified or modified.

• The statistical and analytical plans and methods for Phase III were updated.

Amendment 1

The following summarizes major changes made to the protocol in an amendment dated August 15, 2007.

• Clinical chemistry assessments were added at the 6 hr time point.

• Additional blood pressure monitoring was added.


23



• AE and SAE reporting and processes were clarified.

• Clarification was made of when to report Disease-Related Events to the clinical database or on an SAE report form.

• Urine collection and urinalysis were clarified.

• Windows were established for some study-defined assessments. • Blood collection for neurohormonal assessments was described.


24



3 SYNOPSIS

Title of study: A Phase II/III, Multicenter, Randomized, Double-blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety of Relaxin in Subjects With Acute Heart Failure Protocol number: RLX.CHF.003 (Pre-RELAX-AHF/ RELAX-AHF) Investigators: Multiple Study centers: Multiple centers in North America, Europe, South America and Israel Study period: Up to 48 hr treatment, short-term follow-up through Day 5 and at Day 14, and intermediate term follow-up at 60 and 180 days

Phase of development: II/III

Objectives: The overall objectives of this study are:

1. to test the ability of relaxin to improve symptoms (dyspnea and signs of heart failure) in patients hospitalized with acute heart failure (AHF), normal to elevated blood pressure and mild to moderate renal impairment;

2. to evaluate the safety of relaxin in these patients; and

3. to evaluate the ability of relaxin to reduce intermediate term mortality/morbidity in these patients.

The study will be conducted in 2 phases.

The Pre-RELAX-AHF Phase II pilot portion of the study has been completed. This amendment focuses on the RELAX-AHF main Phase III portion of the study. Sections of existing text pertaining to the pilot phase are retained for reference, but are displayed in italicized type.

Pre-RELAX-AHF Pilot Phase: The objectives of the pilot phase are to evaluate the effects of IV relaxin, in addition to standard therapy, on symptom relief and renal improvement (or prevention of impairment) in patients with AHF, hypertension and mild to moderate renal impairment; to select a dose of IV relaxin for further study in the RELAX-AHF study; to assess the distribution of the primary and secondary endpoints in the study population, as well as the adequacy of the power calculations; and to evaluate the overall safety of IV relaxin in these patients. RELAX-AHF Main Phase: The objectives of this phase of the study are to confirm the efficacy of IV relaxin, in addition to standard therapy, in improving symptoms of heart failure, dyspnea, and in preventing intermediate term re-admission for HF or renal failure and cardiovascular death in subjects hospitalized for AHF with normal to elevated blood pressure and mild to moderate renal impairment.

The study’s safety objective is to assess the overall safety of IV relaxin in this patient population.

Number of subjects: Up to 330 (pilot phase) and 1160 subjects (main phase)


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Diagnosis and main criteria for inclusion and exclusion: Subjects who meet all of the following inclusion criteria and none of the exclusion criteria at screening will be eligible for the study. The screening period is defined as that interval that begins at the time the informed consent is signed and ends with the qualification of the subject for entry into the study (i.e. when subject is determined to meet all eligibility criteria).

Inclusion Criteria

1. Able to provide written informed consent

2. Male or female ≥ 18 years of age, with body weight < 160 kg

3. Systolic blood pressure > 125 mmHg at the start of screening and at the end of screening

4. Hospitalized for AHF. AHF is defined as including all of the following measured at any time between presentation (including the emergency department [ED]) and the end of screening:

a. Dyspnea at rest or with minimal exertion

b. Pulmonary congestion on chest radiograph

c. BNP ≥ 350pg/mL or NT-pro-BNP ≥ 1400 pg/mL

5. Able to be randomized within 16 hours from presentation to the hospital, including the ED

6. Received IV furosemide of at least 40 mg (or equivalent) at any time between admission to emergency services (either ambulance or hospital, including the ED) and the start of screening for the study

7. Impaired renal function defined as an estimated glomerular filtration rate (eGFR) on admission between 30-75 mL/min/1.73 m2, calculated using the simplified Modification of Diet in Renal Disease (sMDRD) equation

Exclusion Criteria

1. Pregnant or breast-feeding women (women of child bearing potential must have the results of a negative pregnancy test recorded prior to study drug administration)

2. Administration of intravenous radiographic contrast agent within 72 hours prior to screening or acute contrast-induced nephropathy at the time of screening

3. Temperature >38°C (oral or equivalent) or sepsis or active infection requiring IV anti-microbial treatment

4. Current (within 2 hours prior to screening) or planned (through the completion of study drug infusion) treatment with any IV therapies, including vasodilators (including nesiritide), positive inotropic agents and vasopressors, or mechanical support (intra-aortic balloon pump, endotracheal intubation, mechanical ventilation, or any ventricular assist device), with the exception of IV furosemide (or equivalent), or of IV nitrates at a dose of < 0.1 mg/kg/hr if the patient has a systolic BP > 150 mmHg at screening


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5. Current or planned ultrafiltration, hemofiltration, or dialysis

6. Known significant pulmonary disease

7. Known significant valvular disease (including any of the following: severe aortic stenosis [AVA < 1.0 or mean gradient > 50 on prior or current echocardiogram], severe aortic regurgitation, or severe mitral stenosis)

8. Any organ transplant recipient, or patient currently listed for imminent transplant (i.e., does not exclude patients on an administrative transplant waiting list), or admitted for any transplantation

9. Major surgery within 30 days

10. Hematocrit < 25% or blood transfusion in the prior 14 days or active, life-threatening GI bleeding; or active menorrhagia or metrorrhagia

11. Major neurologic event, including cerebrovascular events, in the prior 60 days

12. Clinical diagnosis of acute coronary syndrome within 45 days prior to screening (including the present admission) as determined by both clinical and enzymatic criteria

13. Troponin > 3 times the upper limit of normal (including "borderline/intermediate") between presentation and the end of screening.

14. AHF due to significant arrhythmias (including any of the following: ventricular tachycardia, bradyarrhythmias with ventricular rate <45 beats per minute or any second or third degree AV block or atrial fibrillation/flutter with ventricular response of >120 beats per minute)


16. Known hepatic impairment

17. Non-cardiac pulmonary edema, including suspected sepsis

18. Administration of an investigational drug or implantation of investigational device, or participation in another trial, within 30 days before screening or previous treatment with relaxin

19. Inability to follow instructions or comply with follow-up procedures

20. Known hypersensitivity to relaxin or similar substances or to any of the excipients

Pre-RELAX-AHF

The pilot phase (“Pre-RELAX AHF”) is designed to evaluate the efficacy and safety of relaxin, as compared to placebo, administered for up to 48 hours in subjects with AHF, hypertension and mild to moderate renal impairment, and to select the most appropriate dose of relaxin for further study, as well as to assess the power calculation based on distribution of the endpoints in the


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overall study population. After signing an Ethics Committee or Institutional Review Board-approved Informed Consent Form, subjects will be asked to undergo screening procedures for study eligibility. Three hundred thirty (330) eligible subjects will be randomized 3:2:2:2:2 to placebo or one of 4 doses of relaxin: 10, 30, 100 or 250 µg/kg/day. Study drug treatment will be administered as an IV infusion for 48 hours unless at any time during dosing, the subject’s systolic blood pressure is < 100 mm Hg or is decreased by > 40 mm Hg from baseline in 2 consecutive measurements 15 min apart, at which point, study drug treatment will be terminated. Patient-reported AHF symptoms will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline, and a Visual Analog Scale (VAS) describing symptom severity at each point in time. These evaluations will be done at 6, 12 and 24 hours from start of study drug infusion and then daily up to Day 7 and then at 14 days after the start of study drug infusion. Subjects will have clinical evaluations, including AHF symptom assessments, vitals signs, physical examination emphasizing signs of HF, as well as an assessment of need for further IV HF treatment and worsening HF events at least daily to the earlier of Day 7 or discharge, and then at Day 14. Blood will be collected at 6 hours for clinical chemistry assessment, and blood and urine specimens will be collected at 12 and 24 hours during the first day of treatment for routine safety assessments and to evaluate renal function, followed by blood evaluations daily to the earlier of Day 7 or discharge and then at Day 14. Clinical and laboratory evaluations, as detailed above, will be mandatory at Days 5 and 14. If the patient is discharged from the hospital prior to these visits, these evaluations (Days 5 and 14) will be performed as outpatient visits.

All subjects will receive phone calls at days 30 and 60 from start of study drug infusion to assess the occurrence of adverse events and serious adverse events (Day 30 only), mortality and hospital readmission for HF or renal dysfunction. Patients enrolled early in the trial will receive a follow-up phone call at 180 days to ascertain vital status. At the time that the last patient enrolled in the study reaches the 60 day follow up, the 180-day telephone contact will be performed in all patients not contacted prior to that time.

Safety will be assessed by recording medical history, monitoring adverse events and vital signs, and performing physical examinations and routine clinical laboratory tests as per the protocol, as well as those felt to be clinically indicated by the investigator. Adverse events (AE) and serious adverse events (SAE) will be collected up to day 30 from start of study drug infusion.

The Pre-RELAX-AHF Phase II portion of this Phase II/III study has been completed. The pilot phase met its objectives of evaluating the efficacy of IV relaxin in patients with AHF; of selecting a dose of IV relaxin for further study in the Phase III RELAX-AHF study; of assessing the distribution of the primary and secondary endpoints in the study population and the adequacy of the power calculations; and of evaluating the overall safety of IV relaxin in these patients.

RELAX-AHF

The main phase of the study (“RELAX-AHF”) is designed to confirm the efficacy and evaluate the safety of relaxin at 30 µg/kg/day, which is the optimal dose selected based on the safety and efficacy results of the Pre-RELAX-AHF phase, versus placebo infused for up to 48 hours in


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subjects hospitalized with AHF. Enrollment has been paused temporarily between Pre-RELAX-AHF and RELAX-AHF to conduct the analysis and/or make any necessary protocol modifications. In this phase of the study, eligible subjects will be randomized 1:1 to receive either IV placebo or relaxin. A total of 1160 patients will be enrolled in RELAX-AHF in order to attain approximately 1100 efficacy evaluable (FAS) patients.

After signing an Ethics Committee or Institutional Review Board-approved Informed Consent Form, subjects will be asked to undergo screening procedures for study eligibility. Study drug will be administered as an IV infusion for 48 hours. If at any time during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration. If at any time during dosing, the subject’s systolic blood pressure is < 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion will be permanently terminated. All patients will be evaluated at screening and baseline and thereafter at 6, 12 and 24 hours, daily while hospitalized through Day 4, and at Days 5, 14 and 60 for symptoms and signs of heart failure. Patient-reported AHF symptoms will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline, and a 100-mm Visual Analog Scale (VAS) describing symptom severity at each point in time. These evaluations will be done at baseline (VAS only), 6, 12 and 24 hours from start of study drug infusion and then daily while hospitalized through Day 4, and at Days 5 and 14. Subjects will have clinical evaluations, including AHF symptom assessments, vitals signs, physical examination emphasizing signs of HF, as well as an assessment of need for further IV HF treatment and worsening HF events at least daily to the earlier of Day 4 or discharge, and then at Days 5 and Days 14 and 60 (except for assessment of need for further IV HF treatment and worsening HF events, which are not conducted at Day 60). Blood and urine samples will be collected at baseline and at 24 hours (Day 1) and 48 hours (Day 2) for routine safety assessments and to evaluate renal function. Blood samples will also be collected on Days 3, 4, 5, 14 and 60. Clinical and laboratory evaluations, as detailed above, will be mandatory at Days 5, 14 and 60. If the patient is discharged from the hospital prior to these visits, these evaluations will be performed as outpatient visits.

Just prior to the time that the last patient enrolled in the study reaches the Day 60 follow-up, patients who have not completed the study will be contacted to ascertain an interim vital status. All patients will be contacted by phone at Day 180 to ascertain vital status. AEs will be collected through Day 5 and SAEs collected through Day 14.

Study drug, dose and mode of administration: The study drug is relaxin, provided as a 1 mg/mL solution in 5 mL vials (3.5 mL fill). Matching placebo is provided in identical vials. Both relaxin and placebo are administered as a continuous IV infusion in combination with saline or a compatible IV infusion solution as described in the Pharmacy Manual. Study drug kits prepared for each treatment arm will be identical to one another to maintain the blind. Doses are administered on a weight adjusted basis.

In the Pre-RELAX-AHF phase, patients will be randomized on a 3:2:2:2:2 fashion to receive:


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• Placebo (n = 90)

• Relaxin, 10 µg/kg/day (n = 60)




In the main phase of the study, patients will be randomized in a 1:1 fashion to receive either IV placebo or IV relaxin at 30 µg/kg/day.

Duration of study drug infusion: Study drug infusion will commence as soon as possible after randomization and will be administered via continuous IV infusion for 48 hours. If at any time during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the 48 hour study drug administration. Study drug administration will be permanently discontinued at any time if in 2 consecutive measurements, 15 minutes apart, systolic blood pressure is reduced to < 100 mm Hg.

Reference therapy, dose and mode of administration: The placebo, a 20 mM sodium acetate solution with a pH of 5.0, will be infused IV in a manner identical to that of relaxin. The primary efficacy endpoint is:

Proportion of patients with marked or moderate improvement in subject-reported dyspnea score using the Likert 7-point scale at both 12 and 24 hours following the start of study drug infusion in the absence of worsening heart failure symptoms and signs between 3 and 24 hours following the start of study drug infusion.

The secondary efficacy endpoints are:

• Proportion of patients with renal impairment defined as a ≥ 25% increase in serum creatinine from baseline to Day 5

• Time from study drug initiation to death or rehospitalization for HF or renal dysfunction through Day 60 in the combined RELAX-AHF-1 and -2 studies

Additional exploratory efficacy analyses are:

• Change in dyspnea score by VAS at 6, 12, 24 and 48 hours and 5 days from baseline, calculated as area under the curve.

• Proportion of patients with marked or moderate improvement in dyspnea compared to baseline by Likert scale assessed at 6 hours and then daily to Day 5 and at Day 14.

• Time to moderately or markedly better self-assessed dyspnea on the 7-point Likert scale at Day 5

• Time to worsening heart failure through Day 5


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• Proportion of patients with uncontrolled blood pressure (systolic >150 mmHg or diastolic > 90 mmHg) at Day 2 and through Day 5 or at time of discharge, whichever occurs first.

• Changes in heart failure signs and symptoms from baseline to Day 5

• Proportion of patients with renal impairment (defined as above) compared to baseline at 12, 24 and 48 hours, 3, 4, 7 days and 14 days

• Proportion of patients with renal impairment (defined as > 0.3 mg/dL increase in serum creatinine) compared to baseline at 12, 24 and 48 hours, 3, 4, 5 days and 14 days

• Mean change in serum creatinine (SCr) from baseline to 24 and 48 hours, 5 and 14 days

• Time to the first SCr increase of ≥ 25% mg/dL above baseline through Day 5

• Time to the first SCr increase of ≥ 0.3 mg/dL above baseline through Day 5

• Proportion of subjects with increases in SCr of 0.2, 0.4, and 0.5 mg/dL over baseline at any time from the start of study drug infusion through Day 14

• Proportion of subjects with increases in SCr of 20%, 30% and 40% over baseline at any time from the start of study drug infusion through Day 14

• Mean change in BUN from baseline to Day 5 and Day 14

• Total dose of IV loop diuretics from start of study drug infusion through Day 5 or discharge if earlier

• Length of hospital stay from start of study drug infusion

• Time to death or heart failure rehospitalization through Day 60

• Days alive and out of hospital through Day 60 from start of study drug infusion

• Time to death up to Day 180 from start of study drug infusion

• Changes in renal, neurohormonal or inflammatory markers believed to play a role in AHF from baseline through Day 5 and Day 14

The two primary efficacy endpoints for the Phase III RELAX-AHF are:

• Area Under the Curve (AUC) representing the change in patient-reported dyspnea from baseline measured by a 100-mm Visual Analog Scale (VAS) from baseline through Day 5

• Moderately or markedly better patient-reported dyspnea relative to the start of study drug on the 7-point Likert scale at 6, 12 and 24 hours (at all 3 time points)

The secondary efficacy endpoints for RELAX-AHF are:

• Days alive out of hospital through Day 60

• Cardiovascular death or rehospitalization due to heart failure or renal failure through Day


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60

Additional endpoints to be collected as supportive evidence for relaxin efficacy in RELAX-AHF are described in section 7.4.1 of the protocol and in the Statistical Analysis Plan.

Safety: Safety will be assessed by comparing the relaxin group to the placebo group with regard to the frequency of adverse events, and changes in vital signs, physical examination findings, AHF-related signs and symptoms, and clinical laboratory test results (chemistry, hematology, and urinalysis).

Adverse Events (AEs) and Serious Adverse Events (SAEs) are assessed up to Day 30 in the Pre-RELAX-AHF phase and through Day 5 and Day 14, respectively, in RELAX-AHF.

Statistical Methods: The sample size in the Pre-RELAX-AHF pilot phase is not expected to provide power to detect statistically significant differences; the point estimates and overall pattern of results will be used to guide the dose selection for RELAX-AHF. For each of the primary and key secondary endpoints, a global statistical comparison will be made between the pooled 30, 100, and 250 µg/kg/day and the pooled 10 µg/kg/day and placebo groups. Each active dose group will be compared with placebo at the two-sided 0.05 significance level, without adjustment for multiple comparisons. Results from the pilot phase will not be combined with results of the main phases to evaluate the efficacy of the selected relaxin dose. The sample size required for the main phase will be re-estimated based on the observed rate in the placebo group. A total of 90 patients in the placebo group of the pilot phase will provide a 95% confidence interval of approximately ± 10 % about a success rate of 50%.

The primary global null hypothesis is that both the mean area under the dyspnea VAS change from baseline curve (VAS AUC) from baseline to Day 5 and the proportion of subjects with moderately to markedly better dyspnea at 6, 12, and 24 hours are the same in both treatment groups. The Type I error rate for this global null hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach. The VAS AUC means will be compared between treatment groups using a t-test as the primary analytic method. The proportions with moderately or markedly better dyspnea at 6, 12, and 24 hours will be compared between treatment groups using a chi-square test. Assuming a standard deviation of 2700 mm-hr for the dyspnea VAS AUC, 25% of placebo subjects with moderately or markedly better dyspnea at 6, 12, and 24 hours, and a correlation of 0.25 between the two endpoints, the study has approximately 81% power to detect a mean difference on the dyspnea VAS AUC of 468 mm-hr and/or a relative risk of 1.3 (absolute difference of 7.5%) on the proportion moderately or markedly better at the two-sided 0.05 significance level using the Hochberg approach.

The secondary global null hypothesis is that both the mean number of days alive out of hospital through Day 60 is the same in both treatment groups, and the hazard for CV death or rehospitalization for heart failure or renal failure through Day 60 is the same in both groups. The Type I error rate will be controlled across the two global null hypotheses at the two-sided 0.05


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level by sequential testing: the secondary global null hypothesis will be tested given rejection of the primary global null hypothesis. The Type I error rate for the secondary global hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach. Groups will be compared with respect to the number of days alive and out of hospital using a Wilcoxon rank sum test as the primary analytic method. Treatment groups will be compared with respect to the hazard ratio for CV death or HF rehospitalization using a log-rank test. If the primary global null hypothesis is rejected at the two-sided 0.00125 significance level (equivalent to that required for two trials conducted at the two-sided 0.05 significance level), the single study will be considered to support the efficacy of relaxin in this indication. A result significant at the two-sided 0.05 significance level, but not significant at the two-sided 0.00125 level, will be confirmed in a subsequent study.

Tests of exploratory efficacy analyses and safety analyses will be performed without adjustment for multiple comparisons at the two-sided 0.05 significance level.


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RLX.CHF.003 (PRE-RELAX-AHF / RELAX-AHF) Page 17 of 99 AMENDED PROTOCOL VERSION 05 CLEAN


3.1 Abbreviations and Terms

AE adverse event ACE angiotensin converting enzymes ADHERE Acute Decompensated National Heart Failure Registry ADR adverse drug reaction AHF acute heart failure ALT alanine transaminase ANCOVA analysis of covariance AST aspartate transaminase AUC area under the curve AV Atrioventricular AVA aortic valve area BNP brain natriuretic peptide BP blood pressure BUN blood urea nitrogen º C degrees Celsius CEC Clinical Endpoints Committee CFR Code of Federal Regulations CHF congestive heart failure CI cardiac index cm Centimeter CRA clinical research associate CRF CRT-D CRT-P

Case Report Form cardiac resynchronization therapy defibrillator cardiac resynchronization therapy pacemaker

CV Cardiovascular DBP diastolic blood pressure DCF Data Clarification Form dL Deciliter DRE disease-related event DSMB Data Safety Monitoring Board EC Ethics Committee ED Emergency Department ECG Electrocardiogram eGFR estimated glomerular filtration rate ERB Ethics Review Board


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FDA Food and Drug Administration GCP Good Clinical Practice GFR glomerular filtration rate GGT gamma-glutamyl-transferase HDL high-density lipoprotein HF heart failure HJR hepatic jugular reflex hr ICD

Hour implantable cardiac defibrillator

ICF Informed Consent Form ICH International Conference on Harmonization IND Investigational New Drug IRB Institutional Review Board IV Intravenous IVRS Interactive Voice Response System JVP jugular venous pulse kg Kilogram L Liter LDL low-density lipoprotein LVEDP left ventricular end diastolic pressure LVEF left ventricular ejection fraction MedDRA Medical Dictionary for Regulatory Activities m Meter mg Milligram min Minute mL Milliliter mm Millimeter mM Millimolar mmHg millimeters mercury µg Microgram NA not applicable NAV not available ND not done NT-pro-BNP N-terminal fragment of pro-brain natriuretic peptide NYHA New York Heart Association PCWP pulmonary capillary wedge pressure


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pg Picograms pH hydrogen ion concentration PK Pharmacokinetics RBF renal blood flow REB Research Ethics Board SAE Serious Adverse Event SAS Statistical Analysis System SAP Statistical Analysis Plan SBP systolic blood pressure SC Subcutaneous SCr serum creatinine SD standard deviation SGOT serum glutamic oxalo-acetic transaminase SGPT serum glutamic pyruvate transaminase SOP Standard Operating Procedure SUSAR suspected unexpected serious adverse reaction SVR systemic vascular resistance sMDRD simplified Modification of Diet in Renal Disease T1/2 half-life Tmax time to maximum concentration UNK unknown U.S. United States VAS visual analog scale

WMA World Medical Association

Definitions Study drug: For the purposes of this study, study drug refers to relaxin or placebo.


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4 TABLE OF CONTENTS

1 TITLE Page ....................................................................................................................... 1 1.1 Signature Page ............................................................................................................ 2

2 CONTACT INFORMATION ........................................................................................... 3 Amendment 5 to protocol RLX.CHF.003................................................................................. 4 3 SYNOPSIS ........................................................................................................................ 8

3.1 Abbreviations and Terms ......................................................................................... 17 4 Table of Contents ............................................................................................................ 20 5 Introduction ..................................................................................................................... 23

5.1 Acute Heart Failure .................................................................................................. 23 5.2 Relaxin as a Potential Therapeutic ........................................................................... 24 5.3 Previous Clinical Studies Using Relaxin ................................................................. 25

5.3.1 Pre-RELAX-AHF Pilot Phase in AHF Subjects ............................................. 25 5.3.2 Phase II Study in Compensated CHF Subjects ............................................... 26 5.3.3 Investigator-Sponsored Renal Study ............................................................... 27 5.3.4 Relevant Information from Other Clinical Studies ......................................... 27

5.4 Study Rationale ........................................................................................................ 30 6 STUDY OBJECTIVES ................................................................................................... 31 7 Investigational Plan ......................................................................................................... 31

7.1 Overall Study Design and Plan ................................................................................ 32 7.2 Selection of Study Population .................................................................................. 34

7.2.1 Inclusion Criteria ............................................................................................ 34 7.2.2 Exclusion Criteria ........................................................................................... 35 7.2.3 Prior and Concomitant Therapy ...................................................................... 36 7.2.4 Subject Identification ...................................................................................... 37

7.3 Study Drug ............................................................................................................... 37 7.3.1 Investigational Products .................................................................................. 37 7.3.2 Selection of Doses Used in the Study ............................................................. 38 7.3.3 Method of Assigning Subjects to Study Drug and Blinding........................... 40 7.3.4 Study Drug Administration ............................................................................. 40 7.3.5 Discontinuation of Patient Dosing .................................................................. 41 7.3.6 Study Drug Administration Compliance ........................................................ 41

7.4 Efficacy and Safety Assessments ............................................................................. 41 7.4.1 Efficacy Endpoints .......................................................................................... 41 7.4.2 Safety Assessments ......................................................................................... 44

7.5 Pharmacokinetics ..................................................................................................... 45 7.6 Study Procedures ...................................................................................................... 46

7.6.1 Screening......................................................................................................... 46 7.6.2 Baseline (Day 0 - Prior to start of study drug infusion) .................................. 46 7.6.3 Study Drug Infusion Period ............................................................................ 47 7.6.4 Post-Study Drug Infusion Follow-Up Period (Day 3 through Day 180) ........ 48 7.6.6 Early Withdrawal from the Study ................................................................... 50

7.7 Data Quality Assurance ............................................................................................ 50 8 ADVERSE EVENTS ...................................................................................................... 51

8.1 Definitions ................................................................................................................ 51


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8.2 Adverse Events ......................................................................................................... 51 8.3 Serious Adverse Events (SAE) ................................................................................ 52 8.4 Disease-Related Events (DRE) ................................................................................ 53 8.5 Laboratory AEs and SAEs ....................................................................................... 54 8.6 Collection of AEs, and SAEs: Method, Frequency, and Time Period .................... 54 8.7 Documentation and Reporting of AEs and SAEs .................................................... 54 8.8 Follow-up of AEs and SAEs .................................................................................... 57 8.9 Electrocardiogram (ECG) sub-study ........................................................................ 58

9 Statistical and Analytical Plan ........................................................................................ 58 9.1 Pre-RELAX-AHF..................................................................................................... 58 9.2 Objectives and primary hypotheses of RELAX-AHF .............................................. 59 9.3 General analytic methods ......................................................................................... 59 9.4 Analysis populations ................................................................................................ 60

9.4.1 Intent-to-treat (ITT) set ................................................................................... 60 9.4.2 Full analysis set (FAS) .................................................................................... 60 9.4.3 Safety set ......................................................................................................... 60

9.5 Demographic and baseline characteristics ............................................................... 60 9.6 Extent of exposure to study drug .............................................................................. 60 9.7 Use of concomitant medication ................................................................................ 61 9.8 Primary efficacy endpoints ....................................................................................... 61

9.8.1 Area under the change from baseline dyspnea VAS curve from baseline to Day 5 ......................................................................................................................... 61 9.8.2 Moderately or markedly better dyspnea at 6, 12, and 24 hours ...................... 61

9.9 Secondary efficacy endpoints ................................................................................... 62 9.9.1 Days alive and out of hospital through Day 60 ............................................... 62 9.9.2 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 60 ............................................................................................................... 62

9.10 Planned sample size .............................................................................................. 63 9.11 Examination of covariates .................................................................................... 64 9.12 Additional efficacy endpoints ............................................................................... 64

9.12.1 Change from baseline in dyspnea score by VAS at 6, 12, 24 and 48 hours, Day 3, Day 4, Day 5, and Day 14 ........................................................................................... 64 9.12.2 Area under the curve for the dyspnea VAS change from baseline from baseline to Day 14, from Day 1 to Day 5, and from Day 1 to Day 14 ........................... 64 9.12.3 Markedly or moderately better dyspnea by Likert scale at 6, 12, and 24 hours and then daily to Day 5 and at Day 14 ............................................................................ 65 9.12.4 Time to moderately or markedly better dyspnea through Day 5 .................... 65 9.12.5 Time to worsening heart failure through Day 5 and Day 14 .......................... 65 9.12.6 Total doses of IV and oral loop diuretics through Day 5 or discharge if earlier ......................................................................................................................... 65 9.12.7 Change in weight from randomization through Day 5 and 14........................ 65 9.12.8 Length of hospital stay .................................................................................... 65 9.12.9 All cause death or rehospitalization due to heart failure or renal failure through Day 60 ............................................................................................................... 66 9.12.10 Days alive and out of hospital through Day 30 ............................................... 66 9.12.11 Cardiovascular death through Day 180 ........................................................... 66


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9.12.12 Other heart failure signs and symptoms over time ......................................... 66 9.12.13 Days in the ICU/CCU ..................................................................................... 66 9.12.14 All-cause death through Day 30 ..................................................................... 66 9.12.15 All-cause death or worsening heart failure or rehospitalization for heart failure through Day 30 ............................................................................................................... 67 9.12.16 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 30 ............................................................................................................... 67 9.12.17 Cardiovascular death or rehospitalization due to heart failure or renal failure through 30 days after discharge from the index hospitalization ..................................... 67

9.13 Sequence of planned analyses .............................................................................. 67 9.13.1 Interim analyses .............................................................................................. 67 9.13.2 Final Analyses and Reporting ......................................................................... 68

9.14 Safety outcomes .................................................................................................... 68 9.14.1 Mortality (death to Day 180) .......................................................................... 68 9.14.2 Adverse events ................................................................................................ 68 9.14.3 Laboratory data ............................................................................................... 69

9.15 Changes in the Conduct of the Study or Planned Analyses ................................. 70 10 PHARMACOECONOMICS AND QUALITY OF LIFE ASSESSMENTS.................. 70 11 Compensation, Insurance, and Indemnity ....................................................................... 71 12 Ethics............................................................................................................................... 71

12.1 Institutional Review Board, Research Ethics Board or Ethics Committee .......... 71 12.2 Ethical Conduct of the Study ................................................................................ 72 12.3 Subject Information and Informed Consent ......................................................... 72 12.4 Subject Withdrawal of Consent Form .................................................................. 72

13 Investigators and Study Administrative Structure .......................................................... 73 14 INVESTIGATIONAL PRODUCT ACCOUNTABILITY ............................................ 73 15 Laboratory Requirements ................................................................................................ 73 16 Case Report Forms (CRF) .............................................................................................. 74 17 Study Monitoring and Auditing ...................................................................................... 74 18 Retention of Records ....................................................................................................... 75 19 Use of Information and Publication ................................................................................ 75 20 References ....................................................................................................................... 77 21 APPENDICES ................................................................................................................ 79

21.1 Schedule of Events ............................................................................................... 79 21.1.1 Detailed Schedule of Laboratory Assessments: .............................................. 82

21.2 World Medical Association Declaration Of Helsinki........................................... 84 21.2.1 Introduction ..................................................................................................... 84 21.2.2 Basic Principles For All Medical Research .................................................... 85 21.2.3 Additional Principles For Medical Research Combined with Medical Care .. 87

21.3 Patient Reported Symptoms and Physician Assessments ..................................... 89 21.3.1 Patient Reported Symptoms ............................................................................ 89 21.3.2 Physician Assessment ..................................................................................... 91

21.4 Disease-Related Events (DRE) ............................................................................. 95 21.5 RLX.CHF.003 (RELAX-AHF) ECG Sub-study .................................................. 96

21.5.1 Background/Rationale for the ECG Sub-Study .............................................. 96 21.5.2 Objectives ....................................................................................................... 96


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21.5.3 Selection of Sub-Study Population – Inclusion/Exclusion Criteria ................ 96 21.5.4 Study Procedures ............................................................................................ 97 21.5.5 ECG evaluations ............................................................................................. 98 21.5.6 Data collection and analysis ............................................................................ 98 21.5.7 ECG analysis ................................................................................................... 99

5 INTRODUCTION

5.1 Acute Heart Failure

Acute heart failure (AHF) has emerged as a major public health problem because of its high frequency, significant burden of mortality and morbidity and its substantial economic burden. The Acute Decompensated National Heart Failure Registry (ADHERE) has contributed greatly to our understanding of this condition. This national registry of patients admitted with AHF has identified a number of the novel characteristics which have important implications for future scientific investigation and drug development related to this syndrome.

ADHERE has demonstrated that patients with heart failure (HF) due to left ventricular systolic dysfunction and reduced systolic blood pressure (SBP) constitute only a minority of those admitted with AHF (Adams, 2005). In fact, patients with AHF are most commonly hypertensive at the time of admission and often have preserved (normal), not depressed, left ventricular contractile function. In contrast, most physiological studies and the substantial majority of prior clinical trials of AHF have focused on patients with left ventricular systolic dysfunction and either normal or reduced blood pressure. These findings were recently confirmed in publications from two registries. In the OPTIMIZE registry, Georghiade et al. (Gheorghiade, 2006) concluded that systolic hypertension is common in patients hospitalized for heart failure, that the mean SBP at admission was 143 mm Hg and that 51% of patients had preserved systolic function. In a small registry that captured all emergency room admissions with AHF for 3 months, the first blood pressure recorded was found to be even higher, i.e. > 160 mmHg systolic (Milo-Cotter, 2007). Thus our understanding of the pathophysiology of AHF with hypertension, the most common patient population hospitalized for HF, is incomplete and the best treatment strategy for this illness is unknown.

Dyspnea is by far the most common cause for patients to seek medical care during an acute heart failure episode (ADHERE 2007). Hence, fast, significant and persistent improvement of dyspnea is one of the most important treatment targets in patients with AHF. In the PROTECT Pilot phase study (Cotter 2008), patients admitted with acute heart failure as ascertained by high BNP at screening had only < 50% resolution of dyspnea at 24 hours from admission. Hence, current therapy can ameliorate dyspnea in only some (but not most) patients and improved dyspnea resolution is the main therapeutic target in AHF.

Similarly, recent data from the PROTECT pilot study and other studies (Stevenson 2008, Allen 2008) have demonstrated that besides rapid resolution of dyspnea, patients’ priorities during an AHF admission include prevention of worsening in HF symptoms and signs either during admission or after discharge (leading to re-admission), spending as much time as


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possible at home (both shorter initial length of stay and prevention of re-admission) and living longer.

5.2 Relaxin as a Potential Therapeutic

Relaxin is a naturally occurring polypeptide hormone (5963 mw) that is believed to orchestrate many of the maternal physiological responses to pregnancy. These include increases in renal function (Davison, 1981), decreases in systemic vascular resistance (Capeless, 1989), and increases in cardiac output mediated largely by increased stroke volume (Capeless, 1989). In numerous non-clinical studies, relaxin has demonstrated the ability to prevent or reverse the effects of two vasoconstrictors, angiotensin (Ang) II and endothelin, important in the pathophysiology of AHF. In rat studies, renal vasoconstriction induced by Ang II, measured as decreases in glomerular filtration rate (GFR) and renal blood flow (RBF), was reversed by infusion of relaxin (Danielson, 1999). Acute infusion of relaxin also restored arterial compliance, cardiac output, and systemic vascular resistance that were dysregulated by infusion of Ang II in rats (Debrah, 2005). In isolated rat aorta and mesenteric artery, relaxin pretreatment mitigated the maximum contractile response to endothelin-1 by approximately 40% (Dschietzig, 2003).

Gene expression for relaxin and the relaxin receptor has been detected in rat renal, mesenteric and thoracic aorta (Novak, 2006), and in human saphenous vein, mammary artery and vessels in the skin (Kohsaka, 1998). These data suggest that there may be a vessel-derived relaxin ligand-receptor system that can act locally to effect arterial function. Perhaps more importantly, the presence of relaxin receptors in blood vessels strongly suggests that relaxin engages these receptors when given therapeutically. Many studies have attributed relaxin’s vasodilatory activity to nitric oxide as the ultimate effector molecule (Bani, 1998; Danielson, 1999; Dschietzig, 2003), and it is also likely that vasodilation is mediated via the endothelial endothelin type B receptor (Dschietzig, 2003; Danielson, 2000).

Relaxin gene expression may also be upregulated in atria and ventricles from patients with CHF, compared to normal subjects (Dschietzig, 2001). Elevated left ventricular end diastolic pressure (LVEDP) in isolated rat hearts stimulated relaxin gene expression, providing a possible mechanism for the finding of enhanced relaxin expression in ventricles from CHF patients.

Because of the nonclinical data described above, relaxin was tested in a small, pilot, open label, dose ranging study in patients with compensated CHF (Protocol RLX.CHF.001). Relaxin administration was associated with decreases in pulmonary capillary wedge pressure (PCWP) and systemic vascular resistance (SVR) and with increases in cardiac index (CI) during infusion, with a return to baseline values by 24 hr post-dosing. Administration of relaxin was also associated with decreases in serum creatinine, BUN and uric acid, consistent with its ability to increase GFR. Relaxin showed excellent safety in these patients and appeared to be most efficacious in the lower dose groups tested (Section 5.3.1).

The results of this pilot study of relaxin in CHF patients are augmented by data from previous studies of systemic sclerosis patients who were chronically dosed with relaxin. In


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these studies, increases in markers of renal function, including predicted creatinine clearance, and small decreases, within normal limits, in systolic and diastolic blood pressure were observed throughout the 6 months of continuous dosing (Section 5.3.3).

Together, these nonclinical and clinical studies support the hypothesis that relaxin may relieve systemic and renal vasoconstriction. Therefore, the present initiative was designed to provide information about the efficacy and safety of relaxin, an endogenously produced peptide that may produce vasodilation and be renal protective. This combination, if supported by clinical data, would potentially constitute a major advance in AHF treatment, since it addresses two of the most important goals in AHF treatment---hemodynamic improvement and renal protection.

5.3 Previous Clinical Studies Using Relaxin

5.3.1 Pre-RELAX-AHF Pilot Phase in AHF Subjects

The Pre-RELAX-AHF Phase II portion of the study has been completed with a total enrollment of 234 patients from 8 countries. Subjects were enrolled into five dosing groups: placebo and relaxin at 10, 30, 100 and 250 µg/kg/day and followed for up to 180 days. Enrollment was halted following a protocol-specified interim analysis of data up to Day 14 when 2/3 of the subjects (n = 209) had been randomized. The data obtained at the interim analysis fulfilled the study objectives of evaluating the efficacy of IV relaxin in patients with AHF; of being able to select a dose of IV relaxin for further study in the RELAX-AHF study; of assessing the distribution of the primary and secondary endpoints in the study population and the adequacy of the power calculations; and of evaluating the overall safety of IV relaxin in these patients.

The final dataset includes 234 patients who have been followed through an average of 4.5 months post-dosing. Baseline characteristics at study entry were balanced across study groups. The 30 µg/kg/day relaxin dose was optimal in a number of independent measures of patient improvement in dyspnea and HF signs, symptoms and outcomes. Dyspnea improvement measured by the Likert scale indicated that relaxin rapidly improved shortness of breath by 6 hr which was sustained at 12 and 24 hours, with 43% of patients reporting moderate or marked improvement in the 30 µg/kg/day group, compared to 23% in the placebo group (p = 0.037). Measurement by the VAS scale indicated that dyspnea relief in the relaxin-treated groups was sustained, as improvements compared to placebo were observed from 6 hr through Day 14. The largest magnitude of improvement in dyspnea across all time points was observed in the 30 µg/kg/day group with supportive trends in the 10 and 100 µg/kg/day groups. A difference of 888 mm-hour (approximately 7.4 mm difference across time points on average) between the 30 µg/kg/day and placebo groups on the dyspnea VAS AUC from baseline to Day 5 was observed (p=0.10).

Similar trends, with the 30 µg/kg/day dose group being optimal, were seen with multiple outcomes including greater weight loss, lower IV loop diuretic use and lower incidence of in-hospital worsening heart failure in the relaxin groups compared with placebo. The median days alive and out of hospital through Day 60 was higher in the relaxin groups than in the


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placebo group: 46.0 days in the placebo group and 51.0, 49.5, 51.0, and 50.0 days in the relaxin 10, 30, 100, and 250 µg/kg/day groups, respectively. Kaplan-Meier estimates of the proportion with CV death or HF rehospitalization through Day 60 were 17.2, 10.1, 2.6, 8.4 and 6.2 %, respectively, in the placebo and 4 relaxin dose groups (p = 0.053 for the 30 µg/kg/day group vs. placebo). Estimates of the 60-day risk of CV death, with an average follow up of 4.5 months, were 6.9, 2.5, 0.0, 2.9, and 6.2%, respectively.

Relaxin was safe and well tolerated at all doses. AEs and SAEs were balanced among dose groups. Blood pressure decreases were manageable and most resolved spontaneously. Two serious adverse events of hypotension were reported, both in the 250 µg/kg/day group. In the 250 µg/kg/day dose group, asymptomatic increases from baseline in serum creatinine were observed at Day 14, consistent with expectations of evidence of nascent dose limiting toxicity in the highest dose group.

These results support the selection of relaxin 30 µg/kg/day as the dose for evaluation in the Phase III RELAX-AHF portion of this study. These results also confirm the choice of efficacy endpoints for measurement in Phase III RELAX-AHF.

5.3.2 Phase II Study in Compensated CHF Subjects

A single site, open-label study of relaxin in patients with compensated CHF has been completed at the Charite Hospital in Germany. Protocol RLX.CHF.001 was a safety and dose-finding study of IV relaxin at doses ranging from 10 to 960 µg/kg/day. Hemodynamics and clinical chemistry parameters were evaluated to define relaxin doses for further study in HF. The study was designed with 3 parts (Parts A, B, C), with the first 2 parts evaluating dose escalation within a 24-hour period. Part A evaluated 4 patients at doses of 10, 30, and 100 µg/kg/day; for each subject the dose was increased every 8 hours for a total of 24 hours and then subjects were observed post-dosing for another 24 hours. Part B evaluated 6 subjects at doses of 240, 480 and 960 µg/kg/day in the same manner as in Part A. Part C evaluated 6 subjects at the highest safe dose of relaxin from Groups A and B, which was 960 µg/kg/day, for 24 hours by continuous IV infusion.

The study enrolled 16 subjects with compensated CHF, NYHA Class II-III due to ischemic heart disease, hypertensive heart disease, or dilated cardiomyopathy with LVEF <35%, PCWP >16 mm Hg and CI < 2.5 L/min/m2. All 16 subjects completed dosing and the Day 9 follow-up visit.

Trends in improvement in a number of hemodynamic parameters were observed during dosing in Parts A-C. There were decreases in PCWP and SVR and increases in CI, all of which achieved statistical significance at certain time points. Mean changes from baseline in PCWP reached maximums of -5.0, -1.7, and -4.0 mmHg during the 24 hr dosing period in Parts A, B, and C, respectively. Mean changes from baseline in SVR reached maximums of -288, -150, and -395 dynes.sec/cm5 during the dosing period in Parts A, B, and C, respectively. CI achieved maximum increases of +0.40, +0.47, and +0.78 during relaxin dosing in Parts A, B, and C, respectively. No consistent changes in heart rate were observed either during or post-dosing.


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Serum markers of renal function reflected the increase in kidney filtration believed to be mediated by relaxin. Decreases in creatinine, BUN, and uric acid were observed during dosing in all dose groups, with decreases in all of groups achieving statistical significance at some time points. Mean creatinine changes from baseline reached maximums of -0.14, -0.12, and -0.09 mg/dL during dosing in Parts A, B, and C, respectively. Mean changes from baseline in BUN reached maximums of -5.75, -8.66, and -5.0 mg/dL during dosing in Parts A, B, and C, respectively. Mean changes from baseline in uric acid, an independent marker of renal function, reached maximums of -1.05, -1.08, and -0.76 mg/dL during dosing in Parts A, B, and C, respectively. Small increases in creatinine and BUN were observed post-dosing in the higher dose groups. Although these increases were not associated with clinical symptoms, the study of the higher doses will not be continued since these observations may represent dose limiting toxicity.

There were 7 adverse events (headache, insomnia, benign prostatic hyperplasia, angina pectoris, hypotonia, and hemoptysis) reported in 5 of the16 study subjects; all of them were mild and were considered unrelated to study drug. There was 1 SAE, angina pectoris, reported in a subject from Group A after completion of the Day 9 follow-up visit. This SAE was not associated with ECG findings or elevations of enzyme/cardiac markers. The event was considered unrelated to study drug. Consistent with results observed in other studies of relaxin, small decreases from baseline (5-10%), within normal limits, in hematocrit and hemoglobin were observed across the dose groups. Small drops, within normal limits, were observed in platelets, creatinine phosphate, total cholesterol, bilirubin, calcium, albumin, and total protein during dosing. All laboratory values returned to baseline by the next evaluation at the Day 9 follow up visit.

5.3.3 Investigator-Sponsored Renal Study

In a physician-sponsored open-label research study of renal function in normal volunteers conducted in Great Britain, 11 healthy male and female subjects, including one subject who received two treatments, were dosed with an IV infusion of relaxin at 0.5 – 2.0 µg/kg/hr for 5 hr (Smith, 2006). GFR and RBF were measured using inulin and PAH, respectively, according to established methods. RBF increased by 43% but an increase in GFR was not recorded. It is possible that the compensatory mechanisms in a normal kidney in a normal volunteer, the low dose of relaxin, the short period of infusion, or all, played a role. Small increases in sodium clearance (by 20%), sodium fractional excretion (by 25%) and sodium urinary excretion (42%) were observed, although no changes in plasma sodium were seen. Serum osmolality, mean arterial pressure and urinary flow rate were unchanged with relaxin infusion. No serious adverse events or adverse reactions to study drug infusion were recorded in this study. Clinical chemistry and hematology assessments were not performed in this study.

5.3.4 Relevant Information from Other Clinical Studies

Besides the HF and renal studies described in the previous sections, Phase I-II/III studies in which systemic administration of relaxin was tested comprised 353 subjects, including systemic sclerosis patients (continuous subcutaneous [SC] infusion for up to one year, n =


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257), pregnant women at term (24 hr IV infusion, n = 40), patients with fibromyalgia (continuous SC infusion for 8 weeks, n = 10), women undergoing egg donation (continuous SC infusion for 2 weeks, n = 21), and healthy female volunteers (IV bolus, n = 25) (Data on file, Corthera). These studies provide evidence of the safety of relaxin and of relaxin pharmacology, including improvements in markers of renal function and signs of systemic vasodilatation in reductions in blood pressure. The adverse events reported in the systemic sclerosis patients, who were dosed with relaxin by continuous SC infusion for periods of up to one year, were serious but consistent with the underlying disease. In all subjects other than those with systemic sclerosis, relaxin demonstrated an excellent safety profile.

Renal function. While the Phase II/III study in systemic sclerosis patients (Protocol RLXN.C.005) did not meet its primary efficacy endpoint of improvement in composite skin score, systemic exposure to relaxin across the 8 systemic sclerosis studies provides evidence of relaxin pharmacological activity. In these studies, predicted creatinine clearance and decreases in serum creatinine and uric acid showed changes consistent with improved renal function with up to one year of continuous SC dosing.

In the pivotal Phase II/III systemic sclerosis study (n = 231), predicted creatinine clearance, which was a prospectively defined endpoint and measured according to the Cockroft and Gault formula, ranged from 115.5 to 128.0 mL/min at baseline. Placebo-treated subjects (n = 95) showed mean changes from baseline of -4.1 to +4.3 mL/min throughout the 6 months of dosing. Subjects treated with relaxin at 25 µg/kg/day (n = 92) demonstrated mean changes from baseline between +6.4 to +13.4 mL/min, with 6 of the 7 time points measured during the 6 month treatment period, achieving statistically significant differences from placebo (by ANCOVA, adjusting for baseline values). Subjects receiving 10 µg/kg/day relaxin (n = 44) also showed mean decreases from baseline ranging from +4.6 to +10.6 mL/min, with 5 of 7 time points measured during the 6 month treatment period achieving statistically significant differences from placebo. Serum creatinine values showed a maximum change from baseline of -0.6 mg/dL across both relaxin groups, consistent with improved renal function, while the maximum change from baseline in the placebo group was -0.2 mg/dL. Several time points showed statistically significant differences between the relaxin and placebo groups (by ANCOVA, adjusting for baseline values).

Similar changes in predicted creatinine clearance were observed with relaxin dosing in the Phase II systemic sclerosis study (Protocol RLXN.C.003, n = 68). Mean changes from baseline in creatinine clearance in the placebo group (n = 19) ranged from -1.4 to +8.2 mL/min across the 6 months of dosing. In the 100 µg/kg/day relaxin dose group (n = 26), mean changes from baseline ranged from +11.5 to +25.2 mL/min over the 6 months of dosing, with mean changes at all 8 time points measured being significantly greater than mean changes observed in the placebo group (by ANCOVA, adjusting for baseline values).

Serum uric acid, which is freely filtered by the kidney, is another measure of renal function. Baseline values of uric acid were 4.8 – 4.9 mg/dL across the treatment groups in the Phase II/III systemic sclerosis study. Mean changes from baseline in the placebo group were -1.4 to +4.9 mg/dL across the 7 time points tested. Mean changes from baseline in the 10


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µg/kg/day relaxin group ranged from -0.23 to -0.36 mg/dL and in the 25 µg/kg/day from -0.28 to -0.35 mg/dL. Mean changes from baseline at 2 of the 7 time points in the 10 µg/kg/day group and at 6 of the 7 time points in the 25 µg/kg/day group, measured over 6 months of dosing, were statistically significantly greater than mean changes in the placebo group (by ANCOVA, adjusting for baseline values). These data show that renal function, using a metric independent of creatinine, is improved with relaxin administration.

None of the responses observed in predicted creatinine clearance, serum creatinine or uric acid showed evidence of tachyphylaxis across the 6 months of continuous dosing.

Blood pressure. In the Phase II/III systemic sclerosis study, small but statistically significant decreases in both SBP and diastolic blood pressures (DBP) were seen at multiple time points throughout the 6 months of treatment in the relaxin groups compared to the placebo group, and are consistent with the moderate vasodilatation that relaxin is believed to mediate during pregnancy (Capeless, 1989). Mean SBP in the systemic sclerosis patients at baseline ranged from 114 - 119 mm Hg across the dose groups. In the placebo group, mean changes from baseline ranged from -3.0 to +0.4 mm Hg at the 10 time points measured during the 6 months of dosing. In the 10 µg/kg/day relaxin group, mean changes from baseline ranged from -1.5 to +2.1 and in the 25 µg/kg/day relaxin group, mean changes from baseline ranged from -1.4 to -5.7 mm Hg. Mean changes from baseline were statistically different at one of the 10 time points in the placebo group, none of the time points in the low relaxin dose group and at 7 of the 10 time points in the high relaxin dose group. Mean changes from baseline in diastolic blood pressure reached a maximum in the 25 µg/kg/day dose relaxin group of -3.3 mmHg and were statistically significantly different from baseline at 8 of the 10 time points. Mean DBP changes from baseline in the placebo and low dose relaxin group achieved a maximum of -1.6 mmHg and were not statistically different from baseline at any time point.

A post-hoc analysis of the data from the Phase II/III systemic sclerosis study was conducted to examine the responses to relaxin in the subset of patients that was hypertensive at study entry. There were 8 patients in the placebo group and 10 patients in the 25 µg/kg/day relaxin group that were hypertensive (SBP > 140 mmHg) both at screening and at baseline. Mean SBP at baseline measured 155 and 151 mmHg in the placebo and relaxin groups, respectively. Mean changes from baseline ranged from -3.0 to -14.5 mmHg in the placebo group and from -3.2 to -23.0 mmHg in the relaxin group. Mean changes at 3 of the 10 time points in the placebo group and at 8 of the 10 time points in the relaxin group were statistically significantly different from baseline. Relaxin administration was associated with an average change from baseline of -12.4 mmHg in the hypertensive subjects, compared to an average change from baseline of -4.4 mmHg in the group as a whole. This result suggests that relaxin may have a greater BP-lowering effect in subjects that are hypertensive than in subjects that are normotensive and is consistent with findings in hypertensive animals (St. Louis, 1985).

Electrolytes and osmolality. A decrease in serum sodium, potassium and chloride, as well as in osmolality, were observed in the Phase II/III systemic sclerosis study and may also reflect the natural biological effects of relaxin (Weisinger, 1993). The electrolyte changes were small in magnitude and mean values remained within the normal range.


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Safety. The adverse events reported in the systemic sclerosis patients, who were dosed with relaxin by continuous SC infusion for periods of up to one year, were serious but were consistent with the natural history of the disease (Medsger, 1996; Mayes, 2003; Steen, 2003). Increases above baseline in serum creatinine, uric acid and BUN were observed in the relaxin groups post-dosing at the 4 week follow up visit. At this visit, mean SBP measured 116, 123, and 122 mmHg in the placebo, 10 and 25 µg/kg/day relaxin dose groups, respectively, with the difference from baseline in the high relaxin dose group statistically significantly higher than at baseline. There were 7 SAEs, including 2 deaths, in the trial that related to post-dosing hypertension/increased creatinine. Renal failure with or without hypertension, referred to as “scleroderma renal crisis” occurs in 15% of systemic sclerosis patients (DeMarco, 2002) and is complicated by the presence of Raynaud’s phenomenon, a vasospastic condition involving the kidney, which occurs in the majority of patients. All 7 of the SAEs occurred in patients who were dosed for > 19 weeks. It is likely that the duration of exposure and the susceptibility of these patients to renal vasospasm contributed to the SAEs. No SAEs related to post-dosing hypertension or increased creatinine have occurred in any other subject populations treated with relaxin. No SAEs related to hypotension have been reported in relaxin-treated subjects in any of the systemic sclerosis or other trials.

Across all systemic sclerosis studies, menorrhagia and metrorrhagia were observed more frequently in relaxin-treated subjects, compared to placebo subjects, and are believed to reflect relaxin’s pharmacological effects on the endometrium, including vasodilatation and angiogenesis (Unemori, 1999; Palejwala, 2002). Hemoglobin and hematocrit decreases were associated with relaxin treatment and are believed to at least in part be due to these endometrial effects. A hemodilutional effect of relaxin may also play a role (Sunn, 2002). The occurrence of menorrhagia, metrorrhagia and anemia were occasionally clinically significant following several weeks of dosing in the systemic sclerosis studies. In a few instances, after prolonged exposure to relaxin (> 3 weeks) and perhaps due to the underlying vascular nature of systemic sclerosis, anemia became clinically significant (SAEs related to anemia: 11/257 (4.2%) in relaxin groups; 3/124 (2.4%) in placebo groups). SAEs related to anemia have not been observed in other subject populations receiving relaxin.

5.4 Study Rationale

As the ADHERE and OPTIMIZE registries have demonstrated, many AHF patients are hypertensive at admission. Dyspnea improvement, prevention of in-hospital worsening HF and re-admissions due to heart failure, shorter hospital stay and preventing mortality were all shown in several studies (ADHERE, Stevenson 2008, Allen 2008) to be important treatment targets for patients admitted with AHF. The RELAX-AHF program was designed to test the efficacy and safety of relaxin in patients with AHF, elevated SBP and mild to moderate renal insufficiency. The doses selected for testing in the Pre-RELAX-AHF pilot phase were based on previous experience in humans, both in heart failure and other diseases. These studies have suggested that doses as low as 10 µg/kg/day may induce vasodilatation and improvements in creatinine clearance. On the other hand, the pilot dose escalating CHF study (Protocol RLX.CHF.001, Section 5.3.1) demonstrated some reduced efficacy and possibly reduced intermediate-term renoprotection at the relaxin dose of 960 µg/kg/day.


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Hence, in the Pre-RELAX-AHF pilot phase, relaxin doses of 10 -250 µg/kg/day were examined. Based on the results of the pilot phase, a safe and efficacious dose of 30 µg/kg/day was chosen for further study in the main phase of the RELAX-AHF study. Based on the results of the Pre-Relax-AHF program (see above) it was determined that the dose of 30 µg/kg/day is most likely to induce a fast, significant and sustained improvement in dyspnea accompanied by reduction of worsening heart failure and a reduction in the number of days the patient stays in the hospital. In addition, this dose appears most likely to decrease the need for re-admission for heart failure and potentially reduce cardiovascular mortality. These potential beneficial effects will be examined in the phase III RELAX-AHF study.

6 STUDY OBJECTIVES

The overall objectives of this study are to test the ability of relaxin to improve symptoms (dyspnea and signs of heart failure) in patients hospitalized with acute heart failure (AHF), normal to elevated blood pressure and mild to moderate renal impairment; to evaluate the safety of relaxin in these patients; and to evaluate the ability of relaxin to reduce longer-term mortality/morbidity in these patients. The study was designed to be conducted in 2 phases.

The Pre-RELAX-AHF Phase II pilot portion of the study has been completed. This amendment focuses on the RELAX-AHF main Phase III portion of the study. Sections of existing text pertaining to the pilot phase are retained for reference but are displayed in italicized type. Pre-RELAX-AHF Pilot Phase: The objectives of the pilot phase are to evaluate the effects of IV relaxin, in addition to standard therapy, on symptom relief and renal improvement (or prevention of impairment) in patients with AHF, hypertension and mild to moderate renal impairment; to select a dose of IV relaxin for further study in the RELAX-AHF study; to assess the distribution of the primary and secondary endpoints in the study population, as well as the adequacy of the power calculations; and to evaluate the overall safety of IV relaxin in these patients. RELAX-AHF Program The objectives of this phase of the study are to confirm the efficacy of IV relaxin, in addition to standard therapy, in improving symptoms of heart failure, dyspnea, and in preventing intermediate term re-admission for HF or renal failure or cardiovascular death in subjects hospitalized for AHF with normal to elevated blood pressure and mild to moderate renal impairment.

The study’s safety objective is to assess the overall safety of IV relaxin in this patient population.

7 INVESTIGATIONAL PLAN

7.1 Overall Study Design and Plan

RLX.CHF.003 is a double-blind, randomized, placebo-controlled study and will be conducted in 2 phases. The pilot phase (“Pre-RELAX-AHF”) will enroll 330 subjects and


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evaluate 4 doses of relaxin compared with placebo in patients hospitalized with AHF, hypertension and mild to moderate renal impairment. Enrollment will pause at the completion of Pre-RELAX-AHF to (1) select the most appropriate dose of relaxin for further study; (2) examine the distribution of the primary efficacy endpoint in the study population and evaluate the appropriateness of the study size and power calculations; and (3) evaluate the safety of relaxin in the study population and appropriateness of the safety program to be used in the main phase of the study (“RELAX-AHF”). The RELAX-AHF phase will start after the completion of these evaluations and will enroll 2,000 subjects.

Pre-RELAX-AHF

Pre-RELAX-AHF is designed to evaluate the efficacy and safety of relaxin, as compared to placebo, administered for up to 48 hours in subjects hospitalized with AHF, hypertension and mild to moderate renal impairment, and to select the most appropriate dose of relaxin for further study. Potential study candidates will be patients hospitalized with AHF, SBP > 125 mmHg at the time of screening, and renal impairment, measured as a predicted creatinine clearance of 30-75 mL/min (sMDRD formula) who require IV diuretic therapy.

After signing an Ethics Committee (EC) or Institutional Review Board (IRB)-approved Informed Consent Form (ICF), subjects will be asked to undergo screening procedures for study eligibility. Eligible, subjects will be randomized 3:2:2:2:2 to one of the following treatment assignments:

Placebo (n = 90)

Relaxin, 10 µg/kg/day (n = 60)




Eligible subjects must be randomized no more than 16 hours from the time of arrival to the ED. Patients randomized to the placebo arm will receive standard therapy for AHF plus placebo. Patients randomized to an active study drug arm will receive standard therapy plus one of 4 doses of active study drug. Study drug treatment will be administered as an IV infusion for 48 hours unless at any time during dosing in 2 consecutive measurements 15 min apart, the subject’s SBP is < 100 mm Hg or decreased by > 40 mmHg from baseline, at which point, study drug treatment will be terminated. (See note below regarding Duration of Treatment). Patient-reported AHF symptoms will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline and a VAS describing symptom severity at each point in time. These evaluations will be done at 6, 12 and 24 hours from start of study drug infusion and then daily to Day 7 and then at 14 days after the start of study drug infusion.

Subjects will have clinical evaluations including AHF symptom assessments, vitals signs, physical examination emphasizing signs of HF, as well as an assessment of need for further IV HF treatment and of the occurrence of worsening HF events at least daily to the earlier of


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Day 7 or discharge, and then at 14 days. Blood will be collected at 6 hours for clinical chemistry, and blood and urine specimens will be collected at 12, 24, and 48 hours from study drug initiation for routine safety assessments and to evaluate renal function, followed by blood evaluations daily to the earlier of Day 7 or discharge and then at Day 14. Clinical and laboratory evaluations, as detailed above, will be mandatory at Days 5 and 14. If the patient is discharged from the hospital prior to these visits, these evaluations (Day 5 and 14) will be performed as outpatient visits.

All subjects will receive phone calls at Days 30 and 60 from start of study drug infusion to assess the occurrence of adverse events and serious adverse events (Day 30 only), mortality and hospital readmission for HF or renal dysfunction. Patients enrolled early in the trial will receive a follow-up phone call at 180 days to ascertain vital status. At the time that the last patient enrolled in the study reaches the 60-day follow up, the 180-day telephone contact will be performed in all patients not contacted prior to that time.

Safety will be assessed by recording medical history, monitoring adverse events and vital signs, and performing physical examinations and routine clinical laboratory tests as per the protocol, as well those felt to be clinically indicated by the investigator. Adverse events (AE) and serious adverse events (SAE) will be collected up to Day 30 from start of study drug infusion.

RELAX-AHF

The main phase of the study (“RELAX-AHF”) is designed to confirm the efficacy and evaluate the safety of relaxin at 30 µg/kg/day versus placebo infused for up to 48 hours in subjects hospitalized with AHF. Enrollment has been paused temporarily between the Pre-RELAX-AHF and RELAX-AHF to conduct the analysis and/or make any necessary protocol modifications. In this phase of the study, eligible subjects will be randomized 1:1 to receive either IV placebo or relaxin. A total of1160 patients will be enrolled in RELAX-AHF in order to attain approximately 1100 efficacy evaluable (FAS) patients.

Potential study candidates will be patients hospitalized with AHF, SBP > 125 mmHg at the time of screening, and mild to moderate renal impairment, measured as an estimated glomerular filtration rate (eGFR) of 30-75 mL/min/1.73 m2 (sMDRD formula) who require IV diuretic therapy.

After signing an Ethics Committee (EC) or Institutional Review Board (IRB) approved Informed Consent Form (ICF), subjects will be asked to undergo screening procedures for study eligibility.

Eligible subjects must be randomized within 16 hours from presentation (including the ED). Patients will be randomized in a 1:1 ratio to placebo or relaxin 30 µg/kg/day in a double-blind manner. Patients randomized to the placebo arm will receive standard therapy for AHF plus placebo. Patients randomized to the active study drug arm will receive standard therapy for AHF plus active study drug. Study drug will be administered as an IV infusion for 48 hours unless at any time during dosing in 2 consecutive measurements 15 min apart, the subject’s SBP is < 100 mm Hg at which point, study drug will be terminated. If at any time


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during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration.

Patient self-reported AHF symptoms will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline and a 100-mm VAS describing symptom severity at each point in time. These evaluations will be done at baseline (VAS only), 6, 12 and 24 hours from start of study drug infusion, daily while hospitalized through Day 4, and at Day 5 and Day 14.

Subjects will have clinical evaluations including AHF symptom assessments, vitals signs, physical examination emphasizing signs of HF, as well as an assessment of need for further IV or oral HF treatment and of the occurrence of worsening HF events at least daily while hospitalized through Day 4, at Day 5, and then at Day 14 and Day 60 (except for assessment of need for further IV HF treatment and worsening HF events, which are not conducted at Day 60). Blood and urine samples will be collected at baseline and at 24 hours (Day 1) and 48 hours (Day 2) for routine safety assessments and to evaluate renal function. Blood samples will also be collected on Days 3, 4, 5, 14 and 60. Clinical and laboratory evaluations, as detailed above, will be mandatory at baseline, and at Days 5, 14 and 60. If the patient is discharged from the hospital prior to these visits, these evaluations will be performed as outpatient visits.

Just prior to the time that the last patient enrolled in the study reaches the Day 60 follow-up, patients who have not completed the study will be contacted to ascertain an interim vital status. All subjects will receive a phone call at Day 180 from start of study drug infusion to ascertain vital status. Safety will be assessed by recording medical history, monitoring adverse events and vital signs, and performing physical examinations and routine clinical laboratory tests as per the protocol, as well any tests felt to be clinically indicated by the investigator. Adverse events (AE) will be collected from signing of the informed consent form through Day 5 and serious adverse events (SAE) will be collected through Day 14.

7.2 Selection of Study Population

Subjects will be selected from among patients who have been admitted with AHF who have normal to elevated blood pressure and have mild to moderate renal insufficiency at screening. All patients should have dyspnea at rest, have elevated BNP levels, radiographic evidence of pulmonary congestion, and have received an IV dose of furosemide prior to screening (in the ambulance, ED or at admission to the department). Subjects must be randomized within 16 hours of presentation to the hospital. All subjects must meet all of the inclusion criteria and none of the exclusion criteria listed below.

7.2.1 Inclusion Criteria

Subjects must fulfill all of the following criteria at screening to be eligible for the study. The screening period is defined as that interval that begins at the time the informed consent is signed and ends with the qualification of the subject for entry into the study (i.e. when subject is determined to meet all eligibility criteria).

1. Able to provide written informed consent


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2. Male or female ≥ 18 years of age, with body weight < 160 kg

3. Systolic blood pressure > 125 mmHg at the start of screening and at the end of screening

4. Hospitalized for AHF. AHF is defined as including all of the following measured at any time between presentation (including the emergency department [ED]) and the end of screening:

a. Dyspnea at rest or with minimal exertion

b. Pulmonary congestion on chest radiograph

c. BNP ≥ 350 pg/mL or NT-pro-BNP ≥ 1400 pg/mL

5. Able to be randomized within 16 hours from presentation to the hospital, including the ED

6. Received IV furosemide of at least 40 mg (or equivalent) at any time between admission to emergency services (either ambulance or hospital, including the ED) and the start of screening for the study

7. Impaired renal function defined as an estimated glomerular filtration rate (eGFR) on admission between 30-75 mL/min/1.73 m2, calculated using the simplified Modification of Diet in Renal Disease (sMDRD) equation

7.2.2 Exclusion Criteria

1. Pregnant or breast-feeding women (women of child bearing potential must have the results of a negative pregnancy test recorded prior to study drug administration)

2. Administration of intravenous radiographic contrast agent within 72 hours prior to screening or acute contrast-induced nephropathy at the time of screening

3. Temperature >38°C (oral or equivalent) or sepsis or active infection requiring IV anti-microbial treatment

4. Current (within 2 hours prior to screening) or planned (through the completion of study drug infusion) treatment with any IV therapies, including vasodilators (including nesiritide), positive inotropic agents and vasopressors, or mechanical support (intra-aortic balloon pump, endotracheal intubation, mechanical ventilation, or any ventricular assist device), with the exception of IV furosemide (or equivalent), or of IV nitrates at a dose of < 0.1 mg/kg/hr if the patient has a systolic BP > 150 mmHg at screening

5. Current or planned ultrafiltration, hemofiltration, or dialysis

6. Known significant pulmonary disease

7. Known significant valvular disease (including any of the following: severe aortic stenosis [AVA < 1.0 or mean gradient > 50 on prior or current echocardiogram], severe aortic regurgitation, or severe mitral stenosis)


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8. Any organ transplant recipient, or patient currently listed for imminent transplant (i.e., does not exclude patients on an administrative transplant waiting list), or admitted for any transplantation

9. Major surgery within 30 days

10. Hematocrit < 25% or blood transfusion in the prior 14 days or active, life-threatening GI bleeding; or active menorrhagia or metrorrhagia

11. Major neurologic event, including cerebrovascular events, in the prior 60 days

12. Clinical diagnosis of acute coronary syndrome within 45 days prior to screening (including the present admission) as determined by both clinical and enzymatic criteria

13. Troponin > 3 times the upper limit of normal (including "borderline/intermediate") between presentation and the end of screening

14. AHF due to significant arrhythmias (including any of the following: ventricular tachycardia, bradyarrhythmias with ventricular rate <45 beats per minute or any second or third degree AV block or atrial fibrillation/flutter with ventricular response of >120 beats per minute)


16. Known hepatic impairment

17. Non-cardiac pulmonary edema, including suspected sepsis

18. Administration of an investigational drug or implantation of investigational device, or participation in another trial, within 30 days before screening or previous treatment with relaxin

19. Inability to follow instructions or comply with follow-up procedures

20. Known hypersensitivity to relaxin or similar substances or to any of the excipients

7.2.3 Prior and Concomitant Therapy

To be eligible for the study, all subjects must have received IV furosemide of at least 40 mg (or equivalent) at any time between presentation to emergency services (either ambulance, emergency room) or hospital and screening for the study.

Subjects will not be enrolled in the study if concomitant therapy for AHF includes current (within 2 hours prior to screening) or planned (through the completion of study drug infusion) treatment with any IV therapies, including vasodilators (including nesiritide), positive inotropic agents and vasopressors, or mechanical support (intra-aortic balloon pump, endotracheal intubation, mechanical ventilation, or any ventricular assist device), with the exception of IV furosemide (or equivalent), or IV nitrates at a dose of < 0.1 mg/kg/hr if the patient has a systolic BP > 150 mmHg at screening. Use of IV furosemide (40 mg or


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equivalent) is an inclusion criterion; further doses of IV furosemide can be administered at any time after enrollment. Administration of intravenous radiographic contrast agent is also not allowed within 72 hours prior to screening or at any time during the study.

Major classes of medication taken by a subject in the 30 days prior to study drug initiation and on a daily basis while hospitalized through Day 4 and at Days 5, 14 and 60 will be recorded. Only those medications currently being taken or that were taken within 24 hours prior to the visit, will be collected. These include loop diuretics, other diuretics, ACE inhibitors, angiotensin receptor antagonists, beta blockers, hydralazine, nitrates, calcium channel blockers, digoxin, non-steroidal anti-inflammatory agents, COX-2 inhibitors, aminoglycoside antibiotics, inotropes, vasodilators and others.

The investigator may prescribe any additional medications during the study as dictated by the patients’ condition. Administration of standard treatment should in no instance be delayed or withheld due to patient’s participation in the study. Standard treatment includes, but is not limited to, administration of the major classes of medications, as described above, as well as administration of oxygen, analgesics, anxiolytics and sedatives, as needed. Any medication added or discontinued during the study should be recorded on the CRF.

7.2.4 Subject Identification

Each subject will be identified by a unique subject identification code assigned at the time of randomization. This unique identifier will be written on all CRF pages. In legal jurisdictions where use of initials is not permitted, a substitute identification code will be used.

7.3 Study Drug

7.3.1 Investigational Products

The study drug is relaxin, provided as a 1 mg/mL solution in 5 mL vials (3.5 mL fill). Matching placebo is provided in identical vials. Both relaxin and placebo are administered as a continuous IV infusion in combination with saline or a compatible IV infusion solution as described in the Pharmacy Manual. Study drug should be administered in a dedicated IV line. Blinded study drug kits prepared for each treatment arm will be identical to one another to maintain the blind. Blinded kits will contain 1 or 2 vials of relaxin or placebo. Each blinded kit will be uniquely numbered and will contain enough study drug for 24 hours of infusion. Drug product packaging will be stamped with the expiration date and will be labeled with the study number. Kits should be stored in a refrigerator at 2-8° C in a secure area accessible only to qualified staff authorized to prepare study drug. All study drug should be stored and inventoried according to applicable state, federal, and local regulations and study procedures.

Study drug kits will be allocated by kit number to each subject via Interactive Voice Response System (IVRS). The study drug will be mixed according to instructions provided in the Pharmacy Manual. All site personnel will be blinded to treatment assignments. Compatible IV tubing must be used as described in the Pharmacy Manual. Doses are administered on a weight-adjusted basis.


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Subjects who are eligible to receive the second 24 hours of study drug will require the preparation of a study drug from a second kit (requested and allocated via IVRS).

Qualified clinical research personnel will prepare study drug by mixing the contents of the blinded kits to achieve the proper concentration of relaxin or to prepare placebo as described in the Pharmacy Manual. Study drug will be dispensed only by study staff qualified to perform that function under applicable local laws and regulations for the study site, according to the protocol and Schedule of Events (see Appendix 21.1).

Unused study drug must be kept in a secure location for final accountability and reconciliation by Corthera or their representative, such as a clinical trial monitor. The monitor will account for all study drug in a formal reconciliation process prior to destruction on-site or returning drug to the depot. Only those sites with an approved SOP for Study Drug destruction will be allowed to destroy study drug on site once a final reconciliation has taken place. Return of study drug or study drug materials to a central depot for final accountability and destruction must occur at Corthera’s request and must be accounted for on a Study Drug Returns Form provided by Corthera or representative. All study drug and study drug materials should be stored, inventoried, reconciled, and destroyed or returned according to applicable state and federal regulations and study procedures.

7.3.2 Selection of Doses Used in the Study

Protocol RLX.CHF.001: The selection of relaxin doses in the Pre-RELAX study were based on observations from previous human studies with relaxin and the pilot study of relaxin in subjects with compensated CHF (Protocol RLX.CHF.001). In RLX.CHF.001 Part A, 4 patients were treated sequentially for 8 hours each with relaxin at 0.42, 1.25, and 4.2 µg/kg/hr (equivalent to 10, 30, and 100 µg/kg/day). Six patients were enrolled into Part B, in which relaxin was sequentially administered for 8 hours each with relaxin at 10, 20, and 40 µg/kg/hr (equivalent to 240, 480, and 960 µg/kg/day). In Part C, the highest safe dose of relaxin tested in Part B, 40 µg/kg/hr (960 µg/kg/day), was selected for continuous 24 hr IV infusion. All doses were well tolerated.

The results showed trends in improvement in a number of hemodynamic parameters during dosing in Parts A - C (across doses of 10 – 960 µg/kg/day). There were decreases in PCWP and SVR, and increases in CI, all of which achieved statistical significance at certain time points. However, in the highest dose group, trends reflecting a worsening beyond baseline of hemodynamics, as well as of creatinine and BUN, were observed following cessation of dosing.

Because desirable hemodynamic and renal improvements were observed in Part A, a suitable extended dose range for study in this protocol was reasoned to be 10, 30, 100 and 250 µg/kg/day. While the post-dosing changes in hemodynamics, serum creatinine, and BUN in Part C were asymptomatic, it was elected not to include the 2 highest doses of relaxin, 480 and 960 µg/kg/day, for further study in this protocol. Data from the systemic sclerosis trials in which doses of 10-100 µg/kg/day relaxin achieved improvements in renal parameters, as


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well as in blood pressure, also suggest that doses of 10-250 µg/kg/day relaxin will be efficacious in this study.

Pre-RELAX-AHF Pilot Phase: The Pre-RELAX-AHF Phase II portion of the study has been completed with a total enrollment of 234 patients from 8 countries. Subjects were enrolled into five dosing groups: placebo and relaxin at 10, 30, 100 and 250 µg/kg/day and followed for up to180 days. Enrollment was halted following a protocol-specified administrative analysis of data up to Day 14 when 2/3 of the subjects (n = 209) had been randomized. The data obtained at the interim analysis fulfilled the study objectives of evaluating the efficacy of IV relaxin in patients with AHF; of being able to select a dose of IV relaxin for further study in the RELAX-AHF study; of assessing the distribution of the primary and secondary endpoints in the study population and the adequacy of the power calculations; and of evaluating the overall safety of IV relaxin in these patients.

The final dataset includes 234 patients who have been followed through an average of 4.5 months post-dosing. Baseline characteristics at study entry were balanced across study groups. The 30 µg/kg/day relaxin dose was optimal in a number of independent measures of patient improvement in dyspnea and HF signs, symptoms and outcomes. Dyspnea improvement measured by the Likert scale indicated that relaxin rapidly improved shortness of breath by 6 hr which was sustained at 12 and 24 hours, with 43% of patients reporting moderate or marked improvement in the 30 µg/kg/day group, compared to 23% in the placebo group (p = 0.037). Measurement by the VAS scale indicated that dyspnea relief in the relaxin-treated groups was sustained, as improvements compared to placebo were observed from 6 hr through Day 14. The largest magnitude of improvement in dyspnea across all time points was observed in the 30 µg/kg/day group with supportive trends in the 10 and 100 µg/kg/day groups. A difference of 888 mm-hour (approximately 7.4 mm difference across time points on average) between the 30 µg/kg/day and placebo groups on the dyspnea VAS AUC from baseline to Day 5 was observed (p = 0.10).

Similar trends, with the 30 µg/kg/day dose group being optimal, were seen with multiple outcomes including greater weight loss, lower IV loop diuretic use and lower incidence of in-hospital worsening heart failure in the relaxin groups compared with placebo. The median days alive and out of hospital through Day 60 was higher in the relaxin groups than in the placebo group: 46.0 days in the placebo group and 51.0, 49.5, 51.0, and 50.0 days in the relaxin 10, 30, 100, and 250 µg/kg/day groups, respectively. Kaplan-Meier estimates of the proportion with CV death or HF rehospitalization through Day 60 were 17.2, 10.1, 2.6, 8.4 and 6.2 %, respectively, in the placebo and 4 relaxin dose groups (p = 0.053 for the 30 µg/kg/day group vs. placebo). Estimates of the 60-day risk of CV death, with an average follow up of 4.5 months, were 6.9, 2.5, 0.0, 2.9, and 6.2%, respectively.

Relaxin was safe and well tolerated at all doses. AEs and SAEs were balanced among dose groups. Blood pressure decreases were manageable and most resolved spontaneously. Two serious adverse events of hypotension were reported, both in the 250 µg/kg/day group. In the 250 µg/kg/day dose group, asymptomatic increases from baseline in serum creatinine were


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observed at Day 14, consistent with expectations of evidence of nascent dose limiting toxicity in the highest dose group.

These results support the selection of relaxin 30 µg/kg/day as the dose for evaluation in the Phase III RELAX-AHF portion of this study. These results also confirm the choice of efficacy endpoints for measurement in Phase III RELAX-AHF.

7.3.3 Method of Assigning Subjects to Study Drug and Blinding

In Pre-RELAX-AHF, subjects will be randomized 3:2:2:2:2 to receive an IV infusion of one of the following treatment assignments for up to 48 hours:






In RELAX-AHF, subjects will be randomized in a 1:1 fashion to receive an IV infusion of one of the following treatment assignments for up to 48 hours:



This will be a double-blind study. Subject numbers will be assigned sequentially at each study site through the IVRS. The subjects will be assigned a study drug kit through the IVRS based on the randomization plan. Each study drug kit will be labeled with a unique kit identification number. The two types of vials (relaxin and placebo) are designed to be indistinguishable. Study drug will be prepared from the vials contained in the blinded kits into a 250 mL IV bag of D5W according to instructions in the Pharmacy Manual. All study personnel will remain blinded to treatment assignments.

In the event of a medical emergency, the investigator should contact the medical monitor or other authorized Corthera representative to discuss the necessity of unblinding the subject’s treatment assignment. The treatment assignments will be accessible to the investigator should a subject need to be unblinded in an emergency. If unblinding occurs, the investigator must record the reason for unblinding, as well as the date and time of the event.

7.3.4 Study Drug Administration

Eligible subjects will receive a continuous IV infusion of relaxin or placebo for up to 48 hours. The infusion will begin after the ICF has been signed and all screening procedures are completed. Randomization will occur no more than 16 hours after arriving at the ED or hospital. Patients randomized to the placebo arm will receive standard therapy for AHF plus placebo. Patients randomized to an active study drug arm will receive standard AHF therapy plus active study drug. Study drug should be administered as soon as possible after randomization. If at any time during dosing, the subject’s systolic blood pressure is


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decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration. Study drug administration will continue for 48 hours unless the subject’s SBP at any time during dosing in 2 consecutive measurements, 15 min apart, is <100 mm Hg, at which time the study drug infusion will be permanently terminated. BP should be measured with the patient in the same position and with the same equipment at each time point throughout study drug infusion.

7.3.5 Discontinuation of Patient Dosing

Dosing may be discontinued at any time at the discretion of the investigator. Reasons for which the investigator may discontinue study drug administration include, but are not limited to, the following:

• Subject experiences a serious or intolerable AE

• Subject develops a clinically significant laboratory abnormality

If dosing is discontinued for safety reasons, it should not be re-started. Re-administration of study drug that was terminated due to hypotension is not allowed. For non-safety reasons (problems with the infusion line, etc), dosing may be interrupted once for each patient for up to one hour. This time should not be added back to the end of the infusion and the timed protocol assessments should not be adjusted. Time from initiation of study drug infusion to completion should never exceed 48 hours. In the event that study drug administration is discontinued for any reason, the patient will continue to be followed at all study visits defined in the protocol.

7.3.6 Study Drug Administration Compliance

Study drug will be given IV under medical supervision. Infusion times and rates will be recorded in the CRF. Study drug and supplies must be made available for inspection by the clinical trial monitor for accountability.

7.4 Efficacy and Safety Assessments

7.4.1 Efficacy Endpoints

The two primary efficacy endpoints for the Phase III RELAX-AHF are:

• Area Under the Curve (AUC) representing the change in patient-reported dyspnea from baseline measured by 100-mm Visual Analog Scale (VAS) from baseline through Day 5

• Moderately or markedly better dyspnea relative to the start of study drug on the 7-point Likert scale at 6, 12 and 24 hours (at all 3 time points)

The secondary efficacy endpoints for RELAX-AHF are:

• Days alive out of hospital through Day 60

• Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 60


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Additional outcomes to be analyzed as supportive evidence for relaxin efficacy in RELAX-AHF are:

• Change from baseline in dyspnea score by VAS at 6, 12, 24 and 48 hours and at Days 5 and 14

• Area under the curve representing the change in dyspnea VAS from baseline for baseline to Day 14, Day 1 to Day 5 and Day 1 to Day 14.

• Markedly or moderately better dyspnea compared to baseline by Likert scale assessed separately at 6, 12, and 24 hours and then daily to Day 5 and at Day 14.

• Time to moderately or markedly better self-assessed dyspnea on the 7-point Likert scale up to Day 5

• Time to worsening heart failure through Day 5 and Day 14

• Total doses of IV loop diuretics and oral loop diuretics through Day 5 or discharge if earlier

• Change in weight from randomization through Day 5 and Day 14.

• Length of hospital stay

• All cause death or rehospitalization for heart failure or renal failure through Day 60

• Days alive and out of hospital through Day 30

• Cardiovascular death through Day 180

• Other heart failure signs and symptoms (subject-reported general well-being and investigator-assessed dyspnea on exertion, orthopnea, rales, jugular venous pulse and edema) over time

• Days in the ICU/CCU

• All cause death through Day 30

• All cause death or worsening heart failure or rehospitalization for heart failure through Day 30

• Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 30

• Cardiovascular death or rehospitalization due to heart failure or renal failure through 30 days after discharge from the index hospitalization

Additional details are provided in the Statistical Analysis Plan.

7.4.1.1 Patient-Reported Dyspnea and General Well-Being Assessments

Patient-reported dyspnea and general well being assessments will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline and a 100-mm VAS describing symptom severity at each point in time. These evaluations will be done at baseline (VAS only), 6, 12, 24 and 48 hours from start of study drug infusion, and then daily


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while hospitalized up to Day 4 and then at Day 5 and Day14. Patients should provide this information in the same setting and in the same physical position at each assessment. Please refer to Appendix 21.3.1 for additional details.

7.4.1.2 Physician Assessment of Signs and Symptoms of Heart Failure

The investigator will evaluate the symptoms and signs of heart failure, including dyspnea on exertion or at rest, orthopnea, rales, jugular venous pulse (JVP) and peripheral edema (Appendix 21.3.2). These evaluations will be done at baseline, 6, 12, 24 and 48 hours from start of study drug infusion, and then daily while hospitalized through Day 4, at Day5 and then at Days 14 and 60. These evaluations should be done at the same time of day, each day, in the same position, and hospital setting, preferably by the same assessor. To ensure consistency, if the same assessor cannot perform the daily assessments, the second assessor should evaluate the patient together with the first assessor at least once prior to performing the assessments independently.

WHF is defined for this study as worsening signs and/or symptoms of heart failure that require an intensification of intravenous therapy for heart failure or mechanical ventilatory or circulatory support. Such treatment can include the institution or up-titration of IV furosemide, IV nitrates or any other IV medication for heart failure, or institution of mechanical support such as mechanical ventilation, IABP, etc (Cotter 2010; Weatherley 2009). It is important to note that medications for heart failure (such as IV treatment for hypertension control) can be added for reasons other than WHF.

7.4.1.3 Death or Rehospitalization through Day 60

Vital status will be assessed daily while hospitalized up to Day 4, at Days 5, 14 and 60, and through a telephone contact at Day 180 in all patients. In addition, just prior to the time that the last patient enrolled in the study reaches the Day 60 follow-up, patients who have not completed the study will be contacted to ascertain an interim vital status. The occurrence of rehospitalizations for HF or renal failure will be assessed at Days 5, 14 and 60. Rehospitalization will be defined as an unplanned hospitalization (including admission to a hospital or any attendance in an acute care setting, e.g. ED, or in another health care facility) of 24 hours or greater, regardless of whether the patient was admitted to the hospital.

An independent Clinical Endpoints Committee (CEC) will review all deaths and re-hospitalizations (except planned or elective admissions) that occur through Day 60. The CEC will classify the primary reason for each rehospitalization, and the cause of each death through Day 60. Details of rehospitalizations and deaths will be forwarded to the Sponsor or other facility designated by the Sponsor, who will process the information and provide it to the Clinical Endpoints Committee when the requisite information for each event has been collected. The information needed for adjudication and the process to be followed will be detailed in the CEC Charter.

7.4.2 Safety Assessments

Safety will be assessed by comparing the relaxin group to the placebo group with regard to the frequency of AEs, changes in vital signs, physical examination findings, AHF-related


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signs and symptoms, and clinical laboratory test results (chemistry, hematology, and urinalysis).

During the Pre-RELAX-AHF pilot phase, AEs and SAEs were assessed up to day 30. During the RELAX-AHF phase, AEs will be collected through Day 5 and SAEs through Day 14. Subject-related events and information, including specific instructions given, procedures performed, concomitant medications taken, study drug dispensed, and AEs experienced, should be recorded in the appropriate source documents and CRFs.

The Schedule of Events (Appendix 21.1) describes the timing of the required evaluations.

7.4.2.1 Physical Examinations

A directed physical examination with vital signs will be performed at Screening, Baseline, at 6, 12, 24 and 48 hours after the start of study drug infusion, and daily while hospitalized through Day 4, at Day 5 and at Days 14 and 60.

Subjects discharged post study drug infusion (48 hours) but prior to Day 5 will be required to return for a physical examination as an outpatient at Days 5, 14, and 60.

Physical exams should be performed at the same time each day, with the patient in the same position and in the same location. To ensure consistency, if the same assessor cannot perform the daily assessments, the second assessor should evaluate the patient together with the first assessor at least once prior to performing the assessments independently. Body weight will be measured at Screening and at 24 and 48 hours after study drug infusion, and daily until discharge, Day 5, and at Days 14 and 60.

Vital signs will be followed closely for patients whose study drug was down titrated or who were discontinued due to hypotension or for meeting study drug stopping rules.

7.4.2.2 Clinical Laboratory Assessments

Standard laboratory tests, including hematology and chemistry evaluations and urinalysis, will be performed at Screening (local laboratory), at Baseline and at 24, and 48 hours after the start of study drug infusion, and daily while hospitalized through Day 4, at Day5, and at Days 14 and 60.

Clinical hematology evaluations will include measurement of hemoglobin, hematocrit, white blood cell count, red blood cell count, differential cell count, and platelet count. Clinical chemistry evaluations will include measurement of alkaline phosphatase, ALT (SGPT), AST (SGOT), total bilirubin, BUN, calcium, total cholesterol, creatinine, glucose, HDL, LDL, phosphate, potassium, sodium, triglycerides, uric acid, albumin, and total protein. Urinalysis will include measurement of glucose, ketones, pH, protein, and specific gravity.

Blood specimens for neurohormonal or other markers of HF and renal function will be collected at the same time points as other clinical laboratory assessments (baseline and at 24 and 48 hours after the start of study drug infusion, and daily while hospitalized through Day 4, at Day 5, and at Days 14 and 60). Samples collected for neurohormones may also be analyzed for relaxin levels in order to obtain additional pharmacokinetic data.


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All laboratory assessments for this study, except those done at Screening, will be performed by a central laboratory. The central laboratory will provide instructions and supplies to the study staff before study initiation. The instructions will be included in a laboratory manual.

Since results of study-related laboratory assessments will be delayed because of sample transit times, local laboratory evaluations will be performed by the investigators at the site, if deemed necessary, and local laboratory values will be used to guide patient management.

7.4.2.3 Pregnancy Tests

Urine or serum pregnancy tests will be performed for all females of childbearing potential at Screening and if suspected during the study. Any subject with a positive pregnancy test at Screening will be excluded from participating in the study.

7.4.2.4 Data Safety Monitoring Board (DSMB)

The Data Safety Monitoring Board (DSMB) will act in an advisory capacity to Corthera to monitor subject safety and the efficacy of relaxin in AHF subjects who participate in the Pre-RELAX-AHF and RELAX-AHF phases of the study. The DSMB responsibilities are to:

• Review the study protocol, informed consent, Investigator’s Brochure and plans for data safety and monitoring;

• Evaluate the progress of the trial, study data quality, timeliness, subject recruitment, accrual and retention, subjects’ risk versus benefit, and other factors that could affect the study outcome;

• Consider relevant information that may have an impact on the safety of the participants or the ethics of the study;

• Protect the safety of the study participants;

• Make recommendations to Corthera concerning continuation, termination or other modifications of the study based on their observations of the safety of the study.

For ethical reasons and to ensure integrity of the trial, an interim examination of key safety data was performed during the Pre-RELAX-AHF pilot phase. The DSMB will perform similar evaluations during the RELAX-AHF main phase which are described in section 9.13.

Further details regarding data safety monitoring guidelines will be included in the DSMB Charter.

7.5 Pharmacokinetics

No formal pharmacokinetic (PK) analyses will be performed. Relaxin levels will be measured 24 hours after the initiation of study drug dosing to confirm that the appropriate dose was administered. Blood samples collected for neurohormone analysis may also be used for additional PK-related analyses, including the presence of anti-relaxin antibodies.


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7.6 Study Procedures

7.6.1 Screening

Subjects eligible to participate in this study must be randomized within 16 hours of arriving at the ED or hospital. Prospective study candidates will be identified either en route to or upon arriving at the ED/hospital. After identifying a potential subject, an ICF must be signed before performing study-related screening procedures that are not considered standard of care for AHF patients at that site. Procedures that are part of a site’s standard of care for an individual with AHF may pre-date the signed ICF. The AE and SAE reporting period will begin at the time the ICF is signed. See Schedule of Study Events (Section 21.1.1) and Detailed Schedule of Laboratory Assessments (Section 21.1.2) for tabular summaries of study assessments.

The following study activities will be performed at Screening:

• Review and record medical history

• Record concomitant medications

• BNP or NT-pro-BNP measurement (if not done between presentation and screening)

• Troponin measurement (if not done between presentation and screening)

• Electrocardiogram and Chest X-Ray (if not done between presentation and screening)

• Estimate glomerular filtration rate (by sMDRD formula)

• Physical examination with vital signs (including two BP measurements at the beginning and end of screening to ensure subject meets inclusion criteria #3)

• Record body weight and height

• Blood collection for laboratory tests (local laboratory used for screening)

• Pregnancy test, if applicable

• Assessment of AEs and SAEs

• Assess eligibility criteria (inclusion/exclusion criteria)

If the subject meets all eligibility criteria at the end of screening, call the IVRS to randomize the subject.

7.6.2 Baseline (Day 0 - Prior to start of study drug infusion)

• Patient reported dyspnea and general well-being assessments (by VAS only)

• Physician assessment of heart failure signs and symptoms


• Physical examination with vital signs


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• Blood and urine collection for laboratory tests

• Blood collection for neurohormone measurement


7.6.3 Study Drug Infusion Period

Study drug infusion should be initiated as soon as possible after randomization.

Study drug will be administered as an IV infusion for 48 hours, unless the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart. In this case the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration. If at any time during dosing, SBP is reduced to < 100 mm Hg and confirmed with 2 consecutive measurements 15 minutes apart, the study drug infusion will be permanently terminated. Re-administration of study drug that was terminated due to hypotension is not allowed. BP should be measured with the patient in the same position and with the same equipment at each time point throughout study drug infusion.

Assessments During the Study Drug Infusion Period (up to 48 hours)

Acceptable windows for assessments done during this interval are as follows:

• 6 hour time point, a window at 4.5 - 7 hours is allowed

• 12 hour time point, a window at 9-13 hours is allowed

• 24 hour (Day 1) time point, a window at 20-26 hours is allowed

• 48 hour (Day 2) time point, a window at 42-48 hours is allowed

The purpose of the time windows during the study infusion period is to allow for patients to be assessed during their normal waking hours as much as possible.

• Patient reported dyspnea and general well-being assessments (by Likert and VAS) at 6, 12, 24 and 48 hours after the start of infusion.

• Physician assessment of heart failure signs and symptoms at 6, 12, 24 and 48 hours after the start of infusion

• Record concomitant medications at 24 and 48 hours after the start of infusion

• For laboratory tests, blood and urine collection at 24 and 48 hours after the start of infusion

• Blood collection for relaxin levels at 24 hours after the start of infusion

• Blood collection for neurohormone measurement at 24 and 48 hours after the start of infusion

• Physical examination with vital signs recorded at 6, 12, 24 and 48 hours after the start of infusion; blood pressure and heart rate measurements at intervals defined by institutional standard of care for patients receiving vasodilators, but should be


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performed no less than at 30 minutes and then hourly during the first 6 hours of study drug administration and then every 3 hours during the period of study drug infusion, including the night time hours. After discontinuing study drug, blood pressure and heart rate should be measured every 3 hours through 12 hours post-discontinuation, and then every 6 hours through 48 hours post-discontinuation. Should the study drug dose be decreased or the study drug be discontinued prematurely due to blood pressure decrease, then measurements should be taken every half hour through 2 hours following the blood pressure decrease event, and then hourly through 5 hours after event onset. Upon completion of the 5 hour post event onset time point, heart rate and blood pressure measurements should be resumed per the schedule as set forth in the protocol.

• Determination of worsening heart failure at 24 and 48 hours after start of infusion

• Assessment of AEs and SAEs at 24 and 48 hours after start of infusion

• Record vital status at 24 and 48 hours after start of infusion

• Record weight in kilograms at 24 and 48 hours after start of infusion

7.6.4 Post-Study Drug Infusion Follow-Up Period (Day 3 through Day 180)

Days 3 (± 2 hours), 4 (± 2 hours) and 5 (+ 2 days)

Subjects discharged prior to Day 5 will be asked to return to the clinic for Day 5 procedures and will not need to undergo assessments between discharge and Day 5. If performed as an outpatient visit, Day 5 may be performed between Days 5-7. Daily study procedures include the following:

• Patient reported dyspnea and general well-being assessments (by Likert and VAS)


• Blood collection for laboratory tests



• Physical examination with vital signs; blood pressure and heart rate measurements

• Determination of worsening heart failure


• Record occurrence of re-admissions for patients discharged from the hospital prior to the Day 5 assessment

• Record vital status

• Record weight in kilograms

Day 14 Follow-up (+ 2 days)


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Subjects discharged prior to day 14 will be asked to return to the clinic for Day 14 procedures. Study procedures include the following:

• Patient reported dyspnea and general well-being assessments (by Likert and VAS)




• Determination of worsening heart failure

• Blood collection for laboratory tests


• Assessment of SAEs

• Record occurrence of re-admissions for patients discharged from the hospital since the last assessment



• EQ-5D questionnaire

Day 60 Follow-up (+ 10 days)

Subjects will be required to return to the clinic for Day 60 procedures. Study procedures include the following:




• Blood collection for laboratory tests and neurohormone measurement

• Record occurrence of re-admissions for patients discharged from the hospital since the last assessment



• EQ-5D questionnaire

Day 61-179 Interim Follow-up Contact (-30 days /+7 days from last patient's Day 60 visit)

All subjects will be required to return to the clinic for a Day 60 visit. Just prior to the time that the last patient enrolled in the study reaches the Day 60 follow-up, patients who have not completed the study will be contacted to ascertain an interim vital status. The interim follow-up contact may take place up to 30 days prior to the calculated Day 60 visit of the last patient


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enrolled or within 7 days after the actual Day 60 visit of the last patient enrolled. All patients that have an interim follow-up contact at the time the last patient enrolled reached Day 60, will have a second follow-up contact at Day 180.

Day 180 Follow-up Contact (± 7 days)

All subjects will be contacted by telephone at Day 180 to ascertain vital status.

7.6.6 Early Withdrawal from the Study

Subjects may withdraw consent for participation in the study at any time without prejudice. The investigator must withdraw from the study any subject who requests to be withdrawn.

The subject will be asked to notify the investigator as soon as possible about his/her decision and to sign a Withdrawal of Consent form. On this form the subject will be asked to specify the study-related activities from which he/she is withdrawing consent.

Any AE or SAE that caused a subject to withdraw from the study or any existing AE or SAE, clinically significant abnormal laboratory value or vital sign measurement identified at this visit will be followed up by the investigator.

For subjects who withdraw from the study but will allow further assessments, evaluations should take place at the next scheduled time point and be documented on the corresponding CRF.

If a subject fails to return for scheduled visits, a documented effort must be made to determine the reason. If the subject cannot be reached by telephone, appropriate documented measures must be made requesting contact with the clinic. This information should be recorded in the study records.

Corthera reserves the right to discontinue the study at any time for clinical or administrative reasons, and to discontinue participation by an individual investigator or site for poor enrollment or non-compliance.

7.7 Data Quality Assurance

Corthera personnel or their designees will visit the study site prior to initiation of the study to review with the site personnel information about the study drug, protocol and other regulatory document requirements, randomization procedures, CRFs, monitoring requirements, and procedures for reporting AEs, including SAEs.

At visits during and after the study, a CRA will monitor the site for compliance, including accurate and complete recording of data on CRFs, source documents, regulatory documents and drug accountability records. The study will be conducted according to the principles of GCP.

The designee for Biostatistics and Clinical Data Management functions will enter CRF data in duplicate into a study database.

Data quality control and analysis will be performed by Corthera or a designee, based on a pre-defined analysis plan.


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8 ADVERSE EVENTS

8.1 Definitions

Adverse Event (AE): An AE is any untoward medical occurrence in a patient or clinical trial subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment.

Adverse Drug Reaction (ADR): An ADR is defined as all noxious and unintended responses to an investigational medicinal product related to any dose administered. The phrase “responses to a medicinal product” means that a causal relationship between a medicinal product and an adverse event is at least a reasonable possibility.

Drug Related: means that there is a reasonable possibility that the adverse event may have been caused by the test agent.

Unexpected Adverse Drug Reaction: is an adverse reaction, the nature or severity of which is not consistent with the applicable product information i.e. Investigator’s Brochure.

Serious Adverse Event or Experience (SAE): An SAE is any untoward medical occurrence or effect that at any dose results in death, is life-threatening, requires hospitalization or prolongation of existing hospitalization, results in persistent or significant disability or incapacity or is a congenital anomaly or birth defect.

Suspected Unexpected Serious Adverse Reaction (SUSAR): A SUSAR is an investigational medicinal product-related serious adverse reaction or event, the nature or severity of which is not consistent with the applicable product information, i.e. the Investigator’s Brochure. SUSAR reporting is required according Directive 2001/20/EC.

Disease Related Event (DRE): A DRE is an adverse event that occurs during the participation of the subject in the study but is part of the natural history of the underlying disease and therefore is not unexpected for the condition. See Section 8.4 for further clarification and Appendix 21.4 for a list of DREs for this trial.

8.2 Adverse Events

An adverse event is an undesirable medical condition that can be symptoms (e.g., nausea, chest pain), signs (e.g., tachycardia, enlarged liver) or abnormal results of an investigation (e.g., laboratory findings, electrocardiogram). Therefore, an AE would include the following:

• Any event that is new, i.e., was not seen prior to the start of the study

• A condition that is worsened after study drug administration, even though it may have been present prior to the start of the study

• A pre-existing intermittent event or condition that recurs with increased intensity and/or frequency after the start of the study


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• A symptomatic overdose, with overdose being defined as any dose greater than 2 times the per protocol dose

An AE does not include the following:

• An overdose of the study drug if the subject is asymptomatic, however the Sponsor must be notified immediately if an incorrect dose of study drug is administered and the overdose must be documented.

• A medical or surgical procedure scheduled prior to study enrollment or performed during the trial (e.g., surgery, endoscopy, vascular access or interventions, placement of hemodynamic monitoring catheters, angiography, tooth extraction). Such a procedure may be the result of an AE or may result in an AE, but is not an AE itself

• Hospitalization for elective surgery for a pre-existing disease or condition that did not worsen, or for cosmetic surgery

• A pre-existing condition or disease that does not worsen following study drug administration

• Pregnancy will not be considered an AE, but if occurs, will be reported on pregnancy form.

The following are definitions of levels of severity:

• Mild: an awareness of symptoms but easily tolerated

• Moderate: symptoms interfering with daily activities

• Severe: incapacitating, with inability to perform normal daily activities

Should an AE occur, the Investigator must do the following:

(1) Assess whether or not the study drug caused the AE

(2) Determine the severity of the AE

(3) If an AE occurs during study drug administration, record the action taken regarding the study drug (continued or halted administration)

(4) Indicate whether the AE led to discontinuation of the subject from the study

(5) Determine if the AE is serious (see section 8.3 for definition of “serious”)

All AEs that occur in any enrolled subject from the time the informed consent form is signed through Day 5, whether or not related to the study drug, must be recorded on the appropriate CRF form provided.

8.3 Serious Adverse Events (SAE)

For each AE that occurs, the Investigator must determine if it is an SAE according to the following definition. An SAE is an AE that fulfills one or more of the following criteria:

• An event that results in death


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• An event that is immediately life threatening as it occurred. This does not include an event that, had it occurred in a more serious form, might have caused death.

• An event that results in persistent or significant disability or incapacity

• An event that is a congenital abnormality/birth defect

• An event that requires hospitalization or prolongs an existing hospitalization. Hospitalizations are defined as formal admission to the hospital and does not include presentation at an emergency room.

• An event that, though not included in the above list, is an important medical event that may jeopardize the subject or may require medical intervention to prevent one of the outcomes listed above

An SAE does not include the following:

• Complications that occur during hospitalization and do not meet at least one of the above criteria for seriousness are AEs, not SAEs.

• Presentation at an emergency room.

• Hospitalizations for elective surgery for a pre-existing disease or condition that did not worsen, or for cosmetic surgery are not considered to meet SAE criteria.

• Pregnancy will not be considered an SAE, but if it occurs, it will be reported on the pregnancy forms

Medical and scientific judgment should be exercised in deciding whether a case is serious in those situations where important medical events may not be immediately life threatening or result in death, hospitalization, disability or incapacity. These include events that may jeopardize the subject or may require medical intervention to prevent one or more outcomes listed in the definition of serious. Such events should usually be considered as serious.

All SAEs that occur in any subject from the time the informed consent form is signed through Day 14, whether or not related to the study drug, must be reported and, if the subject is enrolled, must be recorded on the appropriate CRF form provided.

8.4 Disease-Related Events (DRE)

Due to the severity and progressive nature of AHF, certain AEs are not unexpected in this patient population. These AEs will be referred to as Disease-Related Events (DRE). The DREs for this study are listed in Appendix 21.4 and will be captured on the CRF AE page as DREs. If the DRE is “serious” (see Section 8.3 for definition of “serious”), “serious” will be marked on the CRF and the DRE will be recorded in the clinical database as an SAE. The Investigator must then determine if it is unexpected in nature, severity or frequency OR resulted in death OR is study drug-related. If the DRE falls into any of these categories, an SAE form must be completed and the study’s Drug Safety representative will be notified immediately, as described in Section 8.7. If the serious DRE is not unexpected in nature, severity or frequency AND does not result in death AND is not study drug-related, this event will be recorded as an SAE only on the CRF AE page.


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8.5 Laboratory AEs and SAEs

Abnormal laboratory findings (e.g., serum chemistry, hematology) or other abnormal assessments (e.g., ECGs, x-rays, blood gases, vital signs, biopsy) resulting in a new diagnosis that is judged by the investigator to be clinically significant must be recorded as AEs. Clinically significant abnormal laboratory findings or other assessments, resulting in a new diagnosis that are detected following study drug administration or that are present at baseline and worsen following the start of the study, constitute AEs. The investigator should exercise his or her clinical judgment in deciding whether an abnormal laboratory finding or other abnormal assessment is clinically significant.

The investigator should make every effort to establish a diagnosis of the event based on the presenting signs, symptoms, and/or other clinical information. In such cases, the diagnosis and not the individual signs/symptoms should be documented as the AE and/or SAE.

8.6 Collection of AEs, and SAEs: Method, Frequency, and Time Period

The methods of detection of AEs and SAEs include, but are not limited to, the use of non-leading verbal questioning of the subject (e.g., how do you feel?), clinical observation by the investigator or study staff, physical examinations, monitoring of vital signs, or laboratory assessments. During the RELAX-AHF phase, AEs will be recorded for each subject from signing of the ICF through Day 5. SAEs will be captured from signing of the ICF through Day 14. Any AEs, whether drug related or not, that are ongoing at Day 14 will be followed to resolution or if non-resolving, until judged to be stable. SAEs, including DREs that are SAEs, will be recorded for each subject after signing of the informed consent and commencement of study procedures through Day 14. Ongoing SAEs, whether drug-related or not, will be followed to resolution. Deaths will be collected up to Day 180 on a separate CRF page.

8.7 Documentation and Reporting of AEs and SAEs

The Investigator must report all AEs and SAEs according to guidelines outlined in the decision tree shown in Figure 8.1 below.

All AEs occurring during the study must be recorded in the subject’s medical records and on the Adverse Event page of the CRF, whether or not the AE is considered by the investigator to be related to the use of the study drug.

All SAEs that occur during the study must be documented in the subject’s medical record, and on the AE page of the CRF. An additional SAE report form must be completed for certain SAEs according to the guidelines presented in Figure 8.1 below. If the event meets one or more of the criteria for serious, the Investigator must determine if it is study drug related. If yes, the Drug Safety representative, who is the first point of contact for the sites and is responsible for obtaining all medically relevant information on the SAE, must be notified immediately by telephone, and the SAE form, which will be provided by the Sponsor, must be completed and sent within 24 hours to the Drug Safety representative by FAX. The SAE Report Form used to report an SAE should be completed as thoroughly as possible and signed by the investigator or designee before being FAXED to the Drug Safety representative.


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The Drug Safety representative will then relay all of the information to the Sponsor’s Medical Monitor. The investigator must provide his assessment of causality to study drug at the time of the initial report. Any additional documentation, including relevant CRF pages or laboratory tests, must also be sent by FAX.

If the SAE is not related to study drug, the Investigator must then determine if it is a DRE (Appendix 21.4). If the SAE is not a DRE, the Drug Safety representative must be notified immediately by telephone and the SAE form completed and sent by FAX with additional relevant documents, as described above. If the SAE is a DRE, the Investigator must determine if it is unexpected in nature, severity or frequency or results in death. If yes, an SAE form must be completed and the Drug Safety representative notified immediately, as described above. If the SAE is a DRE and is not unexpected and does not result in death and is not study drug-related, only the AE CRF page needs to be completed.


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FIGURE 8.1 DECISION ALGORHITHM FOR DOCUMENTATION OF AES AND SAES

CompleteCRF page

Complete SAEform and CRF

page


page


page

Complete CRFpage only

Did an AEoccur?

Is the SAEdrug-related?

Is the SAE aDRE?

Is DREunexpected in

nature, severityor frequency ordid it result in

death?

Does the AEmeet

the "serious"definition?

Yes

Yes

No

Yes

No

Yes

No Yes

No

In summary, an SAE Report Form, in addition to the Adverse Events CRF page, is required


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for SAEs that:

• Are study drug-related OR

• Are not among the DREs listed in Appendix 21.4 OR

• Are among the DREs but are unexpected in nature, severity or frequency, or resulted in death

If at the time of initial reporting, multiple signs and symptoms are present that are temporally and/or clinically related, they may be reported on the same SAE page until a definitive diagnosis is established. The investigator should make every effort to establish a diagnosis of the event based on the presenting signs, symptoms, and/or other clinical information. In such cases, the diagnosis, and not the individual signs/symptoms, should be documented as the AE and/or SAE. If the diagnosis is not known, the clinical signs and symptoms, rather than the clinically significant abnormal laboratory finding, should be recorded on the AE page of the CRF and on the SAE Report Form, as appropriate. If no diagnosis is known and clinical signs and symptoms are not present, then the abnormal finding should be recorded.

Corthera may be required to report certain SAEs to the appropriate regulatory authorities within 7 and/or 15 calendar days of being notified about the event (21 CFR §312.32, ICH E2A, and ICH E6). Therefore, it is important that investigators submit additional information requested by the Medical Monitor, CRA or other designated representative as soon as possible.

In the United States, investigators are responsible for reporting of SAEs to the IRB, Regulatory Ethics Board (REB) or Ethics Committee (EC) in accordance with the standard operating procedures of the regulatory bodies at each institution. Adequate documentation must be provided to the Medical Monitor, your CRA or other designated representative as soon as possible showing that the regulatory authorities were properly notified. Investigators in the US are also responsible for reporting AEs to their respective IRB/EC in accordance with the individual guidelines for that institution.

In the European Union, the Sponsor is responsible for reporting SUSARs to the Ethics Committees. In all other geographic regions, the appropriate regulatory guidelines will apply.

8.8 Follow-up of AEs and SAEs

AEs related to study drug must be followed to resolution or if judged to be non-resolving, until judged to be stable. SAEs related to study drug must be followed until resolution, the condition stabilizes, the event is otherwise explained, or the subject is lost to follow-up. The investigator is responsible to ensure that follow-up includes any supplemental investigations as may be indicated to elucidate as completely as possible the nature and/or causality of the AE or SAE. This may include additional diagnostic test results/reports, histopathological examinations, any post-mortem findings, hospital discharge summaries, or consultation with other health care professionals. Corthera may request that the investigator perform or arrange for the conduct of supplemental measurements and/or evaluations. New or updated information should be recorded on a Follow-Up SAE report form.


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In the event of a fatal SAE, documentation of any available postmortem findings, including autopsy, should be provided to Corthera. The investigator should provide a narrative summary of circumstances, events related to the death, and cause of death, if known.

In the event of an SAE leading to hospitalization, every effort should be made by the investigational site to obtain a hospital discharge summary.

In the event that a subject is lost to follow-up prior to the Investigator’s being able to verify that an SAE is resolved, stabilized, or otherwise explained, the site must document in the subject’s medical records, as well as in the CRF, the repeated attempts (at least 3) to contact the subject and/or his/her care provider to retrieve the information. Documentation should include date and mode of communication.

8.9 Electrocardiogram (ECG) sub-study

An ECG sub-study will be conducted at a subset of sites in selected countries (Germany, Hungary, Israel, Italy, Poland, Romania, USA). ECGs will be obtained at baseline, at 12, 24, and 48 hours following the start of study drug infusion, and on Day 3 (if the subject is still in the hospital). The purpose of the sub-study is to collect information that will contribute to the cardiac safety profile of relaxin in the target population. Details of the ECG sub-study procedures are provided in Appendix 21.5.

9 STATISTICAL AND ANALYTICAL PLAN

9.1 Pre-RELAX-AHF

The pilot phase (“Pre-RELAX-AHF”) is designed to evaluate the safety of relaxin, as compared to placebo administered for up to 48 hours in subjects with AHF, hypertension and mild to moderate renal impairment, and to select the most appropriate dose of relaxin for further study, as well as assess the power calculation based on distribution of the endpoints in the overall study population. Three hundred thirty (330) eligible subjects will be randomized 3:2:2:2:2 to placebo or one of 4 doses of relaxin: 10, 30, 100 or 250 µg/kg/day. The sample size in the Pre-RELAX-AHF pilot phase is not expected to provide power to detect statistically significant differences; the point estimates and overall pattern of results will be used to guide the dose selection for RELAX-AHF. For each of the primary and key secondary endpoints, a global statistical comparison will be made between the pooled 30, 100, and 250 µg/kg/day and the pooled 10 µg/kg/day and placebo groups. Each active dose group will be compared with placebo at the two-sided 5% significance level, without adjustment for multiple comparisons. Results from the pilot phase will not be combined with results of the main phases to evaluate the efficacy of the selected relaxin dose. The sample size required for the main phase will be re-estimated based on the observed rate in the placebo group. A total of 90 patients in the placebo group of the pilot phase will provide a 95% confidence interval of approximately ± 10 % about a success rate of 50%.


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9.2 Objectives and primary hypotheses of RELAX-AHF

The objectives of the main phase of the RELAX-AHF program are to evaluate the efficacy of IV relaxin in addition to standard therapy in improving symptoms of heart failure, dyspnea, in increasing days out of hospital, and in preventing intermediate-term cardiovascular death or rehospitalization for heart failure or renal failure in subjects hospitalized for AHF with normal to elevated blood pressure and mild to moderate renal impairment, as well as to assess the overall safety of IV relaxin in this patient population.

The effectiveness of relaxin in relieving patient symptoms will be evaluated by two primary endpoints: the area under the curve representing the change from baseline dyspnea VAS score from baseline to Day 5 (‘VAS AUC’), and markedly or moderately better dyspnea on the Likert scale at 6, 12, and 24 hours. The primary global null hypothesis is that both the mean VAS AUC and the proportion with moderately or markedly better dyspnea at 6, 12, and 24 hours are the same in both treatment groups. The Type I error rate for this global null hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach. Two secondary endpoints will evaluate the effect of relaxin in increasing out-of-hospital days and in preventing cardiovascular death or rehospitalization due to heart failure or renal failure through Day 60. The secondary global null hypothesis is that both the mean number of days alive and out of hospital through Day 60 is the same in both treatment groups, and the hazard for CV death or rehospitalization due to heart failure or renal failure through Day 60 is the same in both groups. The Type I error rate will be controlled across the two global null hypotheses at the two-sided 0.05 level by sequential testing: the secondary global hypothesis will be tested given rejection of the primary global null hypothesis. The Type I error rate for the secondary global null hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach.

If the primary global null hypothesis is rejected at the two-sided 0.00125 significance level (equivalent to that required for two trials conducted at the two-sided 0.05 significance level), the single study will be considered to support the efficacy of relaxin in this indication. A result significant at the two-sided 0.05 significance level, but not significant at the two-sided 0.00125 level, will be confirmed in a subsequent study.

9.3 General analytic methods

The data collected in this study will be listed and summarized as appropriate for each treatment group as described below. Continuous data will be summarized by the mean, standard deviation (SD), median, first and third quartiles, minimum and maximum. Categorical data will be presented by absolute and relative frequencies (n and %) or contingency tables. The baseline date and time will be considered the date and time of initiation of study drug, or randomization if the subject was not treated with study drug. Unless stated otherwise, two-sided p values < 0.05 will be considered statistically significant. All statistical tables and listings and analyses will be produced using SAS® release 9.1 or later (SAS Institute, Inc, Cary, NC, USA) or other validated statistical software.


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9.4 Analysis populations

9.4.1 Intent-to-treat (ITT) set

The Intent-to-Treat (ITT) set will include all randomized subjects, whether or not they were exposed to study drug. Subjects will be analyzed by the group to which they were randomized. Analyses in the ITT set will constitute the main efficacy results for the primary and secondary study efficacy endpoints.

9.4.2 Full analysis set (FAS)

The Full Analysis Set (FAS) will include randomized subjects who receive any amount of study drug. Subjects with major eligibility criteria violations (based on data collected prior to randomization) will be excluded. Subjects will be included in each efficacy analysis based on available assessments, after the data handling conventions have been applied. Subjects will be analyzed in the treatment group to which they were randomized, regardless of actual treatment. Supportive (sensitivity) analyses of the primary and secondary efficacy endpoints will be conducted in the FAS.

9.4.3 Safety set

The safety set will include all randomized subjects who receive any amount of study drug and who have at least one post-baseline safety assessment. Subjects will be analyzed as treated.

9.5 Demographic and baseline characteristics

Subject demographics (age, sex, ethnicity, race, weight and height) and baseline characteristics will be summarized for the ITT set and FAS. Screening BNP, NT pro-BNP estimated creatinine clearance, troponin I and troponin T laboratory values as reported by the investigator will be summarized with standard descriptive statistics. The proportion of subjects with positive troponin I and/or T will be presented. The number and percentages of subjects meeting all eligibility criteria at screening will also be provided. The number and percentage of subjects who were hospitalized for heart failure in the past year and subjects on IV nitrates at the time of randomization will both be presented. Inclusion and exclusion criteria violations will be listed. Medical history data will be summarized by treatment group.

9.6 Extent of exposure to study drug

Overall study drug administration details will be summarized for the ITT set. This will include time from presentation to randomization (hrs), time from randomization to study drug administration (hrs), study drug administered (yes/no), reason study drug not administered, actual study drug received, and the number of days infused (one or two).

Study drug administration will also be summarized in the Safety set. The duration of study drug administration (in hours) and the total volume of study drug administered (estimated from the total time and rate of infusion) will be summarized. In addition, the number of subjects whose study medication dose was lowered or discontinued prematurely, and the reasons for discontinuation, will be summarized by treatment group.


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9.7 Use of concomitant medication

The total dose of IV loop diuretic administered during the initial hospitalization is an additional efficacy endpoint and its analysis is described in section 9.12.6. The use of other medications is reported through Day 14 and at Day 60, and will be summarized by treatment group.

9.8 Primary efficacy endpoints

The analysis of the primary efficacy endpoints will be conducted in the ITT and FAS as defined above. The Type I error rate will be controlled for the global primary null hypothesis at the two-sided 0.05 level using the Hochberg approach to control for the two comparisons of the primary endpoints. The primary result will be considered statistically significant if the test of either endpoint is statistically significant at the two-sided 0.025 level, or if both tests are significant at the two-sided 0.05 level.

If the primary result is significant at the two-sided 0.00125 level (i.e., either test is significant at the two-sided 0.000625 level or both are significant at the two-sided 0.00125 level), the single study will be considered to support the efficacy of relaxin in this indication.

9.8.1 Area under the change from baseline dyspnea VAS curve from baseline to Day 5

The area under the curve representing the change from baseline in dyspnea VAS score from baseline through Day 5 (VAS AUC) will be computed by trapezoidal rule after applying the following data handling conventions. For subjects who die or have a worsening heart failure event (either during the index hospitalization or rehospitalization for heart failure) by Day 5, the worst score observed in any subject at any time point will be carried forward for all time points after the time of onset of the event, regardless of whether the score is missing or not. For post-baseline values otherwise missing, a missing score will be imputed using linear interpolation between the last preceding and first following non-missing values; if no following non-missing value is available, the last available preceding value will be carried forward. A missing baseline score will be imputed as the earliest, non-missing score within 24 hours for the subject minus the average change from baseline in the study population to that time point; post-baseline scores for subjects for whom a missing baseline cannot be thus imputed will be included in the analysis as no change from baseline. Except for subjects who die or who experience worsening heart failure, subjects who are missing all post-baseline dyspnea VAS scores will be included in the analysis as having no change from baseline at any time point. Treatment groups will be compared using a t-test as the primary method. If results suggest noteworthy departures from the assumptions underlying the t-test, supportive analyses such as the Wilcoxon rank sum test or a randomization-based determination of the p-value for the t-test may be conducted.

9.8.2 Moderately or markedly better dyspnea at 6, 12, and 24 hours

Subjects will be classified as having moderately or markedly better dyspnea (+2 or +3 on the 7-point scale) at all 3 time points or not. For subjects who die or have a worsening heart failure event (either during the index hospitalization or rehospitalization for heart failure) by 24 hours, the worst score observed in any subject at any time point will be carried forward


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for all time points after the time of onset of the event, regardless of whether the score is missing or not. A non-missing 24-hour score will be substituted for a missing 12-hour score, and a non-missing 12-hour score will be substituted for a missing 6-hour score; otherwise, a non-missing preceding value will be carried forward. Except for subjects who died or who experienced worsening heart failure, subjects who are missing dyspnea Likert scores at all 3 time points will be considered as not having experienced moderately or markedly better dyspnea at 6, 12, and 24 hours.

The proportion of subjects with moderately or markedly better dyspnea at 6, 12, and 24 hours will be compared between treatment groups using a chi-square test.

9.9 Secondary efficacy endpoints

The Type I error rate for the main phase of the RELAX-AHF program will be controlled across the primary and secondary global hypotheses at the two-sided 0.05 level, using a sequential testing approach. Given statistical significance for the primary global hypothesis at the two-sided 0.05 level, statistical testing will proceed to the secondary efficacy endpoints. The overall significance level for the secondary global null hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach. The secondary result will be considered statistically significant if the test of either of the secondary endpoints is significant at the two-sided 0.025 level, or if both tests are significant at the two-sided 0.05 level. Power for these secondary endpoints is discussed in section 9.10. The analysis of the secondary efficacy endpoints will be conducted in the ITT set and FAS as defined above.

9.9.1 Days alive and out of hospital through Day 60

Days alive and out of hospital will be computed as 61 days minus the days in-hospital through Day 60 (including the index hospitalization and any rehospitalizations) minus the days following a death prior to Day 60. Days in hospital will be computed for the index hospitalization as the discharge date minus the baseline date plus one, and for rehospitalizations as the discharge date minus the admission date plus one. If the discharge date is missing for a hospitalization, then the average hospitalization duration will be imputed from the non-missing hospitalizations over all subjects and applied to the hospitalizations missing the discharge date. For these subjects the average hospitalization duration will be truncated at the earliest of days to next additional hospitalization, days to known death, days to the end of the 60 day period, or average hospitalization duration. Subjects with incomplete follow-up will have the average proportion of days alive and out of hospital applied to the missing follow-up period. Treatment groups will be compared using the Wilcoxon rank sum test as the primary method. Supportive analyses using the t-test or its randomization-based counterpart may also be conducted.

9.9.2 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 60

The days from the baseline date to the date of the first event will be used for analysis. Subjects who die from a non-cardiovascular cause before Day 60 without an event will be


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censored at the time of death; subjects otherwise without an event will be censored at the earlier of the last contact date or Day 60. The Clinical Event Committee’s classification of the cause of death or the reason for rehospitalization (heart failure or renal failure) will be used, if available at the time of analysis, otherwise the investigator’s classification will be used. Kaplan-Meier estimates of the event rates will be presented, and treatment groups will be compared using a log-rank test.

9.10 Planned sample size

A total of 1160 subjects will be enrolled in order to obtain approximately 1100 efficacy evaluable (FAS) subjects. This sample size is adequate to detect a clinically meaningful treatment effect for either primary efficacy endpoint at the two-sided 0.05 significance level

Power for the primary and secondary efficacy endpoints was estimated using simulations. Based on correlations among the endpoints observed in Pre-RELAX-AHF, the simulations incorporated the following correlations among the four primary and secondary efficacy outcomes:

VAS AUC Likert 60 day CV Death/ HF

Rehosp

DOOH through d60

VAS AUC 1 0.25 0 0.25

Likert 0.25 1 0 0

60 day CV Death/ HF Rehosp

0 0 1 0.5

DOOH through d60

0.25 0 0.5 1

Rehosp = re-hospitalization; DOOH = days alive and out of hospital

In Pre-RELAX-AHF, a difference of 888 mm-hour on the dyspnea VAS AUC between the 30 µg/kg/day and placebo groups with a pooled standard deviation of 2700 mm-hour was observed; 23.0% in the placebo group and 40.5% in the 30 µg/kg/day group had moderately or markedly better dyspnea at 6, 12, and 24 hours. To estimate power, we assumed a standard deviation of 2700 mm-hour for the VAS AUC and that 25% of placebo subjects would have moderately or markedly better dyspnea at 6, 12, and 24 hours. Minimum clinically relevant effect sizes representing, respectively, a mean difference of 468 mm-hour (4 mm on average) and a relative risk of 1.3 (absolute difference of 7.5%) were assumed. These represent a 28% increase in VAS AUC (468/1679 mm-hour) and a 30% increase in moderately or markedly better dyspnea at 6, 12, and 24 hours (7.5/25%) relative to placebo responses observed in Pre-RELAX-AHF.


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With 1100 subjects, the study has approximately 81% power to detect these minimum clinically relevant effects at the two-sided 0.05 significance level with adjustment for multiplicity using the Hochberg approach.

In Pre-RELAX-AHF, the Kaplan-Meier estimates of the proportion of subjects with cardiovascular death or rehospitalization due to HF or renal failure through Day 60 was 17.2% in the placebo group and 2.6% in the 30 µg/kg/day group (hazard ratio 0.36), and a difference of 3.7 days between the 30 µg/kg/day and placebo groups with a pooled SD of 12.7 days was observed for days alive and out of hospital through Day 60. In order to estimate power for these secondary efficacy outcomes, we assumed a placebo event rate of 17.2% and a standard deviation of 12.7 days for days alive out of hospital through Day 60. Assuming a minimum clinically relevant effect on the primary endpoints and significance at the two-sided 0.05 level as described above, 1100 subjects provides approximately 87% power to detect a hazard ratio of 0.55 (17.2% versus 9.9%) for cardiovascular death or rehospitalization due to heart failure through day 60 and/or a mean difference of 2.7 days in days alive out of hospital through day 60 at the two-sided 0.05 significance level with multiplicity adjustment using the Hochberg approach.

9.11 Examination of covariates

Because many centers will enroll very few subjects, no treatment-by-center interaction will be evaluated, and the primary analyses will be based on results pooled across all centers without adjustment for center. Sensitivity analyses will be conducted examining the results for the primary and secondary efficacy outcomes adjusted for age, gender, race and geographic region or country. The possible interaction between the effect of relaxin and each of these covariates will be examined. A sensitivity analysis will be conducted to examine the effect of further adjustment for the baseline dyspnea VAS score on the results for the VAS AUC to Day 5.

9.12 Additional efficacy endpoints

The additional efficacy endpoints will be analyzed in the ITT set at the two-sided 0.05 significance level, without adjustment for multiple comparisons. Standard descriptive statistics will be presented for each treatment group at each time point the endpoint was measured.

9.12.1 Change from baseline in dyspnea score by VAS at 6, 12, 24 and 48 hours, Day 3, Day 4, Day 5, and Day 14

Treatment groups will be compared with respect to imputed values at each time point using a t-test.

9.12.2 Area under the curve for the dyspnea VAS change from baseline from baseline to Day 14, from Day 1 to Day 5, and from Day 1 to Day 14

The area under the curve of the change from baseline in dyspnea VAS score (VAS AUC) will be computed by trapezoidal rule after applying the data handling conventions in section 9.8.1. Treatment groups will be compared using t-tests.


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9.12.3 Markedly or moderately better dyspnea by Likert scale at 6, 12, and 24 hours and then daily to Day 5 and at Day 14

For subjects who die or have a worsening heart failure event by Day 14, the worst observed score in any subject at any time point will be carried forward for all time points after the time of onset of the event, regardless of whether the score is missing or not. Treatment groups will be compared regarding the proportion of subjects with moderately or markedly better dyspnea at each time point using a chi-square test.

9.12.4 Time to moderately or markedly better dyspnea through Day 5

The first assessment time point of the patient’s first report of moderately or markedly better dyspnea within 5 days will be compared between active and placebo groups using a Wilcoxon rank sum test. Patients who do not report moderately or markedly better dyspnea by Day 5, or who die or have WHF (either during the initial hospitalization or rehospitalization for HF) by Day 5, will be assigned a value of 6 days.

9.12.5 Time to worsening heart failure through Day 5 and Day 14

The onset of worsening heart failure through Day 5 will be the first assessment point following the onset at which the investigator reported worsening heart failure (either during the initial hospitalization or rehospitalization for heart failure). Days will be assigned by nominal time point. Patients for whom the investigator did not report any worsening heart failure by Day 5 will be assigned a value of 6 days. Patients who die by Day 5 without a prior WHF event will be assumed to have had worsening heart failure on the day of death. Treatment groups will be compared using a Wilcoxon rank sum test.

Time to WHF through Day 14 will be analyzed using survival analysis. The time to WHF in days will be computed as the time from the baseline date to the earlier of the onset of WHF or death; subjects without an event will be censored at the earlier of the last assessment or Day 14. Treatment groups will be compared using a log-rank test.

9.12.6 Total doses of IV and oral loop diuretics through Day 5 or discharge if earlier

The total dose of IV loop diuretic and oral loop diuretic administered during the initial hospitalization or to Day 5 will be compared between treatment groups using a t-test. Doses will be converted to furosemide equivalents for analysis.

9.12.7 Change in weight from randomization through Day 5 and 14

Treatment groups will be compared at each time point with a t-test.

9.12.8 Length of hospital stay

Length of stay will be defined as the discharge date minus the baseline date plus 1 day. Subjects still in the hospital at Day 60 will be censored at Day 60. Subjects who die during the initial hospitalization will be assigned the maximum length of stay (including those censored at Day 60) plus 1 day. Treatment groups will be compared using a Wilcoxon rank sum test.


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9.12.9 All cause death or rehospitalization due to heart failure or renal failure through Day 60

The time from the baseline date to the first event will be used for analysis. The CEC classification of the primary reason for rehospitalization (heart failure or renal failure) will be used for the final analyses. Subjects without an event will be censored at the earlier of the last contact date or Day 60. Kaplan-Meier estimates of event rates will be presented, and groups will be compared using a log-rank test.


Days alive and out of hospital will be computed as 31 days minus the days in-hospital through Day 30 (including the index hospitalization and any rehospitalizations) minus the days following a death prior to Day 30. Days in hospital will be computed for the index hospitalization as the discharge date minus the baseline date plus one, and for rehospitalizations as the discharge date minus the admission date plus one. Subjects with incomplete follow-up will have the average proportion of days alive and out of hospital applied to the missing follow-up period. Treatment groups will be compared using a Wilcoxon rank sum test.

9.12.11 Cardiovascular death through Day 180

The time from baseline to the event will be used for analysis. The CEC classification of the cause of death will be used for events up to Day 60 and the investigator’s classification will be used for events from Day 60 to Day 180. Subjects without an event will be censored at the earlier of the last contact date or Day 180. Subjects who die from non-cardiovascular causes will be censored at the time of death. Kaplan-Meier estimates of event rates will be presented, and groups will be compared using a log-rank test.

9.12.12 Other heart failure signs and symptoms over time

Scores following a death or worsening heart failure event (either during the index hospitalization or rehospitalization for HF) will be imputed as the worst observed score in any subject at any time point. Scores and changes in scores will be summarized for subject-reported general well-being VAS, and physician assessment scores for dyspnea on exertion, orthopnea, edema, rales and jugular venous pulse. Scores for subject-reported general well-being on the Likert scale will be summarized. Treatment groups will be compared at each time point with respect to change in VAS scores using two-sided two-sample t-tests, and with respect to Likert scores using Wilcoxon rank sum tests.

9.12.13 Days in the ICU/CCU

The days of the initial hospitalization from randomization spent in the ICU/CCU will be compared between treatment groups using the Wilcoxon rank sum test.

9.12.14 All-cause death through Day 30

The time from baseline to the event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Treatment groups will be compared using a log-rank test.


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9.12.15 All-cause death or worsening heart failure or rehospitalization for heart failure through Day 30

The time from baseline to the first event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Treatment groups will be compared using a log-rank test.

9.12.16 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 30 The time from baseline to the first event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test.

9.12.17 Cardiovascular death or rehospitalization due to heart failure or renal failure through 30 days after discharge from the index hospitalization The time from discharge from the index hospitalization to the first event will be used for analysis. Subjects who were discharged alive will be included in the analysis; subjects who died before or on the same day as discharge, whose last contact was prior to discharge, or whose discharge date is missing will be excluded. Subjects without an event will be censored at the earlier of the last contact date, 30 days from discharge, or the date of the Day 60 follow-up. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test.

9.13 Sequence of planned analyses

9.13.1 Interim analyses

A DSMB will be established to oversee this study (see Section 7.4.2.4). For ethical reasons and to ensure trial integrity, an interim examination of key safety data will be performed at regular intervals during the course of the trial. More details on the objectives and frequency of these interim analyses can be found in the DSMB charter.

An interim examination of key safety data was performed during the Pre-RELAX pilot phase when approximately 200 patients had completed their Day 14 assessment. Similar evaluations will be performed during the RELAX-AHF main phase when approximately 200, 400, 600, and 800 subjects have completed their Day 14 assessment. The primary objective of these analyses will be to evaluate the accumulating safety data. The Sponsor requests that the DSMB not recommend stopping the trial early on the basis of either futility or a statistically significant finding for either or both of the primary outcomes. As the DSMB monitors the safety data from the trial, however, they may require a review of the efficacy data to provide an overall assessment of risk to benefit in order to assist them in making their recommendations. For each review of the primary outcomes, the DSMB will “spend” an alpha of 0.00001.

For each DSMB meeting, the Unblinded Statistical Center will provide Open and Closed Reports. Open Reports, available to all who attend the DSMB meeting, will include pooled data on recruitment and baseline characteristics, as well as pooled data on eligibility violations, completeness of follow-up and compliance. Closed Reports will include data


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separated by treatment group on recruitment, baseline characteristics, eligibility violations, completeness of follow-up, compliance, adverse events and symptom severity, and laboratory data. The Open and Closed reports will consist primarily of tables, figures, and data listings as described in the DSMB Charter. The report will include summary text to accompany the presentations. Upon request of the Sponsor, the Unblinded Statistical Center may modify the content of the Open report. Upon request of the DSMB, the Unblinded Statistical Center may modify the content of the Closed report.

Each month, the Unblinded Statistical Center will provide the DSMB Chair with blinded reports of individual SAEs and frequencies of reported SAEs by treatment group. The DSMB Chair may modify this schedule, request additional data summaries, and may convene additional meetings of the DSMB at his discretion.

9.13.2 Final Analyses and Reporting

The clinical database will be locked twice. The first database lock will occur when all subjects have completed the 60 day evaluation period of the study and all data has been monitored, is query-clean and final. All follow-up between Day 60 and Day 180 available at the time, including Day 180 and interim contacts, will be included in the Day 60 database. All planned analyses summarizing results by treatment group will be performed after the last subject has completed the 60 day evaluation period of the study. For this analysis, group summaries will be presented. Individual subject treatment assignments (e.g., listings) will be provided prior to the Day 180 lock only if required for regulatory submission(s); if required, these will be provided only to personnel identified by the Sponsor, who have no direct role in study operations. These results will be shared with limited group of people to be identified by the Sponsor.

A second database lock will occur after all subjects have reached day 180 of the study. Analyses of subject accountability and length of following, and mortality through Day 180 will be updated. Listings of individual subject data, and any other information regarding individual subjects’ treatment assignments, will be provided only after the Day 180 database is locked, unless required for regulatory submission(s).

9.14 Safety outcomes

9.14.1 Mortality (death to Day 180)

Product-limit (Kaplan-Meier) estimates of mortality rates at relevant time points (e.g., 5, 14, 30, 60, and 180 days) will be presented in the Safety set, along with estimates of the hazard ratio (from Cox regression) comparing active to placebo with 95% confidence limits. The DSMB will examine mortality and other safety data at regular intervals throughout the trials’ course as described above. Preliminary estimates of Day 180 mortality rates will be provided at the Day 60 analysis.

9.14.2 Adverse events

The incidence of AEs recorded through Day 5 and the incidence of SAEs recorded through Day 14 will be presented for the Safety set. Incident AEs will be considered those AEs with an onset date and time after the initiation of study drug. Adverse events with an onset between informed consent and study drug initiation will be listed separately.


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Adverse events will be coded using MedDRA version 9 or later. All reported AEs will be summarized by system organ class (SOC) and preferred term. DREs will be indicated with an asterisk. SAEs will be summarized separately for the interim analyses by source: Safety Surveillance Database, and CRF Database. SAEs will be reconciled between these two databases, and for the final report, SAE data will be summarized from only the CRF database (which in the final database will reflect all SAEs included in the Safety Surveillance Database). In addition, AEs will be summarized by time period of onset: from study drug initiation to Day 5, or from Day 6 to Day 14. Additionally, a summary of AEs by preferred term and severity, using the worst reported severity grade for each event for the subject, will be provided. For analysis purposes, all AEs defined as “definite”, “probable” or “possible” will be considered as related. If the relationship to study drug is unknown or missing, the AE will be considered to be drug-related. All study-drug-related AEs, AEs with an outcome of death, AEs leading to discontinuation of treatment, and study-drug-related SAEs, SAEs with an outcome of death, and SAEs leading to study drug discontinuation will be summarized by percentages and frequencies. Percentages will be based on the number of subjects in the Safety set with at least one AE form completed. For the analysis by time period of onset, the percentages will be based on the number of subjects in the Safety set with the corresponding AE form completed. Odds ratios and associated 95% confidence intervals comparing active treatment with placebo will be presented for those events with 5 or more events in the combined treatment groups.

9.14.3 Laboratory data

Mean laboratory values, and changes in laboratory values from baseline, will be presented by treatment group, and treatment groups compared using both t-tests and Wilcoxon rank sum tests, in the Safety set. Shift tables will be prepared for each laboratory parameter showing the number and percentage of subjects in each treatment group with laboratory values that fall outside pre-determined ranges at each follow-up day by the classification of subjects at baseline. Laboratory values will be presented for each subject in a data listing with an indication of whether the value is above (H) or below (L) the normal reference range.

9.14.3.1 Creatinine and eGFR changes

The proportions of subjects with ≥ 0.3 mg/dL increase, ≥ 0.5 mg/dL increase, and ≥ 1.0 mg/dL increase and >50% increase from baseline in serum creatinine levels at each time point, and at any time point, will be presented. In addition, the proportion of subjects with >= 0.3 mg/dL increase, >=0.5 mg/dL increase, and >= 1.0 mg/dL increase at each of (a) Day 5, (b) Day 14, (c) Day 60, (d) both Day 5 and Day 14, and (e) Day 5 and Day 14 and Day 60 will be presented by treatment groups and compared using chi-squared tests.

The proportion of subjects with decreases from baseline GFR, estimated using the sMDRD formula, of >25%, >40%, >50%, and >30 mL/min/1.73 m2 at each time point, and at any time point, will be presented. 9.14.3.2 Hemoglobin, hematocrit, and RBC count changes

The proportions of subjects with >20% decrease from baseline in hemoglobin, hematrocrit, and RBC count at each time point, and at any time point, will be presented by treatment group.


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9.14.3.3 ALT, AST, alkaline phosphatase, and total bilirubin changes

The proportions of subjects with the following increases from baseline at each time point, and at any time point, will be presented by treatment group: (1) ALT or AST > 3xULN, (2) ALT or AST >5xULN, (3) ALT or AST >8xULN; (4) ALT or AST>3xULN and TB>2xULN; (5) TB>3xULN, and AST or ALT≤3xULN, and alk phos ≤1.5xULN; (6) AP>3xULN, and AST, ALT, and TB are within normal range.

9.14.3.4 Vital signs

Descriptive statistics will be provided for values and change from baseline at each assessment time point for pulse, respiratory rate, body temperature, and weight. Treatment groups will be compared for changes from baseline using t-tests.

9.14.3.5 Blood pressure decrease events

The number and proportion of subjects who experience a confirmed blood pressure decrease event during study drug administration will be provided. Among subjects who experience a confirmed blood pressure decrease event, the events will be further characterized. Summaries will be provided separately for those confirmed events that resulted in study drug dose reduction, and those that resulted in study drug discontinuation. The possible interaction between the effect of relaxin and the effect of IV nitrate administration within the first 48 hours will be examined.

9.14.3.6 Pregnancy test results

Any pregnancies reported will be listed.

9.15 Changes in the Conduct of the Study or Planned Analyses

Only Corthera may modify the protocol. Any change in study conduct requested by the investigator will be made only after consultation with Corthera, who will then issue a formal protocol amendment to implement the change. The only exception may occur when the investigator considers that a subject’s safety is compromised without immediate action. In these circumstances, immediate approval of the chairman of the IRB, REB or EC must be sought, and the investigator should inform Corthera and the full IRB, REB or EC within 2 working days after the emergency occurs. With the exception of minor administrative or typographical changes, the IRB, REB or EC must review and approve all amendments. Amendments that have an impact on subject risk or the study objectives, or require revision of the ICF, must receive approval from the IRB, REB or EC prior to their implementation.

When a protocol amendment substantially alters the study design or the potential risks or burden to subjects, the ICF will be amended and approved by the IRB, REB or EC, and all active subjects will again provide informed consent.

Any revisions to the planned analyses will be documented in the Statistical Analysis Plan.

10 PHARMACOECONOMICS AND QUALITY OF LIFE ASSESSMENTS


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For the main phase of the study, feasibility of pharmacoeconomic and quality of life assessments will take place.

Data for a pharmacoeconomic analysis will be collected from CRFs, most notably, Hospital Discharge, Rehospitalization and Death CRFs. Data on medical resource use will be collected for all enrolled patients at discharge from the index hospitalization, and at the Day 14 and Day 60 follow-up visits. For US sites only, a form developed for this study should be completed and faxed to Corthera Inc., or its designee for each of the three resource use assessments time points (at discharge from the index hospitalization, Day 14 and Day 60). The endpoints and methods of the pharmacoeconomic analysis will be detailed separately in the statistical analysis plan.

The EQ-5D quality of life assessment will be completed by all study patients at the Day 14 and Day 60 follow-up visits.

Additional details regarding pharmacoeconomic and quality of life assessments are provided in the Study Operations Manual.

11 COMPENSATION, INSURANCE, AND INDEMNITY

Study subjects will not be paid for being in this study.

There will be no charge to study subjects to be in this study. Corthera will pay all costs of tests, procedures, and treatments that are part of the study (not part of the standard of care). In addition, Corthera may defer the cost of travel if the company determines that the costs will impose a hardship on study subjects. Corthera will not pay for any hospitalizations, tests, or treatments for medical problems of any sort, whether or not related to the study subject’s disease that are not part of this study. Costs associated with hospitalizations, tests, and treatments should be billed and collected in the way that such costs are usually billed and collected.

The investigator should contact Corthera immediately upon notification that a subject has been injured by the study drug or by procedures performed as part of the study. Any subject who experiences a study-related injury should be instructed by the investigator to seek medical treatment. The subject should be given the name of a person to contact to seek further information about, and assistance with, treatment for study-related injuries. The informed consent form shall provide the conditions under which Corthera will pay for reasonable and necessary medical services to treat study-related injuries.

12 ETHICS

12.1 Institutional Review Board, Research Ethics Board or Ethics Committee

Prior to study initiation, it is the Principal Investigator’s (PI) responsibility to ensure that the Institutional Review Board (IRB), Research Ethics Board (REB) or Ethics Committee (EC) is properly constituted and compliant with all United States Food and Drug Administration (FDA) requirements and local regulations. The PI or Corthera (as appropriate) will provide


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the IRB, REB or EC with all appropriate material, including the protocol, the informed consent form (ICF), and any other written information provided to the subjects. The study will not be started at a given site and study drug supplies will not be shipped to the site until appropriate documents from the IRB, REB or EC confirming unconditional approvals of the protocol, the ICF and all subject recruitment materials are obtained by the PI, and copies are received at Corthera. The approval documents must refer to the study by protocol title and Corthera study number, and identify documents reviewed as well as the approval date. Appropriate reports on the progress of the study will be made to the IRB, REB or EC and Corthera by the PI in accordance with applicable governmental regulations and in agreement with the SOPs being followed for this study.

12.2 Ethical Conduct of the Study

This study will be conducted in accordance with the current versions of the following:

• Applicable national and local regulations, including the appropriate sections of the U.S. Code of Federal Regulations

• ICH (International Conference on Harmonization) Guideline for Good Clinical Practice (GCP)

• Declaration of Helsinki

Specifically, this study is based on adequately performed laboratory and animal experimentation; the study will be conducted under a protocol reviewed and approved by an IRB, REB or EC; the study will be conducted by scientifically and medically qualified persons; the benefits of the study are in proportion to the risks; the rights and welfare of the subjects will be respected; the physicians conducting the study do not find the hazards to outweigh the potential benefits and each subject will give his or her written, informed consent before any protocol-required tests or evaluations are performed.

12.3 Subject Information and Informed Consent

A properly written and executed ICF, in compliance with the current national and local regulations, including the appropriate sections of the US Code of Federal Regulations for Protection of Human Subjects, Declaration of Helsinki, ICH GCP Guideline, and other applicable national and local regulations, will be obtained from each subject prior to entering the subject into the study. The investigator will prepare the ICF and other required authorizations, such as HIPAA, and provide the documents to Corthera for approval prior to submission to the IRB, REB or EC. Corthera and the IRB, REB or EC must then approve the documents before they are implemented. A copy of the approved ICF and, if applicable, a copy of the approved subject information sheet must also be received by Corthera or its designee prior to the delivery of study drug.

12.4 Subject Withdrawal of Consent Form

Subjects have the right to withdraw from the study at any time for any reason. However, an excessive rate of withdrawals can render the study un-interpretable; therefore, unnecessary withdrawal of subjects should be avoided. Investigators should make every effort to


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determine the cause of withdrawal and to accommodate the needs of the subject to make it possible for them to continue to participate in the study. A brief Withdrawal of Consent form has been designed for this study to document the extent of a subject's withdrawal from the study (i.e. withdrawal from further study treatment, withdrawal from any further contact, etc.). This form requires IRB/EC approval and should be completed by any subject who decides to prematurely end their participation in the study.

13 INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE

Prior to study initiation, the PI at each site must provide Corthera with a fully executed and signed FDA Form 1572, a Financial Disclosure Form, and other country specific required documents. All sub-investigators must be listed on the 1572 form. Financial Disclosure Forms must also be completed for all sub-investigators listed on the Form 1572. Financial Disclosure forms will be collected as follows: i) prior to the first shipment of study material to that investigational site ii) at any time during the study that there is an addition of a sub investigator or principal investigator at that site, iii) if there is an update to disclosures, and iv) one year after last patient’s study visit.

The study will be administered and monitored by employees or representatives of Corthera. Clinical Research Associates (CRAs) will monitor each site periodically and verify source documentation for each subject.

14 INVESTIGATIONAL PRODUCT ACCOUNTABILITY

The investigator or designee must maintain accurate records (including dates and quantities) of study drug shipments received at the study site, subjects that were infused with the study drug, and study drug accidentally or deliberately destroyed. The investigator or designee must retain all unused or expired study supplies so that the CRA can perform drug accountability. The monitor will account for all study drug in a formal reconciliation process prior to destruction on-site or returning drug to the depot. Only those sites with an approved SOP for Study Drug destruction will be allowed to destroy study drug on site once a final reconciliation has taken place. Return of study drug or study drug materials to a central depot for final accountability and destruction must occur at Corthera’s request and must be accounted for on a Study Drug Returns Form provided by Corthera or representative. All study drug and study drug materials should be stored, inventoried, reconciled, and destroyed or returned according to applicable state and federal regulations and study procedures.

15 LABORATORY REQUIREMENTS

A local laboratory will be utilized to analyze Screening laboratory tests. Laboratory tests for all other assessments at all other time points will be performed by a central laboratory, except those not specified in the protocol but required by the Investigator to assess patient care.

Instructions for labeling and shipping of samples for this study will be provided in the Study Procedures Manual and Laboratory Manual.


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16 CASE REPORT FORMS (CRF)

CRFs will be provided for each subject. The investigator must review and sign the CRFs to verify their accuracy.

CRFs must be filled out legibly and completely using a black ballpoint pen. Unless explicitly allowed in the CRF instructions, blank data fields are not acceptable. If a field is blank because the item was not done, the field will be marked “ND.” If the item is unknown, the field will be marked “UNK.” If the item is not applicable, the field will be marked “NA.” If the item is not available, the field will be marked “NAV.”

If an entry error has been made and the original CRF page is still at the study site, the error may be corrected on the CRF page by making a single straight line through the incorrect data and writing the correct data, allowing the original text to remain legible. Each correction must be initialed and dated by the person making the change. If corrections are made after review and signature by the investigator, he or she must confirm and endorse the changes. ERRORS MAY NOT BE ERASED, AND CORRECTION FLUID (e.g., WHITEOUT) MAY NOT BE USED. The study staff will be queried for clarification regarding illegible or incomplete entries. Do not write in the margins.

Study data on the CRFs must be verifiable to the source data, which requires access to all original recordings, laboratory reports, and subject records unless to the degree specified in the SOPs being followed for this study. The investigator must therefore agree to allow direct access to all source data. Subjects (or their parent or legal guardian) must also allow access to their medical records, and subjects will be informed of this and will confirm their agreement when giving informed consent. If an investigator or institution refuses to allow access to subject records because of confidentiality, arrangements must be made to allow an “interview” style of data verification.

A CRA designated by Corthera will compare the original CRFs with the original source documents at the study site and evaluate the CRFs for completeness and accuracy before submitting them to data management for entry into the database. If necessary, the study site personnel will be contacted for corrections and/or clarifications. Errors to CRF pages that are identified after the original CRF page has been removed by the CRA will be corrected as described in the Study Procedures Manual. The investigator or designee must also review and sign all Data Clarification Forms (DCFs) to verify their accuracy prior to submission to data management.

A copy of the CRF and any accompanying DCFs will be maintained in the investigator's file. Designated site personnel must complete CRFs as soon as possible after a subject visit, and the forms must be available for review at the next scheduled monitoring visit.

17 STUDY MONITORING AND AUDITING

Qualified individuals designated by Corthera will monitor all aspects of the study according to GCP and standard operating procedures for compliance with applicable government


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regulations. The investigator agrees to allow these monitors direct access to the clinical supplies, dispensing, and storage areas and to the clinical files of the study subjects, and, if requested, agrees to assist the monitors. The Principal Investigator and staff are responsible for being present or available for consultation during routine site visits conducted by Corthera or its designee.

Members of Corthera’s Quality Assurance Department, or a designee, may conduct a quality assurance audit of a clinical site. The investigator will be notified if an audit is scheduled and be advised as to the scope of the audit. Representatives of the FDA or other regulatory agencies may conduct an inspection of the study. If notified of such an inspection, the investigator should contact Corthera immediately. The investigator will ensure that the auditors and inspectors have access to the clinical supplies, study site facilities, source documentation, and all study files.

18 RETENTION OF RECORDS

Investigators must retain all study records required by Corthera and by the applicable regulations in a secure and safe facility. The investigator must consult a Corthera representative before disposal of any study records, and must notify Corthera of any change in the location, disposition, or custody of the study files. The investigator or institution must take measures to prevent accidental or premature destruction of essential documents, that is, documents that individually and collectively permit evaluation of the conduct of a study and the quality of the data produced, including paper copies of study records (e.g., subject charts) as well as any original source records that are electronic, as required by applicable regulatory requirements.

Essential documents must be retained for at least 2 years after the last approval of a marketing application in the U.S. or an ICH region and until (1) there are no pending or contemplated marketing applications in the U.S. or an ICH region or (2) at least 2 years have elapsed since the formal discontinuation of clinical development of the study drug. The investigator/institution should retain subject identification codes for at least 15 years after the completion or discontinuation of the study. Subject files and other source data must be kept for the maximum period of time permitted by the hospital, institution, or private practice, but not less than 15 years. These documents should be retained for a longer period, however, if required by the applicable regulatory requirements or by a Corthera agreement. Corthera must be notified and will assist with retention should the investigator/institution be unable to continue maintenance of subject files for the full 15 years. Corthera is responsible for informing the investigator and/or institution when these documents no longer need to be retained.

19 USE OF INFORMATION AND PUBLICATION

The RELAX- AHF program is a randomized, multicenter, double-blind, placebo-controlled study in which data will be collected, analyzed, and interpreted by a contract agency. Two database locks (DBL) are planned for this study, one including Day 60 data, and one


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additionally including Day 180 vital status information. Following the Day 60 DBL, results will be reported by treatment groups, but individual subject treatment assignments will remain blinded.

Data cannot be used for publication or reporting outside of this study until the database is locked at Day 60 or the study discontinued by Corthera, Inc. This is necessary since dissemination of preliminary information may inappropriately affect the objectivity of this study. For this reason, study investigators or other parties will not be allowed to perform subset analyses at any point before the Day 180 DBL. Violation of this will result in automatic expulsion from this study.

Corthera, Inc. recognizes the importance of communicating medical research and scientific data and its obligations to subjects enrolled in the Study. Sponsor will appoint a publications committee (the “Publications Committee”) that will act in accordance with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals guidelines of the International Committee of Medical Journal Editors (http://www.icmje.org; the “Uniform Requirements”).

Sponsor will register the Study with www.clinicaltrials.gov or an equivalent registry. All publications regarding the Study shall be consistent with the Uniform Requirements.


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20 REFERENCES

Adams, K. F., G. C. Fonarow, C. L. Emeran, T. H. LeJemtel, M. R. Costanzo, W. T. Abraham, R. L. Berkowitz, M. Galvao, D. P. Horton and A. S. A. C. a. Investigators. Characteristics and outcomes of patients hospitalized for heart failure in the United States: rationale, dosing and preliminary observations from the first 100,000 cases in the Acute Decompensated Heart Failure National Registry (ADHERE). Am Heart J. 149: 209-216; 2005.

Allen, LA., J.E. Yager, M.J. Funk, W.C. Levy, J.A. Tulsky, M.T. Bowers, G.C. Dodson, C.M. O’Connor, G.M. Felker. Discordance between patient-predicted and model-predicted life expectancy among ambulatory patients with heart failure. JAMA 299:2533-2542, 2008.

Bani, D., P. Failli, M. G. Bello, C. Thiemermann, T. Bani Sacchi, M. Bigazzi and E. Masini. Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension 31: 1240-7; 1998.

Capeless, E. L. and J. F. Clapp. Cardiovascular changes in early phase of pregnancy. Am J Obstet Gynecol 161: 1449-53; 1989.

Cotter, G., H.C. Dittrich, B.D. Weatherley, D.M. Bloomfield, C.M. O’Connor, M. Metra. The PROTECT pilot study: a randomized, placebo-controlled, dose-finding study of the adenosine A1 receptor antagonist Rolofylline in patients with acute heart failure and renal impairment. J. Card. Fail. 14:631-640, 2008.

Cotter G, Metra M, Weatherley BD, Dittrich HC, Massie BM, Ponikowski P, Bloomfield DM, O’Connor CM. Physician-Determined Worsening Heart Failure: a novel definition for early worsening heart failure in patients hospitalized for acute heart failure—association with signs and symptoms, hospitalization duration,and 60-day outcomes. Cardiology 115: 29–36; 2010.

Danielson, L. A., L. J. Kercher and K. P. Conrad. Impact of gender and endothelin on renal vasodilation and hyperfiltration induced by relaxin in conscious rats. Am J Physiol Regul Integr Comp Physiol 279: R1298-304.; 2000.

Danielson, L. A., O. D. Sherwood and K. P. Conrad. Relaxin is a potent renal vasodilator in conscious rats. J Clin Invest 103: 525-33; 1999.

Davison, J. M. and M. C. B. Noble. Serial changes in 24-hour creatinine clearance during normal menstrual cycles and the first trimester of pregnancy. Br J Obstet Gynecol 88: 10-17; 1981.

Debrah, D. O., K. P. Conrad, A. Jeyabalan, L. A. Danielson and S. G. Shroff. Relaxin increases cardiac output and reduces systemic arterial load in hypertensive rats. Hypertension 46: 745-50; 2005.

DeMarco, P. J., M. H. Weisman, J. R. Seibold, D. E. Furst, W. K. Wong, E. L. Hurwitz, M. Mayes, B. White, F. Wigley, W. Barr, L. Moreland, T. A. Medsger, Jr., V. Steen, R. W. Martin, D. Collier, A. Weinstein, E. Lally, J. Varga, S. R. Weiner, B. Andrews, M. Abeles and P. J. Clements. Predictors and outcomes of scleroderma renal crisis: the high-dose versus low-dose D-penicillamine in early diffuse systemic sclerosis trial. Arthritis Rheum 46: 2983-9; 2002.

Dschietzig T, Teichman SL, Unemori E, Wood S, Richter C, Bohmer J, Baumann G, Stangl K. A Pilot Safety and Dose-Finding Trial of Intravenous Recombinant Human Relaxin in Compensated Congestive Heart Failure. J Cardiac Failure 2009; (In press).


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Dschietzig, T., C. Bartsch, C. Richter, M. Laule, G. Baumann and K. Stangl. Relaxin, a pregnancy hormone, is a functional endothelin-1 antagonist: attenuation of endothelin-1-mediated vasoconstriction by stimulation of endothelin type-B receptor expression via ERK-1/2 and nuclear factor-kappaB. Circ Res 92: 32-40; 2003a.

Dschietzig, T., C. Richter, C. Bartsch, M. Laule, F. P. Armbruster, G. Baumann and K. Stangl. The pregnancy hormone relaxin is a player in human heart failure. Faseb J 15: 2187-95; 2001b.

Gheorghiade, M., W. Abraham, N. M. Albert, B. H. Greenberg, C. M. O'Connor, L. She, W. Gattis Stough, C. W. Yancy, J. B. Young and G. C. Fonarow. Systolic Blood Pressure at Admission, Clinical Characteristics, and Outcomes in Patients Hospitalized with Acute Heart Failure. JAMA 296: 2217-2226; 2006.

Kohsaka, T., G. Min, G. Lukas, S. Trupin, E. T. Campbell and O. D. Sherwood. Identification of specific relaxin-binding cells in the human female. Biol Reprod 59: 991-9; 1998.

Maca, JP, Gallo P, Branson M, Maurer W. Reconsidering some aspects of the two-trials paradigm. Journal of Biopharmaceutical Statistics 12: 107-119, 2002.

Mayes, M. D., J. V. Lacey, Jr., J. Beebe-Dimmer, B. W. Gillespie, B. Cooper, T. J. Laing and D. Schottenfeld. Prevalence, incidence, survival, and disease characteristics of systemic sclerosis in a large US population. Arthritis Rheum 48: 2246-55; 2003.

Medsger, T. A., Jr., and V. D. Steen (1996). Classification, prognosis. Systemic Sclerosis. P. J. Clements and D. E. Furst. Baltimore, Williams and Wilkins: 51-64.

Novak, J., L. J. Parry, J. E. Matthews, L. J. Kerchner, K. Indovina, K. Hanley-Yanez, K. D. Doty, D. O. Debrah, S. G. Shroff and K. Conrad. Evidence for local relaxin ligand-receptor expression and function in arteries. FASEB J. 20: 2353-2362; 2006.

Palejwala, S., L. Tseng, A. Wojtczuk, G. Weiss and L. T. Goldsmith. Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells. Biol Reprod 66: 1743-8; 2002.

Steen, V. D. Scleroderma renal crisis. Rheum Dis Clin North Am 29: 315-33; 2003. Sunn, N., M. Egli, T. C. Burazin, P. Burns, L. Colvill, P. Davern, D. A. Denton, B. J.

Oldfield, R. S. Weisinger, M. Rauch, H. A. Schmid and M. J. McKinley. Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat. Proc Natl Acad Sci USA 99: 1701-6; 2002.

Unemori, E. N., M. E. Erikson, S. E. Rocco, K. M. Sutherland, D. A. Parsell, J. Mak and B. H. Grove. Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women. Hum Reprod 14: 800-6; 1999.

Weatherley BD, Milo-Cotter O, Michael Felker G, Uriel N, Kaluski E, Vered Z, O’Connor CM, Adams KF, Cotter G. Early worsening heart failure in patients admitted with acute heart failure—a new outcome measure associated with longterm prognosis? Fundam Clin Pharmacol 23: 633–9; 2009.

Weisinger, R. S., P. Burns, L. W. Eddie and E. M. Wintour. Relaxin alters the plasma osmolality-arginine vasopressin relationship in the rat. J Endocrinol 137: 505-10; 1993.


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21 APPENDICES

21.1 Schedule of Events

RLX.CHF.003

Study Drug Infusion a

Daily Post Treatment

Assessments b Follow-Up c

Time-point / Visit Screend Baseline(Day 0)

6 hre 12 hr e

24 hr e

(Day 1)

48 hr e

(Day 2) Day 3, 4, 5 b Day 14 c Day 60 Day 180

Informed consent X

Inclusion/exclusion criteria X

Medical history X

BNP or NT-pro-BNP X

Troponin X

Electrocardiogram and Chest X-Ray o X

Digital ECGs (only for subjects enrolled in the ECG sub-study)n

X X X X X

Estimated Glomerular Filtration Rate (sMDRD)

X

Pregnancy test X

Body weight measurement X X X X X X

Height X

Patient-reported dyspnea and general well-being assessments

Xf X X X X X X

Physician assessment of signs and symptoms of HF

X X X X X X X X


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RLX.CHF.003





6 hre 12 hr e

24 hr e

(Day 1)

48 hr e


Physical examination with vital signs g X X X X X X X X X

Frequent BP and HR measurements h X X X X X

Clinical laboratory tests i X X X X X X X

Relaxin measurement j X

Neurohormone measurement k X X X X X X

Collect concomitant medications X X X X X X X

Collect adverse and serious adverse events l

X X X X X X

Occurrence of re-admission X (Day 5) X X EQ-5D X X Vital statusm X X X X Xm X

Footnotes: a. Study drug treatment will be administered as an IV infusion for 48 hours. If at any time during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg

from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug treatment infusion rate will be decreased by 50% for the remainder of the study drug administration. If at any time during dosing, the subject’s systolic blood pressure is < 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion will be terminated.

b. Acceptable window for Days 3 and 4 (if subject is still in the hospital) is +/- 2 hours (based on 24-hr days relative to the start of study drug administration, or if study drug is not administered, the time of randomization). Patients discharged prior to Day 5 will be asked to return to the hospital for Day 5 procedures. Acceptable window is Day 5-7.

c. Patients discharged prior to Day 14 will be asked to return to the clinic for Day 14 procedures; acceptable window is Day 14-16. The Day 60 assessment will be conducted as an outpatient visit if the patient was discharged prior; acceptable window is Day 60-70. Subjects will be contacted by telephone at Day 180 (± 7 days).

d. Procedures that are part of a site’s standard of care for an individual with AHF may be done during the pre-screening interval and may pre-date the signed ICF.


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e. For all assessments scheduled for the 6 hour time point, a window at 4.5-7 hours is allowed for these measurements, for all the assessments at the 12 hour time point, a window at 9-13 hours is allowed, for all assessments scheduled for the 24 hour time point, a window at 20-26 hours is allowed, and for all assessments scheduled for the 48 hour time point, a window at 42-48 hours is allowed.

f. Only patient self-reported dyspnea and general well-being by VAS will be done at baseline. g. A directed physical examination will consist of an assessment of general appearance as well as vital signs (pulse rate, respiratory rate and body temperature). h. BP and HR measurements should be performed at 30 minutes and then every hour for the first 6 hours of study drug infusion, and then every 3 hours during study drug

infusion, including night time hours. Post-infusion, BP and HR should be measured every 3 hrs until 12 hrs following end of infusion, then every 6 hrs for 48 hours and then every 24 hours until the earlier of Day 5 or discharge. BP and HR should be measured with the patient in the same position and with the same equipment throughout study drug infusion.

i. Clinical laboratory tests include hematology and chemistry. Urinalysis will be conducted at baseline, 24 and 48 hours only. Clinical laboratory tests at Screening will be performed locally. At all other time points, laboratory tests will be performed by a central laboratory.

j. Blood draw for relaxin measurement will be performed at 24 hr following start of drug infusion to confirm proper dosing. k. Blood will be drawn for measurement of neurohormones and other markers related to HF at Baseline, 24 and 48 hours after start of study drug infusion and at Day 3, 4, 5, 14

and 60. l. In the Pre-Relax-AHF phase the reporting period for serious adverse events begins with the signing of the ICF and ends at 30 days after the start of study drug infusion. In

the main phase (Relax-AHF) AEs are reported from the signing of the ICF through Day 5 and SAEs through Day 14. m. Just prior to the time that the last patient enrolled reaches 60 days of follow up, all patients that have not completed the study, will be contacted and vital status information

will be obtained at that time. n. Electrocardiograms will be obtained at baseline and within 2 hours of the following timepoints: 12, 24, and 48 hours following the start of study drug infusion, and on Day 3

(if the subject is still in the hospital). When the ECG acquisition time coincides with other procedures/activities (subject and physician assessments, vital signs, blood collection and/or a meal), subject assessments (SSRS and physician assessment) MUST be acquired first, followed by vital signs, body weight, then ECG, blood collection and the meal. The sub-study will only be conducted at a subset of sites in selected countries (Germany, Hungary, Israel, Italy, Poland, Romania, USA).

o. Chest X-ray can be interpreted by the study physicians or physicians attending to the patient. Intepretation and the person interpreting the radiograph should be recorded in the source. A radiologist’s interpretation is not necessary for the purposes of the study.


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21.1.1 Detailed Schedule of Laboratory Assessments:

RLX.CHF.003 Study Drug

Infusion Daily Post-Treatment

Assessments Follow-Up

Time-point / Visit Screen1 Baseline(Day 0)

24 hr

(Day 1)

48 hr

(Day 2)Day 3, 4, 5 8 Day 14 8 Day 608

BNP or NT-pro-BNP X2

Troponin X2


X2

Pregnancy test X2

Serum Chemistry3 X2, 3 X3 X3 X3 X3 X3 X3

Hematology 4 X2, 4 X4 X4 X4 X4 X4 X4

Urinalysis 5 X5 X5 X5

Relaxin measurement 6 X6

Neurohormone measurement 7 X7 X7 X7 X7 X7 X7

Footnotes: 1. Procedures that are part of a site’s standard of care for an individual with AHF may be done during the pre-screening interval and may pre-date the signed ICF. 2. Inclusion/exclusion criteria performed at local laboratory. 3. Serum chemistry includes: BUN, Creatinine, Total Bilirubin, Phosphate, ALT, AST, Alkaline Phosphatase, Sodium, Potassium, Calcium, Total Cholesterol, Triglycerides,

LDL, HDL, Glucose, Uric Acid, albumin, and total protein. Except for the Screening chemistry, which is done locally, chemistry samples (ambient) will be sent to core lab. At Screening, at a minimum, the following tests should be performed: BUN, Creatinine, Sodium and Potassium.

4. Hematology includes: WBC, RBC, Hematocrit, Hemoglobin, Platelet Count, Differential (Bands, Monocytes, Neutrophils, Eosinophils, Lymphocytes, Basophils). Except for the Screening hematology, which is done locally, hematology samples (ambient) will be sent to the core lab. At Screening, at a minimum, the following tests should be performed: WBC, Hematocrit, Hemoglobin and Platelet Count.

5. Urinalysis sample (ambient) to be sent to the core lab. Urinalysis testing at core lab includes glucose, ketones, pH, protein, and specific gravity.


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6. Sample processed and frozen for future shipment. 7. Consists of 1 plasma sample split into 2 and 1 serum sample split into 2 at each time point. Samples processed and frozen for future shipment. 8. Days 5, 14 & 60 are mandatory laboratory evaluations. If patient is discharged prior to Day 5, Day 14, or Day 60 these evaluations will be performed as outpatient visits.


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21.2 World Medical Association Declaration Of Helsinki Ethical Principles for Medical Research Involving Human Subjects

Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, and amended by the: 29th WMA General Assembly, Tokyo, Japan, October 1975 35th WMA General Assembly, Venice, Italy, October 1983 41st WMA General Assembly, Hong Kong, September 1989

48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 52nd WMA General Assembly, Edinburgh, Scotland, October 2000

53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added) 55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added)

59th WMA General Assembly, Seoul, October 2008 21.2.1 Introduction

1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data. The Declaration is intended to be read as a whole and each of its constituent paragraphs should not be applied without consideration of all other relevant paragraphs.

2. Although the Declaration is addressed primarily to physicians, the WMA encourages other participants in medical research involving human subjects to adopt these principles.

3. It is the duty of the physician to promote and safeguard the health of patients, including those who are involved in medical research. The physician's knowledge and conscience are dedicated to the fulfillment of this duty.

4. The Declaration of Geneva of the WMA binds the physician with the words, “The health of my patient will be my first consideration,” and the International Code of Medical Ethics declares that, “A physician shall act in the patient's best interest when providing medical care.”

5. Medical progress is based on research that ultimately must include studies involving human subjects. Populations that are underrepresented in medical research should be provided appropriate access to participation in research.

6. In medical research involving human subjects, the well-being of the individual research subject must take precedence over all other interests.

7. The primary purpose of medical research involving human subjects is to understand the causes, development and effects of diseases and improve preventive, diagnostic and therapeutic interventions (methods, procedures and treatments). Even the best current interventions must be evaluated continually through research for their safety, effectiveness, efficiency, accessibility and quality.

8. In medical practice and in medical research, most interventions involve risks and burdens.

9. Medical research is subject to ethical standards that promote respect for all human subjects and protect their health and rights. Some research populations are particularly vulnerable and need special protection. These include those who cannot give or refuse consent for themselves and those who may be vulnerable to coercion or undue influence.


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10. Physicians should consider the ethical, legal and regulatory norms and standards for research involving human subjects in their own countries as well as applicable international norms and standards. No national or international ethical, legal or regulatory requirement should reduce or eliminate any of the protections for research subjects set forth in this Declaration.

21.2.2 Basic Principles For All Medical Research

11. It is the duty of physicians who participate in medical research to protect the life, health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal information of research subjects.

12. Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory and, as appropriate, animal experimentation. The welfare of animals used for research must be respected.

13. Appropriate caution must be exercised in the conduct of medical research that may harm the environment.

14. The design and performance of each research study involving human subjects must be clearly described in a research protocol. The protocol should contain a statement of the ethical considerations involved and should indicate how the principles in this Declaration have been addressed. The protocol should include information regarding funding, sponsors, institutional affiliations, other potential conflicts of interest, incentives for subjects and provisions for treating and/or compensating subjects who are harmed as a consequence of participation in the research study. The protocol should describe arrangements for post-study access by study subjects to interventions identified as beneficial in the study or access to other appropriate care or benefits.

15. The research protocol must be submitted for consideration, comment, guidance and approval to a research ethics committee before the study begins. This committee must be independent of the researcher, the sponsor and any other undue influence. It must take into consideration the laws and regulations of the country or countries in which the research is to be performed as well as applicable international norms and standards but these must not be allowed to reduce or eliminate any of the protections for research subjects set forth in this Declaration. The committee must have the right to monitor ongoing studies. The researcher must provide monitoring information to the committee, especially information about any serious adverse events. No change to the protocol may be made without consideration and approval by the committee.

16. Medical research involving human subjects must be conducted only by individuals with the appropriate scientific training and qualifications. Research on patients or healthy volunteers requires the supervision of a competent and appropriately qualified physician or other health care professional. The responsibility for the protection of research subjects must always rest with the physician or other health care professional and never the research subjects, even though they have given consent.

17. Medical research involving a disadvantaged or vulnerable population or community is only justified if the research is responsive to the health needs and priorities of this


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population or community and if there is a reasonable likelihood that this population or community stands to benefit from the results of the research.

18. Every medical research study involving human subjects must be preceded by careful assessment of predictable risks and burdens to the individuals and communities involved in the research in comparison with foreseeable benefits to them and to other individuals or communities affected by the condition under investigation.

19. Every clinical trial must be registered in a publicly accessible database before recruitment of the first subject.

20. Physicians may not participate in a research study involving human subjects unless they are confident that the risks involved have been adequately assessed and can be satisfactorily managed. Physicians must immediately stop a study when the risks are found to outweigh the potential benefits or when there is conclusive proof of positive and beneficial results.

21. Medical research involving human subjects may only be conducted if the importance of the objective outweighs the inherent risks and burdens to the research subjects.

22. Participation by competent individuals as subjects in medical research must be voluntary. Although it may be appropriate to consult family members or community leaders, no competent individual may be enrolled in a research study unless he or she freely agrees.

23. Every precaution must be taken to protect the privacy of research subjects and the confidentiality of their personal information and to minimize the impact of the study on their physical, mental and social integrity.

24. In medical research involving competent human subjects, each potential subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail, and any other relevant aspects of the study. The potential subject must be informed of the right to refuse to participate in the study or to withdraw consent to participate at any time without reprisal. Special attention should be given to the specific information needs of individual potential subjects as well as to the methods used to deliver the information. After ensuring that the potential subject has understood the information, the physician or another appropriately qualified individual must then seek the potential subject’s freely-given informed consent, preferably in writing. If the consent cannot be expressed in writing, the non-written consent must be formally documented and witnessed.

25. For medical research using identifiable human material or data, physicians must normally seek consent for the collection, analysis, storage and/or reuse. There may be situations where consent would be impossible or impractical to obtain for such research or would pose a threat to the validity of the research. In such situations the research may be done only after consideration and approval of a research ethics committee.

26. When seeking informed consent for participation in a research study the physician should be particularly cautious if the potential subject is in a dependent relationship with the physician or may consent under duress. In such situations the informed consent should be sought by an appropriately qualified individual who is completely independent of this relationship.


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27. For a potential research subject who is incompetent, the physician must seek informed consent from the legally authorized representative. These individuals must not be included in a research study that has no likelihood of benefit for them unless it is intended to promote the health of the population represented by the potential subject, the research cannot instead be performed with competent persons, and the research entails only minimal risk and minimal burden.

28. When a potential research subject who is deemed incompetent is able to give assent to decisions about participation in research, the physician must seek that assent in addition to the consent of the legally authorized representative. The potential subject’s dissent should be respected.

29. Research involving subjects who are physically or mentally incapable of giving consent, for example, unconscious patients, may be done only if the physical or mental condition that prevents giving informed consent is a necessary characteristic of the research population. In such circumstances the physician should seek informed consent from the legally authorized representative. If no such representative is available and if the research cannot be delayed, the study may proceed without informed consent provided that the specific reasons for involving subjects with a condition that renders them unable to give informed consent have been stated in the research protocol and the study has been approved by a research ethics committee. Consent to remain in the research should be obtained as soon as possible from the subject or a legally authorized representative.

30. Authors, editors and publishers all have ethical obligations with regard to the publication of the results of research. Authors have a duty to make publicly available the results of their research on human subjects and are accountable for the completeness and accuracy of their reports. They should adhere to accepted guidelines for ethical reporting. Negative and inconclusive as well as positive results should be published or otherwise made publicly available. Sources of funding, institutional affiliations and conflicts of interest should be declared in the publication. Reports of research not in accordance with the principles of this Declaration should not be accepted for publication.

21.2.3 Additional Principles For Medical Research Combined with Medical Care

31. The physician may combine medical research with medical care only to the extent that the research is justified by its potential preventive, diagnostic or therapeutic value and if the physician has good reason to believe that participation in the research study will not adversely affect the health of the patients who serve as research subjects.

32. The benefits, risks, burdens and effectiveness of a new intervention must be tested against those of the best current proven intervention, except in the following circumstances: • The use of placebo, or no treatment, is acceptable in studies where no current proven intervention exists; or • Where for compelling and scientifically sound methodological reasons the use of placebo is necessary to determine the efficacy or safety of an intervention and the patients who receive placebo or no treatment will not be subject to any risk of serious or irreversible harm. Extreme care must be taken to avoid abuse of this option.


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33. At the conclusion of the study, patients entered into the study are entitled to be informed about the outcome of the study and to share any benefits that result from it, for example, access to interventions identified as beneficial in the study or to other appropriate care or benefits.

34. The physician must fully inform the patient which aspects of the care are related to the research. The refusal of a patient to participate in a study or the patient’s decision to withdraw from the study must never interfere with the patient-physician relationship.

35. In the treatment of a patient, where proven interventions do not exist or have been ineffective, the physician, after seeking expert advice, with informed consent from the patient or a legally authorized representative, may use an unproven intervention if in the physician's judgment it offers hope of saving life, re-establishing health or alleviating suffering. Where possible, this intervention should be made the object of research, designed to evaluate its safety and efficacy. In all cases, new information should be recorded and, where appropriate, made publicly available.


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21.3 Patient Reported Symptoms and Physician Assessments

21.3.1 Patient Reported Symptoms

The patient’s self-report of dyspnea and general well-being should be obtained using the following steps:

1. All assessments will be done with patient lying in bed; the head of bed should be at 30º, oxygen off for 3-5 minutes before starting assessment.

2. If oxygen off is not tolerated, put oxygen back on the patient and assess immediately, asking the patient to describe how he or she felt when the oxygen was off.

3. Perform the assessments of the patient’s breathing and general well-being daily at baseline/Day 0 (VAS only); at 6, 12, 24, and 48 hours from start of study drug; then daily up to day 4 during hospitalization, at Day 5, and at Day 14.

4. Use the official certified translation (into a language in which the patient is fluent) of the “Self Report Worksheet” containing the dyspnea and general well-being questions provided to you by the RELAX-AHF study management team.

5. Read the question and responses from the “self report” worksheet to the patient EXACTLY as they appear on the translated self report worksheet. If able, the patient may read along with you.

6. Ask the patient to select the most appropriate response on the Likert scale and to place a horizontal line on the visual analog scale to show how he/she feels.

7. Do not coach the patient to select any particular response. You must remain neutral. 8. Mark the response the patient indicates on the “subject self report worksheet”. Use a

separate form for each assessment. 9. Transcribe the responses onto the “Daily Assessment Worksheet”. 10. Transcribe the responses from the “Daily Assessment Worksheet” onto the CRF. 11. File the worksheets in the patient’s study file

For reference, Daily Subject Assessment sheets are attached at end of this appendix.

• Dyspnea 1. Likert scale: Patient will be presented with the following question: “We would

like to measure how you think your breathing is. Please circle the number next to the description that best indicates how you are breathing right now, compared to when you first started the study drug.”

• 3 = Markedly better • 2 = Moderately better • 1 = Minimally better • 0 = No change • -1 = Minimally worse • -2 = Moderately worse • -3 = Markedly worse


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2. Visual analog scale (VAS): Patient will be presented with the following question: “Please draw a horizontal line on the scale to show how you think your breathing is right now. The number “0” equals the worst your breathing has ever felt and the number “100” equals the best you have ever felt.”

100 = Best your breathing has ever felt 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 = Worst your breathing has ever felt

3. Exertional Dyspnea: The subject should be queried as to the extent of dyspnea noted over the preceding 1-3 hours as follows:

• 0 = No exertional dyspnea (NYHA Class I equivalent) • 1 = Mild exertional dyspnea, occurring with moderate exertion (climbing

stairs or equivalent-NYHA Class II equivalent) • 2 = Moderate exertional dyspnea, occurring with only mild exertion

(walking-NYHA Class III equivalent) • 3 = Severe exertional dyspnea, occurring at rest (NYHA Class IV) • NA = Not evaluable; subject immobile for other reasons

• General well being

1. Likert scale: Patient will be presented with the following question: “We would like to measure how you how you feel right now. Please circle the number next to the description that best indicates how you feel right now, compared to when you first started the study drug.”

• 3 = Markedly better • 2 = Moderately better • 1 = Minimally better • 0 = No change • -1 = Minimally worse • -2 = Moderately worse • -3 = Markedly worse


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2. Visual analog scale (VAS): Patient will be presented with the following question: “Please draw a horizontal line on the scale to show how you feel right now. The number “0” equals the worst you have ever felt and the number “100” equals the best you have ever felt.”

100 = Best you have ever felt 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 = Worst you have ever felt

3. Orthopnea: The subject should be observed after being in the lowest recumbent position for 10-15 minutes or queried in order to determine the minimum number of “pillows” required to obtain/maintain comfort while supine. This should be graded on a 0 - 4 scale as follows:

0 = Comfortable with no pillow or very minimal elevation of head 1 = Comfortable with no less than one pillow to elevate head

(approx 10 cm elevation) 2 = Comfortable with no less than two pillows to elevate head

(approx 20 cm elevation) 3 = Comfortable with head no less than at 30 degree elevation NA = Not evaluable; exam could not be performed

21.3.2 Physician Assessment

1. Peripheral Edema, Pre-sacral Edema: Edema should be examined in any dependent area including the lower extremities or the sacral region. The range to be applied is 0 - 3 (4 point scale).

• 0 = The complete absence of edema, as determined by applying mild digital pressure in all dependent areas and failing to elicit any indentation of skin and subcutaneous tissues.

• 1+ = Detection of limited areas where mild digital pressure elicits an indentation of skin and subcutaneous tissues that resolves over


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approximately 10-15 seconds. Edema of this grade is typically limited to only the lower extremities or only the sacrum, not both.

• 2+ = Detection of moderate surface area in one or both areas (sacrum and lower extremities) where indentations of skin and subcutaneous tissues are easily created with limited pressure and these indentations disappear slowly (15-30 seconds or more).

• 3+ = Large areas of lower extremities (and sacrum if subject has been recumbent), often to mid-calf or higher, having easily produced and slowly resolving (more than 30 seconds) indentations. This extent of edema is sometimes associated with acute or subacute skin changes including weeping of skin and/or skin break down.

2. Rales: Auscultation of the lungs applying a 4-point scale: • 0 = No rales heard, either moist or dry, after clearing with cough

anywhere in the lung fields • <1/3 = Moist or dry rales heard in the lower 1/3 of either or both lung

fields that persist after a cough in attempt to clear • 1/3-2/3 = Moist or dry rales heard throughout the lower half to 2/3 of

either or both lung fields • >2/3 = Moist or dry rales heard throughout both lung fields

3. Jugular venous pulse (JVP): With the subject supine at approximately a 45-degree angle, examination of the JVP is performed and the estimation, in cm H2O, is converted into one of 3 categories:

<6 cm H2O = Complete absence of discernable venous wave throughout respiratory cycle above the clavicle, even with hepatic compression (HJR)

6-10 cm H2O = Venous wave detectable above the clavicle, at least during expiration and possibly throughout respiratory cycle but less than 4 cm above the clavicle (<10 cm H2O). Presence of venous wave only with mild HJR should be graded in this category.

>10 cm H2O = Presence of venous wave throughout respiratory cycle with wave sometimes ≥ 4 cm H2O above clavicle and typically increased with HJR. Subjects with values of 6-10 cm H2O and positive HJR should be graded in this category.

NA = Examination could not be performed/result unobtainable


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Breathing

We would like to measure how you think your breathing is. Please circle the number next to the description that best indicates how you are breathing right now compared to when you first started the study drug.

• 3 = Markedly better

• 2 = Moderately better

• 1 = Minimally better

• 0 = No change

• -1 = Minimally worse

• -2 = Moderately worse

• -3 = Markedly worse

Please draw a horizontal line on the scale to show how you think your breathing is right now. The number “0” equals the worst your breathing has ever felt and the number “100” equals the best your breathing has ever felt.

DAILY SUBJECT ASSESSMENT WORKSHEET


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General well being We would like to measure how you feel right now. Please circle the number next to the description that best indicates how you feel right now compared to when you first started the study drug.

• 3 = Markedly better

• 2 = Moderately better

• 1 = Minimally better

• 0 = No change

• -1 = Minimally worse

• -2 = Moderately worse

• -3 = Markedly worse Please draw a horizontal line on the scale to show how you feel right now. The number “0” equals the worst your have ever felt and the number “100” equals the best you have ever felt.

5101520253035404550

0

556065707580859095100

= Worst imaginable health state

= Best imaginable health state


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21.4 Disease-Related Events (DRE)

Cardiac Disorders Heart Failure Atrial tachycardia Acute decompensated heart failure Tachycardia Acute left ventricular failure Accelerated idioventricular rhythm Acute pulmonary edema Tachycardia paroxysmal Acute right ventricular failure Torsades de pointe Cardiac failure Ventricular arrhythmia Cardiac failure acute Ventricular extrasystoles Cardiac failure chronic Ventricular fibrillation Cardiac failure congestive Non-sustained ventricular tachycardia Cardiac failure congestive acute Sustained ventricular tachycardia Cardiac failure congestive worsening Supraventricular tachycardia Decreased cardiac output Metabolic Disorders Decreased ejection fraction Hyperkalemia Dyspnea Hypokalemia Left ventricular failure Hyponatremia Peripheral edema Pulmonary edema Renal and Urinary Disorders Right ventricular failure Cardio Renal syndrome Worsening pericardial effusion not requiring intervention

Azotemia

Hepatic congestion secondary to CHF Renal insufficiency or worsening renal insufficiency, including acute, chronic, exacerbation, end stage or increase in creatinine with the exception of (a) more than a doubling above baseline, (b) an increase of > 3 mg/mL over baseline, or (3) leading to the need for hemofiltration or dialysis

Angina pectoris Atypical chest pain Chest pain

Arrhythmias Sick sinus syndrome Vascular Disorders Bradyarrhythmia Hypertension Sinoatrial or sinus exit block Asymptomatic hypotension Sinus arrhythmia

Sinus bradycardia Respiratory, thoracic and mediastinal disorders AV or heart block – first degree or transient second degree.

Bilateral pleural effusions

Atrioventricular extrasystoles Bilateral pleural effusions worsening Nodal rhythm or arrhythmia Respiratory distress Paroxysmal arrhythmia Respiratory distress acute Sinus tachycardia Respiratory failure Atrial flutter Respiratory failure acute Respiratory failure worsening


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21.5 RLX.CHF.003 (RELAX-AHF) ECG Sub-study

21.5.1 Background/Rationale for the ECG Sub-Study

Strong scientific rationale suggests that relaxin will not deleteriously affect cardiac repolarization based on the following considerations: (1) Relaxin’s structure is identical to the endogenous peptide hormone, which has relatively high selectivity for its cognate receptor; (2) None of the downstream biological mediators of relaxin’s actions are known to prolong QT; (3) Relaxin has no direct effect on sex steroids that are known to affect cardiac repolarization.

Although no in vitro studies have been performed to assess relaxin’s impact on hERG inhibition or trafficking, the potential for other ion channel effects, or the potential for drug-drug interactions, a four week repeat dose cynomolgus monkey toxicity study (0.1, 1, 3 mg/kg sc) showed no reported changes in ECG parameters (Relaxin Investigator’s Brochure for Congestive Heart Failure, September 24, 2010). In addition, there have been no data to suggest pro-arrhythmogenic activity during infusion of relaxin in small numbers of acute heart failure (AHF) patients. However, there are no specific data on the effects of relaxin on ECG intervals in humans. While the probability of an effect on repolarization is small, there remains the biologic possibility for an effect, necessitating further study in the targeted AHF population, a group of patients which is known to have reduced repolarization reserve and an increased incidence of serious ventricular arrhythmias. In addition, the EMA has recently recommended ECG monitoring in clinical trials evaluating relaxin in the AHF population.

This ECG monitoring sub-study at selected sites is an integrated extension of the RLX.CHF.003 study protocol and all information and study procedures set forth are subject to the guidelines, rules, and regulations provided in the RLX.CHF.003 study protocol. Depending on recruitment, up to 60 subjects enrolled at a subset of sites in selected countries (Germany, Hungary, Israel, Italy, Poland, Romania, USA) in the RELAX-AHF trial may participate in this ECG monitoring sub-study.

21.5.2 Objectives

The objective of the sub-study is to collect information that will contribute to the cardiac safety profile of relaxin in the target population, including any electrophysiologic effects such as QT prolongation or arrhythmogenic effects in RELAX-AHF subjects. There is no study hypothesis being tested in this substudy.

21.5.3 Selection of Sub-Study Population – Inclusion/Exclusion Criteria

Subjects who meet all of the eligibility criteria below are eligible for the ECG monitoring sub-study.

Inclusion Criteria

1. Must provide written informed consent to participate in the RELAX-AHF study and the RELAX-AHF ECG sub-study

2. Must continue to meet all eligibility criteria for and be enrolled in the RELAX-AHF study


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3. Screening ECG for RELAX-AHF must show normal sinus rhythm

Exclusion criteria

Subjects with pacemakers, CRT-P/CRT-Ds, and ICDs are excluded from this substudy. There are no other additional exclusion criteria other than what is currently in the main study.

21.5.4 Study Procedures

Clocks on the ECG machine should be synchronized with the central clock on a daily basis during subject participation. The subject number, initials, date of birth or age, date and time of recording, and the study code “RLX.CHF.003” must appear on each page of the tracings. Any other information identifying the subject (name, medical record number, etc.) should be blackened out to de-identify. In addition, each page of the tracings must be labeled to indicate the timepoint at which the tracing was made: “baseline”, “during infusion” (specify “12 hr,” “24 hr,” or “48 hr”), or “Day 3”. Enrolled subjects will have 2-3 sets of triplicate 12-lead ECGs at each of five time points, as described below. All sets of triplicates will be performed within 2 hours of each of the five timepoints. Protocol-required subject (Likert and VAS) and physician assessments must be obtained FIRST, followed by vital signs, body weight, and then ECGs, blood collection and meals, when applicable.

Summary of ECG recordings:

o Three sets of triplicate “baseline” 12-lead paper ECGs performed prior to study drug administration, at least 5 minutes between each set (perform subject and physician assessments first, then vital signs, ECGs and collect lab samples)

o Two sets of triplicate “during study drug infusion” paper ECGs performed 12 hours following the start of study drug infusion, at least 15 min apart (perform subject and physician assessments first)

o Two sets of triplicate “during study drug infusion” paper ECGs performed 24 hours following start of study drug infusion (following study drug IV bag exchange, at least 15 minutes apart; perform subject and physician assessments first, then vital signs, body weight, ECGs and collect lab samples)

o Two sets of triplicate “during study drug infusion” paper ECGs performed 48 hours following start of study drug infusion, at least 15 min apart (perform subject and physician assessments first, then vital signs, body weight, ECGs and collect lab samples)

o Two sets of triplicate “Day 3” paper ECGs performed 24 hours after completion of the 48 hour infusion, if the subject is still in the hospital, at least 15 minutes apart (perform subject and physician assessments first, then vital signs, body weight, ECGs and collect lab samples)

Each set of ECGs consists of three 12-lead ECG recordings collected at 1-minute intervals digitally using Mortara ELI 150 or equivalent recorder at 1000 Hz resolution.


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Depending on the subject’s clinical status, ECGs should be acquired only after subjects have been quietly at rest in the supine position (or comfortable position based on the subject’s clinical condition) for at least 10 minutes to ensure a stable heart rate and stable baseline. During ECG acquisition, subjects should be perfectly still and not speak. When the ECG acquisition time coincides with other procedures/activities (dyspnea assessments, vital signs, blood collection and/or a meal), subject assessments (VAS and Likert SSRS), and physician assessments MUST be acquired first, followed by vital signs, body weight, and then ECGs, blood collection and the meal. Post acquisition, the ECG operator should check the tracing to ensure that it is of high quality. If it is not, the ECG should be repeated. Original ECG copies will be archived at the study center.

Subjects who experience events requiring discontinuation of study drug infusion (e.g., blood pressure drops to <100 mmHg and/or adverse events) should undergo post-therapy ECGs within 24 hours after cessation of study drug infusion.

Subjects who require down-titration of study drug due to blood pressure events (e.g., 40 mm Hg drop in SBP from baseline but SBP is still >100 mm Hg) but do not discontinue study drug should complete all baseline, during study drug infusion, and Day 3 ECG procedures.

21.5.5 ECG evaluations

Standard 12-lead digital ECGs will be acquired as described above and transmitted to a central, blinded, qualified core laboratory for analysis per their standard procedures. Original ECG copies will be archived at the study center.

21.5.6 Data collection and analysis

Site case report forms will collect data: confirming participation in the ECG sub-study, patient identification number, number and timing of ECGs recorded, daily clock synchronization, and position of the patient.

After independent assessments, paired assessments of ECGs from individual patients taken at baseline, during study drug infusion and post-therapy may be made.

These data will be entered onto a core Lab CRF or directly entered into a core Lab database. The core Lab database will contain site and core Lab data.

The randomization codes for patients enrolled in the ECG sub-study will be provided to the core Lab at the time of the RELAX-AHF Day 180 database lock (for the main study) to allow completion of an unblinded ECG data analysis.

Copies of the ECG tracings will be provided to Corthera in the International Society for Holter and Noninvasive Electrocardiology and FDA compliant formats at the conclusion of the study.

The number of patients to be enrolled in the sub-study (up to 60) was selected empirically. There is no study hypothesis being tested. The data will be analyzed to detect any preliminary indications of an effect of study drug on any ECG parameter.


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21.5.7 ECG analysis

The following ECG parameters will be assessed for each ECG in an independent blinded manner: Rate, rhythm, PR interval, QRS duration, QT interval, and morphology. QT correction will be performed using the Fridericia correction method: QTcF = QT/RR0.33.


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Protocol No. RLX.CHF.003 (Phase 3 RELAX-AHF) CONFIDENTIAL Statistical Analysis Plan v4.0 13Jun2012

STATISTICAL ANALYSIS PLAN

Protocol No. RLX.CHF.003 (Phase 3 RELAX-AHF)

A Phase II/III, Multicenter, Randomized, Double-blind, Placebo-

Controlled Study to Evaluate the Efficacy and Safety of Relaxin in Subjects with Acute Heart Failure

SAP Version: 4.0 SAP Date: 13Jun2012 (Final – Amendment 1)

This document is the proprietary property of Corthera, Inc. Information is confidential and is to be used only in connection with matters authorized by Corthera, Inc. No unpublished information presented in this document is to be disclosed to others without prior written permission from the study sponsor.

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TABLE OF CONTENTS

STATISTICAL ANALYSIS PLAN APPROVAL ..........................................................5

REVISION SUMMARY ...................................................................................................6

ABBREVIATIONS AND DEFINITIONS .....................................................................10

1. PREFACE .............................................................................................................13

2. PURPOSE OF THE ANALYSIS PLAN ............................................................14

3. STUDY SUMMARY ............................................................................................14

3.1 Study Objectives ........................................................................................14 3.2 Study Endpoints .........................................................................................14

3.2.1 Primary Endpoints .........................................................................14 3.2.2 Secondary Endpoints .....................................................................14 3.2.3 Additional Efficacy Outcomes .......................................................15 3.2.4 Safety Endpoints ............................................................................15

4. STUDY METHODS ............................................................................................16

4.1 Overall Study Design and Plan ..................................................................16 4.2 Selection of Study Population ....................................................................21 4.3 Method of Treatment Assignment and Randomization .............................21

5. SEQUENCE OF PLANNED ANALYSES ........................................................21

5.1 Interim Analyses ........................................................................................21 5.2 Final Analyses and Reporting ....................................................................22

5.2.1 Analyses Following Day 60 Database Lock ..................................22 5.2.2 Analyses following Day 180 Database Lock .................................22 5.2.3 Exploratory Analyses .....................................................................23

6. SAMPLE SIZE DETERMINATION .................................................................23

7. ANALYSIS DATA SETS ....................................................................................24

7.1 Intent-to-Treat (ITT) Set ............................................................................24 7.2 Safety Set ...................................................................................................24 7.3 Full Analysis Set (FAS) .............................................................................24 7.4 Per Protocol Sets (PPS) ..............................................................................25

8. GENERAL ISSUES FOR DATA ANALYSIS ..................................................26

8.1 General Analysis Approach .......................................................................26 8.2 Examination of Covariates .........................................................................27 8.3 Data Handling Conventions .......................................................................28

8.3.1 Subject Self-Report of Symptoms and Investigator’s Assessments of Heart Failure Signs and Symptoms ......................28

8.3.2 Days Alive and out of Hospital ......................................................29 8.3.3 Time to Death or Rehospitalization Outcomes ..............................29

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8.3.4 Time to WHF .................................................................................29 8.3.5 Concomitant Medications ..............................................................30 8.3.6 Body Weight ..................................................................................30 8.3.7 Length of hospital stay ...................................................................30 8.3.8 Days in the ICU/CCU ....................................................................30 8.3.9 Adverse Events ..............................................................................30

8.4 Multiple Comparisons and Multiplicity .....................................................30 8.5 Coding Dictionaries ...................................................................................31

9. SUBJECT POPULATION ..................................................................................31

9.1 Subject Disposition, Withdrawals, and Follow-up ....................................31 9.2 Protocol Deviations ....................................................................................32 9.3 Demographics and Screening/Baseline Characteristics .............................32

10. EFFICACY ANALYSES ....................................................................................32

10.1 Primary Efficacy Endpoints .......................................................................33 10.1.1 Area under the change from baseline dyspnea VAS curve from

baseline to Day 5 ............................................................................33 10.1.2 Moderately or markedly better dyspnea at 6, 12, and 24 hours .....33

10.2 Secondary Efficacy Endpoints ...................................................................34 10.2.1 Days alive and out of hospital through Day 60 ..............................34 10.2.2 Cardiovascular death or rehospitalization due to heart failure

or renal failure through Day 60 ......................................................34 10.3 Additional Efficacy Outcomes ...................................................................35

10.3.1 Change from baseline in dyspnea score by VAS at 6, 12, 24, and 48 hours, Day 3, Day 4, Day 5, and Day 14 ...........................35

10.3.2 Area under the curve for the dyspnea VAS change from baseline to Day 14, from Day 1 to Day 5, and from Day 1 to Day 14 ............................................................................................35

10.3.3 Moderately or markedly better dyspnea by Likert scale at 6, 12, and 24 hours and then daily to Day 5 and at Day 14 .....................35

10.3.4 Time to moderately or markedly better dyspnea through Day 5 ...35 10.3.5 Time to WHF through Day 5 and Day 14......................................36 10.3.6 Total doses for both IV and oral loop diuretics from Day 0

through Day 5 or discharge if earlier .............................................36 10.3.7 Change in weight from randomization daily through Day 5

and Day 14 .....................................................................................37 10.3.8 Length of hospital stay ...................................................................37 10.3.9 All cause death or rehospitalization due to heart failure or

renal failure through Day 60 ..........................................................37 10.3.10 Reasons for All Rehospitalizations per Investigator and

CEC ................................................................................................38 10.3.11 Causes of Death per Investigator and CEC ................................38 10.3.12 Days alive and out of hospital through Day 30 ..........................38 10.3.13 Cardiovascular death through Day 180 ......................................38

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10.3.14 Other heart failure signs and symptoms over time .....................38 10.3.15 Days in the ICU/CCU ................................................................39 10.3.16 All cause death through Day 30 .................................................39 10.3.17 All cause death or worsening heart failure or

rehospitalization due to heart failure through Day 30 ...................39 10.3.18 Cardiovascular death or rehospitalization due to heart

failure or renal failure through Day 30 ..........................................39 10.3.19 Cardiovascular death or rehospitalization due to heart

failure or renal failure through 30 days after discharge from the index hospitalization ................................................................39

10.4 Exploratory Analysis of Multiple Endpoints .............................................40

11. EXTENT OF EXPOSURE TO STUDY DRUG ................................................40

12. CONCOMITANT MEDICATION ....................................................................41

12.1 IV Inotrope/Vasoactive Medications .........................................................41 12.2 Other Concomitant Medications ................................................................41

13. SAFETY ANALYSES .........................................................................................42

13.1 Adverse Events ..........................................................................................42 13.2 Mortality (Death to Day 180) ....................................................................43 13.3 Clinical Laboratory Evaluations ................................................................43

13.3.1 Creatinine and eGFR changese ......................................................43 13.3.2 Hemoglobin, hematocrit, and RBC count changes ........................44 13.3.3 ALT, AST, alkaline phosphatase, and total bilirubin changes ......44

13.4 Vital Signs ..................................................................................................44 13.5 Blood Pressure Decrease Events ................................................................44 13.6 Pregnancy Test Results ..............................................................................45

14. REPORTING CONVENTIONS.........................................................................46

14.1 General Reporting Conventions .................................................................46 14.2 Population Summary Conventions ............................................................47

15. CHANGES FROM THE PROTOCOL-SPECIFIED ANALYSES ................48

16. REFERENCES .....................................................................................................49

17. APPENDIX A. STATISTICAL ANALYSIS PLAN FOR PHARMACOECONOMIC EVALUATION .....................................................50

18. APPENDIX B. STATISTICAL ANALYSIS PLAN FOR PHARMACOKINETIC ANALYSIS .................................................................74

19. APPENDIX C. STATISTICAL ANALYSIS PLAN FOR BIOMARKER EVALUATION ....................................................................................................85

20. APPENDIX D. STATISTICAL ANALYSIS PLAN FOR ELECTROCARDIOGRAM SUBSTUDY .........................................................91

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STATISTICAL ANALYSIS PLAN APPROVAL

By signing below, all parties accept that the analysis methods and data presentations are acceptable and that this document is final. Authored By: Beth Davison, PhD Date Vice President, Biometrics Momentum Research, Inc. Approved By: Elaine Unemori, PhD Date Vice President, Clinical Development Corthera, Inc. Christopher Bush Date Program Statistician Novartis Thomas Severin, MD Date Medical Monitor Novartis

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REVISION SUMMARY

Version Date Final

4.0 13 June 2012

3.0 17 January 2012

2.0 07 June 2010

1.0 09 March 2010

Version 4.0 provides revisions based on the dry run review.

Version 3.0 provides revisions based on Amended Protocol Version 03 dated

01 December 2010,Version 04 dated 22March2011, and Version 05 dated 12 January 2012.

Relevant point changes to the statistical analysis plan appear in the table below. Other changes that are informal are not individually identified below. These changes may include clarifications, corrections, and editorial adjustments to improve the clarity and provide consistency.

Section Modification (or Explanation)

Global changes throughout the SAP text.

Reference to the recombinant form of human relaxin-2 was changed from ‘relaxin’ to ‘RLX030’.


Analyses of the pharmacoeconomics, pharmacokinetics, and biomarkers data, and of the ECG substudy, which are to be conducted separately from the main analyses, are included as Appendices to this plan.


All references to change in sample size from 880 to 1160 patients were revised.


Four additional efficacy outcomes described with their analyses for: (1) all cause death through Day 30; (2) all cause death or worsening heart failure or rehospitalization due to heart failure or renal failure through Day 30; and (3) cardiovascular death or rehospitalization due to heart failure or renal failure through Day 30, and (4) cardiovascular death or rehospitalization due to heart failure or renal failure

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through 30 days following discharge from the index hospitalization.


Reference to ‘study drug initiation date’ or ‘study drug administration date’ changed to ‘baseline date’ to be consistent with the definition of the ITT Set being the primary efficacy analysis population.


Clarified that cause of death and primary reason for rehospitalization through day 60 for primary analyses will be as adjudicated by the CEC.

4 Study Methods Revised to include ECG substudy in accordance with protocol amendment 04.

6 Sample Size Determination Revised to reflect increased sample size from 880 to 1160 in accordance with protocol amendment 03 .

7 Analysis Data Sets The intent-to-treat (ITT) set replaces the full analysis set (FAS) as the primary analysis population for the primary and secondary efficacy endpoints. The FAS will be used as the supportive analysis population.

The exploratory analysis set (EAS) is replaced with per-protocol sets 1 and 2 (PPS1 and PPS2) and the description moved from section 10 to section 7, and is limited to the analyses of the primary and secondary efficacy endpoints.

8.1 General Analysis Approach Clarification of efficacy results reported and analyzed on imputed values and not raw data. Clarification of sensitivity analyses using other imputation methods.

8.1 General Analysis Approach Clarification of the baseline time and date.

8.1 General Analysis Approach Rehospitalization definition given in the protocol repeated in the SAP for clarity.

8.2 Examination of Covariates Models for estimating covariate-adjusted treatment effects clarified. Age and region subgroups updated for consistency with program standards.

8.3 Data Handling Conventions Imputation rules clarified.

8.4 Multiple Comparisons and Multiplicity

Adjustment for potential efficacy interim analysis added for consistency with protocol and DSMB Charter.

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10.1 Primary Efficacy Endpoints

Tests of assumptions underlying the analysis of the dyspnea VAS AUC clarified. Imputation for moderately/markedly better dyspnea at 6, 12, 24 hours clarified.

10.4 Exploratory Analysis of Multiple Endpoints

An exploratory analysis using an average z across multiple endpoints of interest added.

13. Safety Analyses Analyses added to quantify potential risks identified in the RLX030 Development Safety Profiling Plan.

17-20. Appendices SAPs for ancillary studies are appended to the main SAP.

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Version 2.0 provides revisions based on FDA’s ADVICE/INFORMATION REQUEST (BB IND 100806), dated 04/23/2010 from Norman L. Stockbridge, MD, PhD, Director, Division of Cardiovascular and Renal Products, Office of Drug Evaluation I, CDER.

Relevant point changes to the statistical analysis plan by section appear in the table below. Other changes that are informal are not individually identified below. These changes may include clarifications, corrections, and editorial adjustments to improve the clarity and provide consistency.

Section Modification (or Explanation)


All references to MITT were removed and replaced with ITT according to FDA’s advice/information request.


All references to Enrolled Set were removed. ITT replaced Enrolled Set where applicable.

4.3 Method of Treatment Assignment and Randomization

Clarified according to FDA’s advice/information request.

7 Analysis Data Sets 7.1 Intent-to-Treat (ITT) Set defined; and Enrolled Set and MITT removed according to FDA’s advice/information request.

8.3.1 Subject Self-Report of Symptoms and Investigator’s Assessments of Heart Failure signs and Symptoms

Data handling rule for missing baseline scores was defined.

10.4 Additional Exploratory Analyses

Data handling rule for missing baseline dyspnea VAS score was specified.

15. Changes From the Protocol-Specified Analysis

Updated to reflect changes in response to FDA’s advice/information request.

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ABBREVIATIONS AND DEFINITIONS

AE Adverse event

ACE Angiotensin converting enzyme

AHF Acute heart failure

ANOVA Analysis of variance

ATC Anatomical therapeutic chemical

ARB Angiotensin receptor blocker

AUC Area under the curve

BDRM Blinded data review meeting

BNP Brain natriuretic peptide

BP Blood pressure

BPDE Blood pressure decrease event

CCU Coronary care unit

CDER Center for Drug Evaluation Research

CEC Clinical Endpoints Committee

CHF Congestive heart failure

CI Confidence interval

cm Centimeter

CRF Case Report Form

CSR Clinical Study Report

CV Cardiovascular

dL Deciliter

DRE Disease-related event

DSMB Data Safety Monitoring Board

EC Ethics Committee

ED Emergency Department

EF Ejection fraction

eGFR Estimated glomerular filtration rate

FAS Full Analysis Set

FDA Food and Drug Administration

GCP Good Clinical Practice

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H High

HF Heart failure

hr Hour

ICF Informed Consent Form

ICH International Conference on Harmonization

ICU Intensive care unit

IRB Institutional Review Board

ITT Intent-to-Treat

IV Intravenous

IVRS Interactive Voice Response System

kg Kilogram

L Low

mcg Microgram

MedDRA Medical Dictionary for Regulatory Activities

mg Milligram

min Minute

mL Milliliter

mm Millimeter

mmHg Millimeters mercury

µg Microgram

ng Nanogram

NT-pro-BNP N-terminal prohormone of brain natriuretic peptide

pg Picograms

pH Negative log10 hydrogen ion concentration

PT Preferred Term

SAE Serious adverse event

SAP Statistical Analysis Plan

SAS Statistical Analysis System

SBP Systolic blood pressure

SD Standard deviation

sMDRD Simplified Modification of Diet in Renal Disease

SOC System Organ Class

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US United States

VAS Visual analog scale

WHF Worsening heart failure

WHO-DD World Health Organization Drug Dictionary

Definitions: Study drug: The human recombinant form of human relaxin-2 will be referred to as RLX030. For the purposes of this study, study drug refers to RLX030 or placebo.

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1. PREFACE

This Statistical Analysis Plan (SAP) describes the planned analysis and reporting for the Phase III portion of the Phase II/III protocol RLX.CHF.003 (Pre-RELAX-AHF/RELAX-AHF):

“A Phase II/III, Multicenter, Randomized, Double-blind, Placebo- Controlled Study to Evaluate the Efficacy and Safety of Relaxin in

Subjects With Acute Heart Failure”

The study, sponsored by Corthera, Inc., is being conducted in two phases: 1) the Phase II Pre-RELAX-AHF (Pilot Phase), which has been completed, and 2) the Phase III RELAX-AHF (Main Phase). This SAP describes the planned analyses in the RELAX-AHF Main Phase. The analyses of the pharmacoeconomics, pharmacokinetics, and biomarkers data, and of the ECG substudy data are not detailed in the main SAP and will be conducted separately. SAPs for these analyses are included in Appendices A, B, C and D, respectively.

The structure and content of this SAP provides sufficient detail to meet the requirements identified by the FDA and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH): Guidance on Statistical Principles in Clinical Trials. All work planned and reported for this SAP will follow internationally accepted guidelines, published by the American Statistical Association and the Royal Statistical Society, for statistical practice.

The following documents were reviewed in preparation of this SAP:

• Protocol RLX.CHF.003 (Pre-RELAX-AHF/RELAX-AHF), Amendments 3 issued 01 December 2010, Amendment 4 issued 22March2011, and Amendment 5 issued 12 January 2012

• Case report forms (CRFs) for Protocol RLX.CHF.003 (Pre-RELAX-AHF/RELAX-AHF).

• RLX.CHF.003 (RELAX-AHF) Unblinding Plan • Clinical Data Management Plan for Protocol RLX.CHF.003 (RELAX-AHF) • Data and Safety Monitoring Board Charter for Protocol RLX.CHF.003 (RELAX-

AHF) and DSMB Report Template • Significant Deviation Definitions for Protocol RLX.CHF.003 • RLX030 Development Safety Profiling Plan • ICH Guidance on Statistical Principles for Clinical Trials.

The reader of this SAP is encouraged to also read the clinical protocol for further details on the conduct of this study, and the operational aspects of clinical assessments and timing for completing a subject in this study.

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2. PURPOSE OF THE ANALYSIS PLAN

The purpose of this SAP is to outline the planned analyses to be completed to support the completion of the Clinical Study Report (CSR) for protocol RLX.CHF.003 (Phase 3 RELAX-AHF). The planned analyses identified in this SAP will be included in regulatory submissions and/or future manuscripts. Also, exploratory analyses not identified in this SAP may be performed to support the clinical development program. Any post-hoc or unplanned analyses, not identified in this SAP will be clearly identified as such if they are included in the respective CSR.

3. STUDY SUMMARY

3.1 Study Objectives

The overall objectives of this study are as follows: 1. To test the ability of RLX030 to improve symptoms (dyspnea) and signs of heart failure

in subjects hospitalized with acute heart failure (AHF), normal to elevated blood pressure and mild to moderate renal impairment;

2. To evaluate the safety of RLX030 in these subjects; and 3. To evaluate the ability of RLX030 to reduce intermediate term mortality/morbidity in

these study subjects

3.2 Study Endpoints

3.2.1 Primary Endpoints

The two primary efficacy endpoints are as follows:

1. Area Under the Curve (AUC) representing the change in subject-reported dyspnea from baseline measured by a 100-mm Visual Analog Scale (VAS) from baseline through Day 5; and

2. Moderately or markedly better subject-reported dyspnea relative to the start of study drug on the 7-point Likert scale at 6, 12 and 24 hours (at all 3 time points)

RLX030 will be considered effective in relieving dyspnea if the statistical test(s) comparing treatment groups (RLX030 versus placebo) is significant either at the two-sided 0.05 significance level for both endpoints, or at the 0.025 significance level for one test (Hochberg approach to control of type I error at 0.05). 3.2.2 Secondary Endpoints

The two secondary efficacy endpoints are as follows: 1. Days alive and out of hospital through Day 60; and 2. Cardiovascular (CV) death or rehospitalization due to heart failure (HF) or renal failure

(RF) through Day 60.

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If the above criterion is met for the primary endpoints (either significant at the 0.025 level or both significant at the 0.05 level), then testing will proceed to the secondary endpoints, with the significance level controlled at the two-sided 0.05 level using the Hochberg approach; that is, significance will be declared if the test for either endpoint is significant at the two-sided 0.025 level or both are significant at the 0.05 level. 3.2.3 Additional Efficacy Outcomes

Additional outcomes to be analyzed as supportive evidence for RLX030 efficacy are as follows:

• Change from baseline in dyspnea score by VAS at 6, 12, 24 and 48 hours and at Days 5 and 14;

• Area under the curve representing the change in dyspnea VAS from baseline for baseline to Day 14, Day 1 to Day 5 and Day 1 to Day 14;

• Markedly or moderately better dyspnea compared to baseline by Likert scale assessed separately at 6, 12, and 24 hours and then daily to Day 5 and at Day 14;

• Time to moderately or markedly better self-assessed dyspnea on the 7-point Likert scale up to Day 5;

• Time to worsening heart failure (WHF) through Day 5 and through Day 14; • Total doses of IV loop diuretics and oral loop diuretics through Day 5 or discharge if

earlier; • Change in body weight from randomization through Day 5 and Day 14; • Length of hospital stay; • All cause death or rehospitalization due to heart failure or renal failure through Day 60; • Days alive and out of hospital through Day 30; • Cardiovascular death through Day 180; • Change from baseline in heart failure signs and symptoms (subject-reported dyspnea and

general well-being using Likert scale and VAS, and physician assessments). • Days of the initial hospitalization from randomization spent in the intensive care

unit/coronary care unit (ICU/CCU) • All cause death through Day 30; and • All cause death or worsening heart failure or rehospitalization due to heart failure through

Day 30. • Cardiovascular (CV) death or rehospitalization due to heart failure (HF) or renal failure

(RF) through Day 30 • Cardiovascular death or rehospitalization due to heart failure or renal failure through 30

days following discharge from the index hospitalization The analysis of changes in NT-proBNP and other biomarkers as measured by a core laboratory are described in the Biomarker SAP (Appendix C). 3.2.4 Safety Endpoints

Safety will be assessed by comparing the RLX030 group to the placebo group with regard to the risk of death from any cause through Day 180, frequency of adverse events, clinical laboratory

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test results (chemistry, hematology, and urinalysis), and changes in vital signs. Adverse Events (AEs) and Serious Adverse Events (SAEs) are assessed through Day 5 and Day 14, respectively.

4. STUDY METHODS

4.1 Overall Study Design and Plan

The Phase III portion of the study (“RELAX-AHF”) is designed to confirm the efficacy and evaluate the safety of RLX030 at 30 µg/kg/day versus placebo infused for up to 48 hours in subjects hospitalized with AHF. Enrollment was paused temporarily between the Pre-RELAX-AHF and RELAX-AHF to analyze the data from Pre-RELAX-AHF and make the necessary protocol modifications. In the Phase III portion of the study, eligible subjects will be randomized 1:1 to receive either IV placebo or RLX030. A total of up to 1160 subjects will be enrolled in RELAX-AHF in order to attain approximately 1100 efficacy evaluable subjects (FAS).

Potential study candidates will be subjects hospitalized with AHF, SBP > 125 mmHg at the time of screening, and mild to moderate renal impairment, measured as an estimated glomerular filtration rate (eGFR) of 30-75 mL/min/1.73 m2 (sMDRD formula) who require IV diuretic therapy.

After signing an Ethics Committee (EC) or Institutional Review Board (IRB) approved Informed Consent Form (ICF), subjects will be asked to undergo screening procedures for study eligibility.

Eligible subjects must be randomized within 16 hours from presentation (including the ED). Subjects will be randomized in a 1:1 ratio to placebo or RLX030 30 µg/kg/day in a double blinded manner. Subjects randomized to the placebo arm will receive standard therapy for AHF plus placebo. Subjects randomized to the active study drug arm will receive standard therapy for AHF plus active study drug. Study drug will be administered as an IV infusion for 48 hours unless at any time during dosing in 2 consecutive measurements 15 min apart, the subject’s SBP is < 100 mm Hg at which point, study drug will be terminated. If at any time during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration.

Subject self-reported AHF symptoms will be collected using both a 7-point Likert scale describing the change in symptom severity from baseline (+3=markedly better, +2=moderately better, +1=minimally better, 0=no change, -1=minimally worse, -2=moderately worse, -3=markedly worse) and a 100-mm VAS describing symptom severity at each point in time. These evaluations will be done at baseline (VAS only), 6, 12 and 24 hours from start of study drug infusion, daily while hospitalized through Day 4, and at Day 5 and Day 14.

Subjects will have clinical evaluations including AHF symptom assessments, vitals signs, physical examination emphasizing signs of HF, as well as an assessment of need for further IV or oral HF treatment and of the occurrence of worsening HF events at least daily while hospitalized through Day 4, at Day 5, and then at Day 14 and Day 60. Blood and urine samples will be collected at baseline and at 24 hours (Day 1) and 48 hours (Day 2) for routine safety assessments and to evaluate renal function. Blood samples will also be collected on Days 3, 4, 5,

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14 and 60. Clinical and laboratory evaluations, as detailed above, will be mandatory at baseline, and at Days 5, 14 and 60. If the subject is discharged from the hospital prior to these visits, these evaluations will be performed as outpatient visits.

Just prior to the time that the last subject enrolled in the study reaches the Day 60 follow-up, subjects who have not completed the study will be contacted to ascertain an interim vital status. All subjects will receive a phone call at Day 180 from start of study drug infusion to ascertain vital status. Safety will be assessed by recording medical history, monitoring adverse events and vital signs, and performing physical examinations and routine clinical laboratory tests as per the protocol, as well as any tests felt to be clinically indicated by the investigator. Adverse events (AE) will be collected from signing of the informed consent form through Day 5 and serious adverse events (SAE) will be collected through Day 14.

An ECG sub-study will be conducted at a subset of sites in selected countries (Germany, Hungary, Israel, Italy, Poland, Romania, USA). ECGs will be obtained at baseline, at 12, 24, and 48 hours following the start of study drug infusion, and on Day 3 (if the subject is still in the hospital).

The schedule of assessments is given in Table 1.

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Table 1. Schedule of Assessments

RLX.CHF.003





6 hre 12 hr e

24 hr e(Day 1)

48 hr e


Informed consent X

Inclusion/exclusion criteria X

Medical history X

BNP or NT-pro-BNP X

Troponin X

Electrocardiogram and Chest X-Ray X Digital ECGs (only for subjects enrolled in the ECG sub-study)n

X X X X X


X

Pregnancy test X

Body weight measurement X X X X X X

Height X

Patient-reported dyspnea and general well-being assessments

Xf X X X X X X

Physician assessment of signs and symptoms of HF

X X X X X X X X

Physical examination with vital signs g X X X X X X X X X

Frequent BP and HR measurements h X X X X X

Clinical laboratory tests i X X X X X X X

RLX030 measurement j X

Neurohormone measurement k X X X X X X

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RLX.CHF.003





6 hre 12 hr e

24 hr e(Day 1)

48 hr e


Collect concomitant medications X X X X X X X

Collect adverse and serious adverse events l X X X X X X

Occurrence of re-admission X (Day 5) X X

EQ-5D X X

Vital statusm X X X X Xm X

Footnotes:

a. Study drug treatment will be administered as an IV infusion for 48 hours. If at any time during dosing, the subject’s systolic blood pressure is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug treatment infusion rate will be decreased by 50% for the remainder of the study drug administration. If at any time during dosing, the subject’s systolic blood pressure is < 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion will be terminated.

b. Patients discharged prior to Day 5 will be asked to return to the hospital for Day 5 procedures. Acceptable window is Day 5-7.

c. Patients discharged prior to Day 14 will be asked to return to the clinic for Day 14 procedures; acceptable window is Day 14-16. The Day 60 assessment will be conducted as an outpatient visit if the patient was discharged prior; acceptable window is Day 60-70. Subjects will be contacted by telephone at Day 180 (± 7 days).

d. Procedures that are part of a site’s standard of care for an individual with AHF may be done during the pre-screening interval and may pre-date the signed ICF.

e. For all assessments scheduled for the 6 hour time point, a window at 4.5-7 hours is allowed for these measurements, for all the assessments at the 12 hour time point, a window at 9-13 hours is allowed, for all assessments scheduled for the 24 hour time point, a window at 20-26 hours is allowed, and for all assessments scheduled for the 48 hour time point, a window at 42-48 hours is allowed.

f. Only patient self-reported dyspnea and general well-being by VAS will be done at baseline.

g. A directed physical examination will consist of an assessment of general appearance as well as vital signs (pulse rate, respiratory rate and body temperature).

h. BP and HR measurements should be performed at 30 minutes and then every hour for the first 6 hours of study drug infusion, and then every 3 hours during study drug infusion, including night time hours. Post-infusion, BP and HR should be measured every 3 hrs until 12 hrs following end of infusion, then every 6 hrs for 48 hours and then every 24 hours until the earlier of Day 5 or discharge. BP and HR should be measured with the patient in the same position and with the same equipment throughout study drug infusion.

i. Clinical laboratory tests include hematology and chemistry. Urinalysis will be conducted at baseline, 24 and 48 hours only. Clinical laboratory tests at Screening will be performed locally. At all other time points, laboratory tests will be performed by a central laboratory.

j. Blood draw for RLX030 measurement will be performed at 24 hr following start of drug infusion to confirm proper dosing.

k. Blood will be drawn for measurement of neurohormones and other markers related to HF at Baseline, 24 and 48 hours after start of study drug infusion and at Day 3, 4, 5, 14 and 60.

l. In the Pre-Relax-AHF phase the reporting period for serious adverse events begins with the signing of the ICF and ends at 30 days after the start of study drug infusion. In the main phase (Relax-AHF) AEs are reported from the signing of the ICF through Day 5 and SAEs through Day 14.

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m. Just prior to the time that the last patient enrolled reaches 60 days of follow up, all patients that have not completed the study, will be contacted and vital status information will be obtained at that time.

n. Electrocardiograms will be obtained at baseline and within 2 hours of the following timepoints: 12, 24, and 48 hours following the start of study drug infusion, and on Day 3(if the subject is still in the hospital). When the ECG acquisition time coincides with other procedures/activities (subject and physician assessments, vital signs, blood collection and/or a meal), subject assessments (SSRS and physician assessment) MUST be acquired first, followed by vital signs, body weight, then ECG, blood collection and the meal. The sub-study will only be conducted at a subset of sites in selected countries (Germany, Hungary, Israel, Italy, Poland, Romania, USA).

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4.2 Selection of Study Population

Subjects will be selected from among subjects who present with AHF who have normal to elevated blood pressure and have mild to moderate renal insufficiency at screening. All subjects should have dyspnea at rest, have elevated BNP levels, radiographic evidence of pulmonary congestion, and have received an IV dose of furosemide prior to screening (in the ambulance, ED or at admission to the department). Subjects must be randomized within 16 hours of presentation to the hospital. All subjects must meet all of the inclusion criteria and none of the exclusion criteria as listed in the protocol. 4.3 Method of Treatment Assignment and Randomization

A total of up to 1160 subjects will be enrolled in RELAX-AHF in order to attain approximately 1100 efficacy evaluable subjects, defined as those subjects in the Full Analysis Set (see Section 7). Eligible subjects will be randomized in a 1:1 fashion to receive an IV infusion of one of the following treatment assignments for up to 48 hours:

• Placebo (n = 580) • RLX030, 30 µg/kg/day (n = 580)

This is a double-blind study. Subject numbers will be assigned sequentially at each study site through the IVRS. The subjects will be assigned blinded study drug kits through the IVRS based on the randomization scheme. Each site is assigned a block of the randomization scheme upon activation; up to one additional block may be assigned automatically by the IVRS using a pre-defined algorithm based on enrollment. Each study drug kit will be labeled with a unique kit identification number. The two types of vials (RLX030 and placebo) are designed to be indistinguishable. Study drug will be prepared from the vials contained in the blinded kits according to instructions in the Pharmacy Manual. All study personnel will remain blinded to treatment assignments. Investigators may unblind a subject’s treatment assignment only by discussing the need with the medical monitor through the IVRS Helpline (see the Unblinding Plan). If the subject’s treatment assignment is unblinded, the event is recorded in the IVRS. Whether any subjects’ assignments were unblinded will be noted in the final clinical study report (CSR), following the full analysis of the Day 180 database. 5. SEQUENCE OF PLANNED ANALYSES

5.1 Interim Analyses

Two interim examinations of key safety data are planned for the purpose of reporting to the Data and Safety Monitoring Board (DSMB). All analyses related to the DSMB will be carried out by an independent statistical group; details are not included in this SAP but may be found in the DSMB Charter and DSMB Report Template.

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5.2 Final Analyses and Reporting

The study will have two database locks. The first database lock will occur when all subjects have completed the 60 day evaluation period of the study and all data has been monitored, is query-clean and final. All follow-up between Day 60 and Day 180 available at the time, including Day 180 and interim contacts, will be included in the Day 60 database. A second database lock will occur when all subjects have reached day 180 of the study and all data collected following the Day 60 database lock has been monitored, is query-clean and final. 5.2.1 Analyses Following Day 60 Database Lock

In accordance with the Unblinding Plan, all analyses summarizing results by treatment group, and planned in the protocol and in this SAP will be performed only after the last subject has completed the 60 day evaluation period of the study. For this analysis, group summaries will be presented. Individual subject treatment assignments (e.g., listings) will be provided prior to the Day 180 lock only if required for regulatory submission(s); if required, these will be provided only to personnel identified by the Sponsor, who have no direct role in study operations. In addition, no database may be locked, treatment code unblinded, or analyses completed until this SAP has been approved. All analyses outlined in the SAP, with the exception of individual data listings, will be carried out after:

• The SAP has been approved;

• The Day 60 database has been authorized by the Sponsor’s clinical team as complete and locked;

• All protocol deviations have been reviewed and the relevant study analysis populations have been identified; and

• The Sponsor has authorized unblinding.

These results will be shared with a limited group of people to be identified by the Sponsor.

After all CRFs have been retrieved and entered into the clinical database, all queries issued and answered to the extent possible, and prior to locking and unblinding the database, a blinded data review will take place. The primary purposes of this review will be to come to agreement on patients to include in the various analysis sets, to establish courses of action to address unresolved data queries, and to resolve other statistically related issues. The minimum participants in this review will include representatives from the clinical, statistical and data management functional groups of the trial. 5.2.2 Analyses following Day 180 Database Lock

Data in the Day 60 database will be considered final after the Day 60 database lock, and no further changes to the Day 60 data are anticipated during the time that the Day 180 data are being collected. After the last subject has completed Day 180, and the data have been query-cleaned, and the database locked, the following analyses will be updated:

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• Subject accountability and length of follow-up • Mortality through Day 180

Listings of individual subject data, and any other information regarding individual subjects’ treatment assignments, will be provided only after the Day 180 database is locked, unless required for regulatory submission(s) as indicated in Section 5.2.1. 5.2.3 Exploratory Analyses

Further exploratory analyses not necessarily identified in this SAP may be performed to support the clinical development program. Any post-hoc or unplanned analyses performed and not identified in this SAP will be clearly identified as such if included in the respective CSR. 6. SAMPLE SIZE DETERMINATION

A total of 1160 subjects will be enrolled in order to obtain approximately 1100 efficacy evaluable (FAS) subjects. This sample size is adequate to detect a clinically meaningful treatment effect for either primary efficacy endpoint at the two-sided 0.05 significance level Power for the primary and secondary efficacy endpoints was estimated using simulations. Based on correlations among the endpoints observed in Pre-RELAX-AHF, the simulations incorporated the following correlations among the four primary and secondary efficacy outcomes: VAS AUC Likert 60 day CV

Death/HF-RF Rehosp

DOOH through Day 60

VAS AUC 1 0.25 0 0.25 Likert 0.25 1 0 0 60 day CV Death/ HF-RF Rehosp

0 0 1 0.5

DOOH through Day 60

.025 0 0.5 1

Rehosp = re-hospitalization; DOOH = days alive and out of hospital In Pre-RELAX-AHF, a difference of 888 mm-hour on the dyspnea VAS AUC between the 30 µg/kg/day and placebo groups with a pooled standard deviation of 2700 mm-hour was observed; 23.0% in the placebo group and 40.5% in the 30 µg/kg/day group had moderately or markedly better dyspnea at 6, 12, and 24 hours. To estimate power, we assumed a standard deviation of 2700 mm-hour for the VAS AUC and that 25% of placebo subjects would have moderately or markedly better dyspnea at 6, 12, and 24 hours. Minimum clinically relevant effect sizes representing, respectively, a mean difference of 468 mm-hour (4 mm on average) and a relative risk of 1.3 (absolute difference of 7.5%) were assumed. These represent a 28% increase in VAS AUC (468/1679 mm-hour) and a 30% increase in moderately or markedly better dyspnea at 6, 12, and 24 hours (7.5/25%) relative to placebo responses observed in Pre-RELAX-AHF.

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With 1100 subjects, the study has approximately 81% power to detect these minimum clinically relevant effects at the two-sided 0.05 significance level with adjustment for multiplicity using the Hochberg approach. In Pre-RELAX-AHF, the Kaplan-Meier estimates of the proportion of subjects with cardiovascular death or rehospitalization due to HF or renal failure through Day 60 was 17.2% in the placebo group and 2.6% in the 30 µg/kg/day group (hazard ratio 0.36), and a difference of 3.7 days between the 30 µg/kg/day and placebo groups with a pooled SD of 12.7 days was observed for days alive and out of hospital through Day 60. In order to estimate power for these secondary efficacy outcomes, we assumed a placebo event rate of 17.2% and a standard deviation of 12.7 days for days alive out of hospital through Day 60. Assuming a minimum clinically relevant effect on the primary endpoints and significance at the two-sided 0.05 level as described above, 1100 subjects provides approximately 87% power to detect a hazard ratio of 0.55 (17.2% versus 9.9%) for cardiovascular death or rehospitalization for heart failure or renal failure through day 60 and/or a mean difference of 2.7 days in days alive and out of hospital through day 60 at the two-sided 0.05 significance level with multiplicity adjustment using the Hochberg approach. 7. ANALYSIS DATA SETS

7.1 Intent-to-Treat (ITT) Set

The Intent-to-Treat Set will include all randomized subjects, whether or not they were exposed to study drug. Subjects will be analyzed by the group to which they were randomized. The ITT will be used to describe subject accountability. Analyses in the ITT Set will constitute the main efficacy results for the primary and secondary study efficacy endpoints. ITT subjects will be included in each efficacy analysis based on available assessments, after the data handling conventions have been applied as described in Section 8.3.1 and Sections 10.1 and 10.3. 7.2 Safety Set

The Safety Set will consist of all randomized subjects receiving any amount of study drug. Subjects will be analyzed according to the treatment actually received. The Safety Set will be used to perform safety analyses. 7.3 Full Analysis Set (FAS)

The Full Analysis Set will include all randomized subjects who (1) were treated with any amount of study drug, and (2) did not have any major pre-randomization eligibility protocol violations from the list below. Subjects will be analyzed by the group to which they were randomized. Supportive (sensitivity) analyses of the primary and secondary efficacy endpoints will be conducted in the FAS. The major pre-randomization eligibility protocol violations that will exclude subjects are:

• Systolic BP < 100 mmHg at screening (substantial violation of Inclusion Criterion #3)

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• Screening BNP < 200 pg/mL or NT-pro-BNP < 800 pg/mL (substantial violation of Inclusion Criterion #4)

• Randomized >18 hours from presentation to hospital (substantial violation of Inclusion Criterion #5)

• Known significant pulmonary disease (violation of Exclusion Criterion #6) • AHF due to significant arrhythmia as defined in protocol (violation of Exclusion Criteria

#14)

7.4 Per Protocol Sets (PPS)

Additional exploratory analyses will be conducted to evaluate the robustness of the findings and to assess the role of compliance and dose adjustment with the treatment regimen in the results. These analyses will exclude further from the FAS population subjects who had any of the major protocol violations from the lists below. Two PP analysis sets will be defined: (1) PPS1 excludes only patients with pre-randomization protocol violations defined below, (2) PPS2 excludes patients with both pre-randomization and post-randomization protocol violations described below. Subjects will be analyzed by the group to which they were randomized. Key primary and secondary efficacy endpoints, as well as Day 180 mortality will be examined in both PPS1 and PPS2. Subjects who are missing the baseline dyspnea VAS score will be excluded from the PP analyses of the VAS AUC to Day 5 endpoint. Pre-randomization protocol violations that will be excluded from these exploratory analyses include:

• Screening blood pressure measurement <125 mm Hg (violation of Inclusion Criterion #3) • BNP < 350 pg/mL or NT-pro-BNP <1400 pg/mL (violation of Inclusion Criterion #4) • Randomized more than 16 hours from presentation (violation of Inclusion Criterion #5) • Use of disqualifying IV vasodilators, positive inotropes, vasopressors or mechanical

support during screening (violation of Exclusion Criterion #4) • Known significant pulmonary disease (violation of Exclusion Criterion #6) • AHF due to known significant valvular disease as defined in protocol (violation of

Exclusion Criterion #7) • AHF due to current (at the time of screening) acute coronary syndrome (violation of

Exclusion Criteria #12, 13) • AHF due to significant arrhythmia as defined in protocol (violation of Exclusion Criteria

#14)

Post-randomization protocol violations that will be excluded from these exploratory analyses include:

• Non-compliance with study drug administration, i.e., receipt of less than 75% of the intended amount of study drug. For the primary Likert dyspnea efficacy endpoint during the first 24 hours, the minimum duration of study drug infusion required is 18 hours. For the 3 remaining efficacy endpoints, the minimum duration of study drug infusion

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required is 36 hours, i.e. 75% of the intended amount. Subjects whose dose of study drug was appropriately reduced or discontinued will not be excluded.

• Failure to reduce or discontinue study drug administration in the presence of a blood pressure decrease event.

8. GENERAL ISSUES FOR DATA ANALYSIS

Statistical analyses will be performed to determine baseline comparability, to compare the results for the primary, secondary, and additional endpoints, and to summarize the safety evaluations between the two treatment groups. Additionally, within each treatment group vital signs and lab parameters will be assessed for changes from baseline.

8.1 General Analysis Approach

• Available data at each time point will be presented. For the efficacy outcomes, the data will be imputed according to the methods described in Section 8.3. The imputed results will be used for all analysis and reporting of efficacy. Further, to examine the affect of data handling conventions on the results, the sensitivity analyses will use other imputation rules as described in Section 10.1.1.

• The assessments identified in the CRF as baseline assessments will be used as the baseline reference for all analyses (Day 0 in all subjects). The baseline date and time will be considered the date and time of initiation of study drug, or randomization if the patient was not treated with study drug.

• All nominal time points will be used for analysis; actual assessment times will not be used to reclassify the time point at which a measure was taken. Time-to-event analyses will be based on actual dates reported.

• All references to index hospitalization refer to the initial hospitalization for AHF.

• Rehospitalization will be defined as an unplanned hospitalization, including admission to a hospital or any attendance in an acute care setting (e.g., ED or in another health care facility) of 24 hours or greater, regardless of whether the patient was admitted to the hospital.

• Appropriate descriptive statistics will be computed and displayed (by time point and other key variables as appropriate) for both continuous and categorical variables. For continuous variables, descriptive statistics will include n (the number of subjects with non-missing data), mean and the two-sided 95% confidence interval (CI; using normal approximation), standard deviation (SD), median, first and third quartiles, minimum and maximum values. For categorical parameters, the number and percentage of subjects within each category will be presented. The denominator for percentages will be based on the number of subjects with non-missing data appropriate for summary purposes. Unless otherwise noted, all percentages will be presented to one decimal place.

• Unless stated otherwise, all significance tests will be two-sided with statistical

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significance assessed at the 5% level. Unless otherwise specified, p-values will be reported to three decimal places. (See Section 14.2).

• Unless otherwise specified, treatment comparisons for continuous outcomes will be made using two-sided two-sample t-tests assuming pooled variances. If the distribution of the responses is markedly non-normal (e.g., highly skewed or contains outliers) then results of the Wilcoxon rank sum tests will be reported as well. Tests for the normal distribution of results will be conducted by treatment using the Shapiro-Wilk test. Because the Shapiro-Wilk test tends to be overly sensitive to departures from normality in large samples sizes, a more restrictive alpha level will be used as the critical value for claiming non-normality (p≤0.005). Equality of variances will be assessed using an F statistic. If the variances are unequal (p≤0.005) then the results of the t-test using the Satterthwaite method will be presented in addition to results using the method for pooled variances.

• Pooling of centers: Because many centers will enroll very few subjects, all analyses will be based on results pooled across all centers without adjustment for center and no treatment-by-center interactions will be evaluated. Individual data listings of all data represented on the CRF will be presented (following the Day 180 database lock). Sort order for data listings will be treatment, site-subject number, visit, and time points where appropriate.

• Version 9.1 (or higher) of the SAS® statistical software package will be used to provide all tables, figures, data listings, and analyses.

8.2 Examination of Covariates

Sensitivity analyses will be conducted examining the results for the primary and secondary efficacy outcomes adjusted for the following covariates:

• Age • Gender • Race (White/Caucasian, other) • Region (South America, Western Europe, Eastern Europe, North America, Israel) or

Country The baseline NT-proBNP and troponin values will be considered as additional covariates; these analyses are described in the Biomarker SAP (Appendix C). Estimates of the treatment effect adjusted for the covariates with 95% confidence limits will be provided. The models for dyspnea VAS AUC to Day 5 and days alive out of hospital will be adjusted for country, while the other models will be adjusted for region. Treatment effects (odds ratio, mean difference, or hazard ratio) will be estimated from regression models: logistic for moderately/markedly better dyspnea at 6, 12, and 24 hours; ANCOVA for the VAS AUC to Day 5 and days alive and out of hospital to Day 60, and Cox for the time to cardiovascular death or heart failure/renal failure rehospitalization to Day 60. Models will include for each endpoint the effects of RLX030 and the covariates (for covariate-adjusted effect estimates).

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Additionally, a sensitivity analysis will be conducted to examine the effect of adjustment for baseline dyspnea VAS score on the results for the VAS AUC to Day 5. Because the association between the baseline VAS and the VAS AUC is likely to be non-linear, adjustment will be made with baseline VAS as a categorical variable, grouped into deciles of the analysis population. The mean difference between treatments with 95% confidence interval will be estimated from an ANCOVA model including the effects of treatment, the above-listed covariates, and baseline VAS. In addition, the possible interaction between the effect of RLX030 and each of the above-listed covariates will be examined based on models (one for each endpoint and covariate) including the effects of RLX030, the covariate, and the RLX030-by-covariate interaction (for covariate stratum-specific effect estimates). Estimates of the treatment effect within each level of the covariate with 95% confidence limits will be provided for each of the following subgroups:

• Age : <65, ≥65; <75, ≥75 years • Gender • Race: (White/Caucasian versus other) • Region: ‘Region 1’=United States, Canada, Israel, France, Germany, Italy, Spain, Poland,

Netherlands; and ‘Region 2’= Argentina, Romania and Hungary • Region: South America (Argentina), Western Europe (France, Germany, Italy,

Netherlands, Spain), Eastern Europe (Hungary, Poland, Romania), North America (USA), and Israel

These analyses will be conducted in the ITT. 8.3 Data Handling Conventions

Missing data will be imputed as described below for individual outcomes. In general for efficacy analyses, for subjects who die or experience WHF, the worst reported score over all subjects, regardless of treatment group, and time points will be imputed for all time points after the onset of the event, regardless of whether the score is missing or not. For post-baseline values otherwise missing, a missing score will be imputed using linear interpolation between the last preceding and first following non-missing values; if no following non-missing value is available, the last available preceding value will be carried forward. Additional analyses may be undertaken to assess the impact of various data handling rules on the results.

8.3.1 Subject Self-Report of Symptoms and Investigator’s Assessments of Heart Failure Signs and Symptoms Subjects who die or have a worsening heart failure event (either during the index hospital or rehospitalization due to heart failure) will have their score imputed to be the worst observed score in any subject at any time point in the respective analysis population carried forward from the day of death or WHF regardless of whether their original data are missing.

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For post-baseline visual analog scale (VAS) values otherwise missing, a missing score will be imputed using linear interpolation between the last preceding and first following non-missing values. If no following non-missing value is available, the last available preceding value will be carried forward. A missing baseline score will be imputed as the earliest, non-missing score within 24 hours for the subject minus the average change from baseline in the study population to that time point; post-baseline scores for subjects for whom a missing baseline cannot be thus imputed will be included in the analysis as no change from baseline. Except for subjects who die or who experience WHF, subjects who are missing all post-baseline dyspnea VAS scores will be included in the analysis as having no change from baseline at any time point. Missing categorical scores will be replaced with the last preceding non-missing value. 8.3.2 Days Alive and out of Hospital

For subjects who die during the initial hospitalization, the number of days alive and out of hospital through Day 30 or Day 60 will be 0 days. For this endpoint, all hospitalizations are to be included. If the discharge date is missing for a hospitalization, then the average hospitalization duration will be imputed from the non-missing hospitalizations over all subjects and applied to the hospitalizations missing the discharge date. For these subjects the average hospitalization duration will be truncated at the earliest of days to next additional hospitalization, days to known death, days to the end of the 30 or 60 day period, or average hospitalization duration. If the subject is not followed for the full 30 or 60 days, then the average proportion of days alive and out of hospital is computed over all subjects with non-missing values and the ratio applied to the remaining days where the status is unknown. If the total days in hospital exceeds the time period of interest following imputation, then the value will be truncated at 30 or 60 days, as appropriate. 8.3.3 Time to Death or Rehospitalization Outcomes

Subjects without the event of interest in the time period of interest, and with incomplete follow-up, will be censored at the last contact date. It is assumed that the day and month of all event dates will be known. If the date’s day of the month is missing such that a determination of whether the event occurred within the reporting window cannot be made, the event will be assumed to have occurred on the earliest day within the month that includes the event in the reporting window. All events through Day 60 will be adjudicated at the time of database lock; the CEC’s classification of the event will be used for analysis. 8.3.4 Time to WHF

Subjects who die by Day 5 or Day 14 without having experienced WHF, for the respective analyses, will be considered as having WHF on the day of death. Subjects who withdraw from the study prior to completion of Day 5 without experiencing a WHF event will be treated as missing for the respective analysis. Time to WHF by Day 14 will be analyzed using survival analysis. For this analysis, subjects who withdraw from assessments prior to Day 14 will be censored at the time of withdrawal; subjects who do not die or do not have WHF by Day 14 will be censored at Day 14.

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8.3.5 Concomitant Medications

Subjects’ use of concomitant medication within pre-specified classes, including the specific medication and daily dose, is recorded for pre-specified medication at each time point, which represents use in the 24 hours preceding the given time point. Lack of reporting at a given time point will be interpreted as no use in the relevant time period, and for IV loop diuretic use, the dose will be imputed as zero. Concomitant medication use is not reported for days out-of-hospital (e.g., Days 3 and 4); for oral loop diuretic use, the Day 5 dose will be imputed for these days. A daily intravenous loop diuretic dose will be imputed as the lesser of 160 mg or twice the last recorded daily dose following a death. A daily oral loop diuretic dose will be imputed as the lesser of 80 mg or twice the last recorded daily oral dose following a death. 8.3.6 Body Weight

Missing values will be imputed using linear interpolation between the last preceding and first following non-missing values. If no following non-missing value is available, the last available preceding value will be carried forward. Subjects who are missing the baseline weight will be excluded from the analysis. 8.3.7 Length of hospital stay

Subjects who die during the initial hospitalization will be assigned the maximum length of stay over all subjects (including those truncated at Day 60) plus 1 day. Subjects missing the discharge date, such that a determination of the length of stay cannot be calculated, will be assigned the mean length of stay over all subjects not missing such determinations. 8.3.8 Days in the ICU/CCU

Subjects who die during the initial hospitalization will be assigned the maximum number of days in the ICU/CCU over all subjects plus 1 day. 8.3.9 Adverse Events

Adverse Events with incomplete start dates will be considered treatment emergent (incident) if it is not obvious from the partial date whether the event began after study drug was initiated.

8.4 Multiple Comparisons and Multiplicity

The primary global null hypothesis is that both the mean area under the dyspnea VAS change from baseline curve (VAS AUC) from baseline to Day 5 and the proportion of subjects with moderately to markedly better dyspnea at 6, 12, and 24 hours are the same in both treatment groups. The Type I error rate for this global null hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach.

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At each interim analysis, the DSMB will assess safety, but may request summaries of the efficacy data to provide an overall assessment of risk versus benefit. For each review of the primary outcomes, an alpha of 0.00001 will be considered to have been spent, and the significance level for the final analysis adjusted accordingly. The secondary global null hypothesis is that both the mean number of days alive and out of hospital through Day 60 is the same in both treatment groups, and the hazard for CV death or HF/RF readmission through Day 60 is the same in both groups. The Type I error rate will be controlled across the two global null hypotheses at the two-sided 0.05 level by sequential testing: the secondary global null hypothesis will be tested given rejection of the primary global null hypothesis. The Type I error rate for the secondary global hypothesis will be controlled for the two comparisons within it at the two-sided 0.05 level using the Hochberg approach. If the primary global null hypothesis is rejected at the two-sided 0.00125 significance level (equivalent to that required for two trials conducted at the two-sided 0.05 significance level), the single study will be considered to support the efficacy of RLX030 in this indication. A result significant at the two-sided 0.05 significance level, but not significant at the two-sided 0.00125 level, will be confirmed in a subsequent study. Tests of additional efficacy outcome analyses and safety analyses will be performed without adjustment for multiple comparisons at the two-sided 0.05 significance level. 8.5 Coding Dictionaries

Verbatim adverse events will be coded into standardized system organ classes (SOC) and preferred terms (PT) using the Medical Dictionary for Regulatory Activities (MedDRA). Concomitant medications will be coded to the Anatomical Therapeutic Chemical (ATC) classification level 4 and preferred terms using the World Health Organization Drug Dictionary (WHO-DD).

9. SUBJECT POPULATION

9.1 Subject Disposition, Withdrawals, and Follow-up

Each subject randomized in the study will be accounted for and summarized by treatment group and overall. The number of randomized subjects in each of the study populations will be tabulated. Also, a summary of subjects randomized by region, country, and investigative site will also be presented by treatment group and overall. The number of subjects alive through the Day 60 visit and number completing the study through the Day 180 contact and the number of subjects withdrawing from the study will be tabulated. Withdrawals will be categorized by reason for withdrawal. For each subject, the length of follow-up (days) will be computed by subtracting the study drug

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initiation date (or the randomization date for subjects not treated with study drug) from the end of study date. Univariate statistics will be presented for length of follow-up by treatment group and overall. Categorical summaries will also be presented for the number and percent of subjects within predefined follow-up intervals as: None; Day 0-5; Day 6-14; Day 15-60; Day 61-180; and Day 180+. 9.2 Protocol Deviations

Protocol deviations that will be considered “significant” are identified in the Significant Deviation Definitions for Protocol RLX.CHF.003. All protocol deviations will be reviewed on an ongoing basis during the study by the Medical Monitor and Corthera clinical and regulatory team, and deviations meeting criteria for ‘significant’ deviations will be identified as such on a master list maintained by the Corthera clinical team. A summary table and a listing for individual subjects will be presented to describe the significant protocol deviations for this study based on the ITT Set. 9.3 Demographics and Screening/Baseline Characteristics

Demographics and screening/baseline characteristics will be summarized using the ITT and FAS populations by treatment group and for all subjects combined. Subject demographics (age, sex, ethnicity, race, weight and height) at baseline will be summarized. Weight and height will be converted to units of the metric system, kilograms (kg) and centimeters (cm) respectively. The number and percentage of subjects who were hospitalized for heart failure in the past year and subjects on IV nitrates at the time of randomization will both be presented. Screening laboratory values for BNP, NT pro-BNP, estimated Glomerular Filtration Rate (eGFR), troponin I and troponin T laboratory values as reported by the investigator will be summarized with appropriate descriptive statistics. The number and percent of subjects with ‘detectable’ versus ‘not detectable’ levels of troponin I and/or T will be presented. “Detectable” levels will be defined as either troponin I or T having any numeric result. The number and percentage of subjects who failed to meet any of the eligibility criteria at screening will also be provided. Medical history findings will be descriptively summarized by the number and percentage of subjects having a particular finding. Most recent ejection fraction will be summarized using descriptive statistics, and as the proportion of subjects with an ejection fraction < 40% versus ≥ 40%. 10. EFFICACY ANALYSES

Statistical analyses will be conducted for all primary and secondary efficacy outcomes for the

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ITT, FAS, and PP Sets. Analyses of exploratory efficacy outcomes will be carried out for the ITT. Efficacy outcomes for the ITT Set will constitute the main outcomes for this study. For all outcomes, appropriate descriptive summaries and statistical inference analyses will be performed and/or plotted and all data will be listed. (Listings will not be provided until following the Day 180 database lock.) All outcomes will be presented descriptively by treatment group and for all subjects combined. 10.1 Primary Efficacy Endpoints

10.1.1 Area under the change from baseline dyspnea VAS curve from baseline to Day 5

The area under the curve representing the change from baseline in dyspnea VAS score from baseline through Day 5 (VAS AUC) will be computed after applying the data handling conventions described in Section . For each subject, the change in dyspnea VAS scores from baseline (Day 0) will be computed by subtracting the baseline score from each post-baseline dyspnea VAS score. The area under the curve for the change from baseline values (mm-hour) will be computed using the trapezoidal rule. Treatment groups will then be compared using a two-sided two-sample t-test as the primary method of analysis. If the distribution of the responses is markedly non-normal (e.g., highly skewed or contains outliers) then results of the Wilcoxon rank sum tests will be reported as well. The normal distribution of results will be assessed by treatment using the Shapiro-Wilk test. Because the Shapiro-Wilk test tends to be overly sensitive with large sample sizes to departures from normality a more restrictive alpha level will be used as the critical value for identifying non-normality (p≤0.005). Equality of variances will be assessed using an F statistic. If the variances are unequal (p≤0.005) then the results of the t-test using the Satterthwaite method will be presented in addition to results using the method for pooled variances. P-values for this comparison will be reported to five decimal places. A sensitivity analysis using the Set will be conducted to examine the effect of data handling conventions on the results. For this analysis, scores for all assessments following a death will be imputed as the worst observed value in the analysis population. Scores for all assessments following the onset of WHF (either during the index hospitalization or rehospitalization due to heart failure) will be imputed as the baseline value for that individual subject; a missing baseline score will be imputed as described in Section 8.3.1. Further missing values will be imputed as described in Section 8.3.1. 10.1.2 Moderately or markedly better dyspnea at 6, 12, and 24 hours

Subjects will be classified as having moderately or markedly better dyspnea (+2 or +3 on the 7-point scale) at all 3 time points (at 6 ,12, and 24 hours) or not. After applying the data handling conventions described in Section , remaining missing values will be imputed as follows: a non-

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missing 24-hour score will be substituted for a missing 12-hour score, and a non-missing 12-hour score will be substituted for a missing 6-hour score; otherwise, a non-missing preceding value will be carried forward. Subjects who have only the 24-hour score present will not have missing values at 6 and 12 hours imputed and will be considered as not having experienced moderately or markedly better dyspnea at 6, 12, and 24 hours. Subjects who are missing Likert scores at all three time points will be considered as not having experienced moderately or markedly better dyspnea at 6, 12, and 24 hours. The proportion of subjects with moderately or markedly better dyspnea at 6, 12, and 24 hours (all three time points) will be compared between treatment groups using a chi-square test; the p-value for this comparison will be reported to five decimal places. The odds ratio and 95% CI will be presented. 10.2 Secondary Efficacy Endpoints


Days alive and out of hospital will be computed as 61 days minus the days in-hospital through Day 60 (including the index hospitalization and any rehospitalizations) minus the days following a death prior to Day 60. Days in hospital will be computed for the index hospitalization as the discharge date minus the baseline date plus one, and for rehospitalizations as the discharge date minus the presentation date plus one. Subjects with incomplete follow-up will have the average proportion of days alive and out of hospital applied to the missing follow-up period as described in Section 8.3.2. Treatment groups will be compared using the Wilcoxon rank sum test as the primary analysis method. In addition, the median of differences and corresponding 95% CI will be presented using the Hodges-Lehmann estimator of shift. Supportive analyses using the two-sided two-sample t-test or its randomization-based counterpart may also be conducted. P-values for this comparison will be reported to five decimal places. 10.2.2 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 60

The days from the baseline date to the date of the first event will be used for analysis. Subjects who die from a non-cardiovascular cause before Day 60 without an event will be censored at the time of death; subjects otherwise without an event will be censored at the earlier of the last contact date or Day 60. The Clinical Event Committee’s classification of the cause of death or the reason for rehospitalization (heart failure or renal failure) will be used for the final analysis. Kaplan-Meier estimates of the rates of the composite event and its components will be presented, and treatment groups will be compared using a log-rank test; the p-value for this comparison will be reported to five decimal places. Kaplan-Meier plots will be presented. Hazard ratios and 95% CIs will be estimated from a Cox regression model.

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10.3 Additional Efficacy Outcomes

The additional efficacy outcomes will be analyzed using the ITT at the two-sided 0.05 significance level, without adjustment for multiple comparisons. Appropriate descriptive statistics will be presented for each treatment group and overall at each time point (or interval) the outcome was computed. 10.3.1 Change from baseline in dyspnea score by VAS at 6, 12, 24, and 48 hours, Day 3, Day 4, Day 5, and Day 14

After applying the data handling rules described above in Section for the actual dyspnea VAS scores, change in dyspnea VAS scores from baseline (Day 0) will be computed for each subject by subtracting the baseline score from each post-baseline dyspnea VAS score. Descriptive statistics of the imputed values at each visit and the change in dyspnea VAS scores from baseline will be presented at each post-baseline visit by treatment group. Treatment groups will be compared for change from baseline at each post-baseline time point using a two-sided two-sample t-test. 10.3.2 Area under the curve for the dyspnea VAS change from baseline to Day 14, from Day 1 to Day 5, and from Day 1 to Day 14

The area under the curve of the change from baseline in dyspnea VAS score (VAS AUC) will be computed by trapezoidal rule after applying the data handling conventions and computations described in Section . Descriptive statistics of the area under the curve for the dyspnea VAS change from baseline scores will be presented at each time interval of interest by treatment group. Treatment groups will be compared by presenting the mean difference and corresponding 95% CI and using two-sided two-sample t-tests. 10.3.3 Moderately or markedly better dyspnea by Likert scale at 6, 12, and 24 hours and then daily to Day 5 and at Day 14

After first applying the data handling conventions described in Section and then Section 10.1.2, the distribution of symptom scores on the 7-point scale will be presented at each time point by presenting the number and percentage of subject having each score. Treatment groups will be compared regarding the proportion of subjects with markedly or moderately better dyspnea at each time point using a chi-square test. 10.3.4 Time to moderately or markedly better dyspnea through Day 5

The time to the subject’s first report of moderately or markedly better dyspnea within 5 days (defined as the first assessment time point when moderately or markedly better dyspnea is first reported) will be compared between treatment groups using a Wilcoxon rank sum test. The 6-hour time point will be assigned a value of 0.25 days, the 12-hour time point 0.5 days, and 24-

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hours 1 day; subsequent time points will be assigned 2, 3, 4, or 5 days based on the nominal time point as appropriate. Subjects who do not report moderately or markedly better dyspnea by Day 5, or who die or have WHF (either during the initial hospitalization or rehospitalization due to heart failure) by Day 5, will be assigned a value of 6 days. Treatment groups will be compared using a Wilcoxon rank sum test. 10.3.5 Time to WHF through Day 5 and Day 14

The onset of WHF through Day 5 will be the first assessment point following onset of the investigator reported WHF (either during the initial hospitalization or rehospitalization due to heart failure), defined as the number of days from the baseline date. Days will be assigned by nominal time point as described above for time to moderately or markedly better dyspnea. Subjects for whom the investigator did not report any WHF by Day 5 will be assigned a value of 6 days. Subjects who die by Day 5 without a prior WHF event will be assumed to have had WHF on the day of death. WHF will be summarized descriptively as the cumulative proportion of subjects with WHF within 6, 12, 24, and 48 hours, and Days 3, 4, and 5 by treatment group. Time to WHF (days) will be summarized descriptively. Treatment groups will be compared using a Wilcoxon rank sum test. Time to WHF through Day 14 will be analyzed using survival analysis. Time to WHF in days will be computed as the time from the baseline date to the earlier of the onset of WHF or death. Subjects without an event by Day 14 will be censored at the earlier of the last assessment or Day 14. Kaplan-Meier estimates of the cumulative event rate at Days 5 and 14 will be presented. Treatment groups will be compared with a log-rank test. The estimated hazard ratio and 95% confidence interval will be computed using Cox regression. 10.3.6 Total doses for both IV and oral loop diuretics from Day 0 through Day 5 or discharge if earlier

Doses for both oral and IV loop diuretics other than furosemide will be converted to furosemide equivalents for analysis as follows:

If diuretic is Multiply mg dosage by

Bumetanide 20

Torasemide 2

Ethacrynic acid 0.8 Descriptive statistics for the daily dose in furosemide equivalents will be presented for each study day by treatment group. Doses will be imputed as zero for study days where no use was reported, except for oral doses, where the Day 5 dose will be assumed for days out of hospital (e.g., Days 3 and 4). The total IV dose recorded from Day 1 (reflecting use on Day 0) through Day 5 will be summed, and

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compared between treatment groups using a two-sided two-sample t-test. 10.3.7 Change in weight from randomization daily through Day 5 and Day 14

Changes in body weight from randomization (baseline/Day 0) will be computed for each study day for each subject by subtracting the baseline weight from the post-baseline weights for each subject. Treatment groups will be compared for changes in body weight at each post-baseline study day using a two-sided two-sample t-test. Missing values will be imputed as described in Section 8.3.6. 10.3.8 Length of hospital stay

Length of stay will be defined as the date of discharge from the initial hospitalization minus the baseline date plus one. Subjects still in the hospital at Day 60 will be censored at Day 60. Analysis conventions described in Section 8.3.7 will be followed. Subjects who die during the initial hospitalization will be assigned the maximum length of stay over all subjects (including values censored at Day 60) plus 1 day. Treatment groups will be compared using a Wilcoxon rank sum test. 10.3.9 All cause death or rehospitalization due to heart failure or renal failure through Day 60

The time from the baseline date to the first event will be used for analysis. The CEC classification of the primary reason for rehospitalization (heart failure or renal failure) will be used for the final analyses. Subjects without an event will be censored at the earlier of the last contact date or Day 60. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test. Further, the estimate of the hazard ratio (from Cox regression) with a 95% confidence interval for comparing treatment groups will be presented.

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10.3.10 Reasons for All Rehospitalizations per Investigator and CEC

Summary tables will present the specific reasons for rehospitalization per the Investigator and CEC determinations with the number of subjects who experienced each reason and Kaplan-Meier estimates of the 60-day event rates, as appropriate, for the ITT and FAS analysis sets.

10.3.11 Causes of Death per Investigator and CEC

Summary tables will present the specific causes of death per the Investigator and CEC determinations with the number of subjects who experienced each death cause and Kaplan-Meier estimates of the 60-day and 180-day event rates, as appropriate. In computing the event rates, subjects who die from a cause other than the cause of interest will be censored on the day of death.

Cause-specific mortality through Day 60 according to the investigator and according to the CEC will be presented in the ITT and Safety analysis sets. Cause-specific mortality through Day 180 using the ‘best available’ classification (CEC where adjudicated and investigator where not adjudicated) will also be presented in the ITT, FAS, and Safety analysis sets.


Days alive and out of hospital will be computed as 31 days minus the days in-hospital through Day 30 (including the index hospitalization and any rehospitalizations) minus the days following a death prior to Day 30. Days in hospital will be computed for the index hospitalization as the discharge date minus the baseline date plus one, and for rehospitalizations as the discharge date minus the presentation date plus one. Subjects with incomplete follow-up will have the average proportion of days alive and out of hospital applied to the missing follow-up period. Treatment groups will be compared using a Wilcoxon rank sum test. In addition, the median of differences and corresponding 95% CI will be presented using the Hodges-Lehmann estimator of shift. 10.3.13 Cardiovascular death through Day 180

The time from baseline to the event will be used for analysis. The CEC classification of the cause of death will be used for events up to Day 60 and the investigator’s classification will be used for events from Day 60 to Day 180. Subjects without an event will be censored at the earlier of the last contact date or Day 180; subjects who die from non-cardiovascular causes will be censored at the time of death. Kaplan-Meier estimates of event rates will be presented, and treatment groups will be compared using a log-rank test. Kaplan-Meier plots will be presented. Hazard ratios and 95% CIs will be estimated from a Cox regression model. 10.3.14 Other heart failure signs and symptoms over time

Scores will be imputed as described in Sections and then Section 10.1.2 (where applicable for Likert scores). Scores and changes in scores for self-reported general well-being will be summarized for each assessment time point using appropriate descriptive statistics. The Likert scores will be presented as both categorical and continuous statistics. Change from baseline using VAS scores will be computed as the baseline value subtracted from the post-baseline

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values. Treatment groups will be compared with respect to change from baseline in VAS scores using two-sided two-sample t-tests; and Wilcoxon rank sum tests will be used for the Likert scores on a continuous range. In addition, treatment groups will be compared for the physician assessment scores for dyspnea on exertion, orthopnea, edema, rales, and jugular venous pulse using Wilcoxon rank sum tests. For each assessment, the scores will be presented as both categorical and continuous statistics. Change from baseline will be computed as the baseline value subtracted from the post-baseline values. Treatment groups will be compared with respect to change from baseline using Wilcoxon rank sum tests. 10.3.15 Days in the ICU/CCU

Data handling rules as described in section 8.3.8 will be applied. The days of the initial hospitalization from randomization spent in the ICU/CCU will be compared between treatment groups using the Wilcoxon rank sum test. 10.3.16 All cause death through Day 30

The time from baseline to the event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Kaplan-Meier estimates of event rates will be presented, and groups will be compared using a log-rank test. 10.3.17 All cause death or worsening heart failure or rehospitalization due to heart failure through Day 30

The time from baseline to the event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test.

10.3.18 Cardiovascular death or rehospitalization due to heart failure or renal failure through Day 30 The time from baseline to the first event will be used for analysis. Subjects without an event will be censored at the earlier of the last contact date or Day 30. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test. Hazard ratios and 95% CIs will be estimated from a Cox regression model.

10.3.19 Cardiovascular death or rehospitalization due to heart failure or renal failure through 30 days after discharge from the index hospitalization The time from discharge from the index hospitalization to the first event will be used for analysis. Subjects who were discharged alive will be included in the analysis; subjects who died before or

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on the same day as discharge, whose last contact was prior to discharge, or whose discharge date is missing will be excluded. Subjects without an event will be censored at the earlier of the last contact date, 30 days from discharge, or the date of the Day 60 follow-up. Kaplan-Meier estimates of rates of the composite event and its components will be presented, and groups will be compared using a log-rank test. Hazard ratios and 95% CIs will be estimated from a Cox regression model. Caution should be used in interpreting the results of this analysis, because randomization can no longer be relied upon to balance the characteristics of the treatment groups at the reference time point. 10.4 Exploratory Analysis of Multiple Endpoints

To evaluate the strength of the evidence for the efficacy of RLX030 therapy across multiple endpoints in this patient population, an average z score will be estimated and treatment groups compared with respect to this average z. Endpoints measured on different scales are transformed to a common metric – a z score. First, data from all treatment groups are pooled together. Second, time-to-event endpoints are transformed to log-rank scores; dichotomous, rank, or continuous variables are not transformed at this step. Third, z-scores are calculated for each subject for each endpoint by subtracting the overall means and dividing by the standard deviations for each endpoint. z-scores are aligned so that small values are ‘good’ and large values ‘bad’. The z-scores across the endpoint are averaged to obtain an average z-score for each subject. The treatment groups are then compared with respect to the average z scores using a Wilcoxon rank sum test.

The average z will be computed for the following 7 endpoints:

• Dyspnea VAS area under the change from baseline curve from baseline to Day 5 • Moderately or markedly better dyspnea at 6, 12, and 24 h by Likert scale • Time to worsening heart failure to Day 5 • All-cause death or rehospitalization for heart failure or renal failure through Day 60 • Days alive out of hospital to Day 60 • All-cause death through day 30

The analysis will be conducted in the ITT Set. Results will be presented graphically using a forest plot with estimated mean treatment differences in z scores and 95% CIs for each endpoint and for the average z score. 11. EXTENT OF EXPOSURE TO STUDY DRUG

For the ITT Set, baseline study drug administration details will be summarized by treatment group and overall for the time from presentation to randomization (hrs), time from randomization to study drug administration (hrs), study drug administered (yes/no), reason study drug not administered, actual study drug received, and the number of days infused (one or two).

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Actual study drug administration details will be summarized using the Safety population for the two time points as Baseline/Day 0 and 24 hours/Day, and together across the 48 hour infusion period. The duration of study drug administration (in hours) and the total volume of study drug administered (estimated from the total time and rate of infusion) will be summarized by treatment group and overall. In addition, the number of subjects whose study medication dose was lowered and the number of subjects whose study medication was discontinued prematurely, and the reason for discontinuation will be summarized by treatment group and overall. 12. CONCOMITANT MEDICATION

12.1 IV Inotrope/Vasoactive Medications

The use of all concomitant medications will be summarized using the ITT Population. For IV Inotrope/Vasocactive Medications, the data as reported by the investigator will be summarized for each of the time periods: 24 hours/Day 1, 48 hours/Day 2, Day 3, Day 4, Day 5, Day 14, and Day 60. Each time point reflects medication use within the preceding 24 hours. In addition to use at each reported time point, cumulative use reported at 24 and 48 Hours, and at 24 Hours to Day 5 will be summarized. For each study day, the number and percent of subjects who took each medication (as pre-printed on the CRF), and statistics summarizing the average dose given, will be presented. For subjects who took ‘other’ iv inotrope/vasoactive medications, only the number and percent of subjects who took these medications will be presented. 12.2 Other Concomitant Medications

The use of other concomitant medications is reported through Day 60. The data as reported by the investigator will be summarized for each of the time periods: 30 days prior to study drug initiation, Baseline/Day 0, 24 hours/Day 1, 48 hours/Day 2, Day 3, Day 4, Day 5, Day 14, and Day 60 and totals. Use of these medications will be summarized for each time point as the number and percent of subjects who took a medication in each class (as pre-printed on the CRF) in each treatment group and in the groups combined. In addition, cumulative use reported at 24 to 48 Hours, and from 24 Hours to Day 5 will be summarized. The average daily dose administered will be summarized at each time point for each medication within the ACE inhibitor, angiotensin inhibitor (ARB), and beta blocker classifications. (Only the number and percent of subjects who took ‘other’ ACEi, ARBs, or beta-blockers will be presented.) In addition, all ‘Other’ medications will be summarized for each time point as the number and percent of subjects who took a medication within each coded ATC class and generic name. The total dose of IV or oral loop diuretics administered during the initial hospitalization is also an additional efficacy outcome and its analysis is described in section 10.3.6.

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13. SAFETY ANALYSES

Safety will be assessed by comparing the RLX030 group to the placebo group with regard to the frequency of AEs, changes in clinical laboratory test results (chemistry, hematology, and urinalysis) and vital signs. All safety analyses will be presented for the Safety population. Estimates of Day 180 mortality will also be reported in the PPS1 and PPS2.

13.1 Adverse Events

The incidence of AEs recorded through Day 5 and the incidence of SAEs recorded through Day 14 will be presented for the Safety population. Incident AEs will be considered those AEs with an onset date and time after the initiation of study drug. Adverse events with an onset between informed consent and study drug initiation will be listed separately.

For presentation, adverse event verbatim text will be coded into a MedDRA (Medical Dictionary for Regulatory Activities) term and classified by MedDRA System Organ Class (SOC) and preferred term. A cross-reference listing, which will relate SOC, preferred term, and verbatim text will be provided. The incidence of all reported AEs will be summarized by system organ class and preferred term. A subject reporting the same event more than once will be counted once when reporting the number of subjects with that particular event. Disease-Related Events (DREs) will be indicated with an asterisk. A list of specified disease-related events (DREs) can be found in the protocol section 21.4.

SAEs will be summarized separately for the interim DSMB analyses by source: Safety Surveillance Database and CRF Database. SAEs will be reconciled between these two databases, and for the final report, SAE data will be summarized from only the CRF database (which in the final database will reflect all SAEs included in the Safety Surveillance Database).

In addition, AEs will be summarized by time period of onset: from study drug initiation to Day 5 and from Day 6 to Day 14. Additionally, a summary of AEs by preferred term and severity, using the worst reported severity grade for each event for the subject, will be provided. For analysis purposes, all AEs defined as “definite”, “probable” or “possible” will be considered as related. If the relationship to study drug is unknown or missing, the AE will be considered to be drug-related. All study-drug-related AEs, AEs leading to discontinuation of study drug, and study-drug-related SAEs, SAEs with an outcome of death, and SAEs leading to study drug discontinuation will be summarized by the number and percentage of subjects reporting particular events. Percentages will be based on the number of subjects in the safety population. For the analysis by time period of onset, the percentages will be based on the number of subjects in the safety population with the corresponding AE form completed. Odds ratios and associated 95% confidence intervals comparing RLX030 with placebo will be presented for those events with 5 or more events in the combined treatment groups.

In addition, adverse event terms identified in the RLX030 Development Safety Profiling Plan as representing potential compound and class related risks and general risks will be grouped

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according to the RLX030 Case Retrieval Strategy. The incidence (absolute and relative frequency) of each potential risk will be summarized by treatment group. In addition, subjects experiencing each of these risks will be listed. Such risks with adverse events grouped by the RLX030 Case Retrieval Strategy include (1) hypotension, (2) renal impairment, (3) hemoglobin/hematocrit transient decrease, (4) menorrhagia/metrorrhagia, (5) malignancy, (6) immunogenicity, and (7) hepatotoxicity.

Complete listings of all adverse events will be provided overall and by time period after the Day 180 database lock; serious adverse events will also be listed separately. For each adverse event listed, the verbatim and coded terms, the corresponding severity, relationship to study drug, seriousness (yes/no), and start and stop dates of the event will be specified and displayed by treatment group and subject number.

13.2 Mortality (Death to Day 180)

Product-limit (Kaplan-Meier) estimates of mortality rates at relevant time points (i.e., 5, 14, 30, 60, and 180 days) will be presented. Subjects who do not die by Day 180 will be censored at the earlier of the last contact date or Day 180. Treatment groups will be compared using a log-rank test. Further, the estimate of the hazard ratio (from Cox regression) with a 95% confidence interval for comparing treatment groups will be presented. Kaplan-Meier plots will be presented. Preliminary estimates of Day 180 mortality rates will be provided at the Day 60 analysis.

13.3 Clinical Laboratory Evaluations

Descriptive summaries of actual and change from baseline will be presented for all continuous clinical laboratory values by treatment and time point for the Safety population. Treatment groups will be compared for changes from baseline using both two-sided two-sample t-tests and Wilcoxon rank sum tests.

Clinical shift tables showing the pattern of change from baseline versus each post-baseline time point will be presented based on the cross-classification of the number of subjects with clinical laboratory values below (low), within (normal), or above (high) normal ranges for each laboratory test.

All laboratory data will be listed for each subject. Laboratory values that are outside the normal ranges will be flagged high (H) or low (L).

13.3.1 Creatinine and eGFR changese

The proportion of subjects with >= 0.3 mg/dL increase, >=0.5 mg/dL increase, >= 1.0 mg/dL increase and >50% increase from baseline in serum creatinine levels at each time point, and at any time point, will be presented. In addition, the proportion of subjects with >= 0.3 mg/dL increase, >=0.5 mg/dL increase, and >= 1.0 mg/dL increase at each of (a) Day 5, (b) Day 14, (c) Day 60, (d) Day 5 and Day 14, (e) Day 5 and Day 14 and Day 60 will be presented by treatment

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group and compared using a chi-square test. The proportion of subjects with decreases from baseline GFR, estimated using the sMDRD formula, of >25%, >40%, >50%, and >30 mL/min/1.73 m2 at each time point, and at any time point, will be presented. 13.3.2 Hemoglobin, hematocrit, and RBC count changes

The proportions of subjects with >20% decrease from baseline in hemoglobin, hematrocrit, and RBC count at each time point, and atany time point, will be presented by treatment group. 13.3.3 ALT, AST, alkaline phosphatase, and total bilirubin changes

The proportions of subjects with the following increases from baseline at each time point, and at any time point, will be presented by treatment group: (1) ALT or AST > 3xULN, (2) ALT or AST >5xULN, (3) ALT or AST >8xULN; (4) ALT or AST>3xULN and TB>2xULN; (5) TB>3xULN, and AST or ALT≤3xULN, and alk phos ≤1.5xULN; (6) AP>3xULN, and AST, ALT, and TB are within normal range. 13.4 Vital Signs

Descriptive statistics will be provided for the actual and change from baseline values at each assessment time point for pulse, respiratory rate, body temperature and weight. Treatment groups will be compared for changes from baseline using the two-sided two-sample t-test. 13.5 Blood Pressure Decrease Events

The number and percentage of subjects who experience a confirmed blood pressure decrease event during study drug administration will be provided. Among subjects who experience a confirmed blood pressure decrease event, the events will be further characterized with respect to impact on study drug administration (dose decreased or study drug discontinued), the timing of event onset relative to study drug initiation, the magnitude of the blood pressure decrease at its onset and the trough blood pressure recorded, the blood pressure measurements after study drug discontinuation, outcome, treatment(s) required, and whether considered symptomatic, i.e. an AE. In addition, these summaries will be provided separately for those confirmed events that resulted in study drug dose reduction, and those that resulted in study drug discontinuation.

These summaries will also be provided for the subsets of subjects who were and were not administered IV nitrates within the first 48 hours following randomization. Furthermore, the possible interaction between treatment and the administration of IV nitrates within the first 48 hours following randomization on confirmed blood pressure decrease events will be examined using a logistic regression model. The logistic regression model will include treatment, IV nitrate use, and the treatment-by-IV nitrate use interaction. Separate two-sided 95% confidence limits about the treatment effects (expressed as odds ratios) for the two IV nitrate use responses

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will be presented along with the overall result.

13.6 Pregnancy Test Results

Any pregnancies reported will be listed.

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14. REPORTING CONVENTIONS

The following reporting conventions will be adopted for the SAP. These conventions will enhance the review process and help to standardize presentation with common notations.

14.1 General Reporting Conventions

• All tables and data listings will be presented in Landscape Orientation, unless presented as part of the text in a CSR, in Courier New, 9 point font.

• Figures will be presented in Landscape Orientation, unless Portrait Orientation suggests that the information presented is easier to interpret.

• Legends will be used for all figures with more than one variable or item displayed.

• Figures will be in black and white (no color). Symbols on figures will not be filled. Lines should be wide enough to see the line after being copied.

• Specialized text styles, such as bolding, italics, borders, shading text will not be used in tables, figures, and data listings unless they add significant value to the table, figure, or data listing.

• Only standard keyboard characters should be used in tables and data listings. Special characters, such as non-printable control characters, printer specific, or font specific characters, will not be used on a table, figure, or data listing. Hexadecimal characters are allowed (e.g., µ,α,β).

• Sentence case (capitalization of the initial word) will be used throughout all tables, figures, and data listings.

• All titles will be centered on a page. The first title line will be the number of the table, figure, or data listing. The second (and if required, third) line will be the description of the table, figure, or data listing. The ICH numbering convention will be used for all TFLs.

• All footnotes will be left justified and at the bottom of a page. Footnotes must be present on the page where they are first referenced.

• The order of presentation of the two treatments for all tables, figures, and data listings will be placebo followed by RLX030.

• Missing values (other than DDMMMYYYY) for both numeric and character variables will be presented as blanks in a table or data listing. Missing date/month/year will be presented as dash (‘-‘) for each space.

• All date values will be presented as DDMMMYYYY (e.g., 29AUG2009) format. A four-digit year is preferred for all dates.

• All observed time values will be presented using a 24-hour clock HH:MM format (e.g., 11:26).

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• Time durations will be reported in mixed HHhr MMm notation (e.g., 5h 32m). The use of decimal notation to present time durations should be avoided (e.g. 0.083h = 5m) unless it is necessary to show the computation of time differences in a table, figure, or data listing, in which case both notations may be used to display the time duration.

• All tables, figures, and data listings will have the name of the program and a date/time stamp on the bottom of each output.

• Each table, figure, and data listing will have “Page x of y” presented on the bottom right-hand side of each page.

14.2 Population Summary Conventions

• Population(s) represented on the tables will be clearly identified in the last title of the Table as “Population: <name of population>” and will be identical in name to that identified in the protocol or SAP.

• Consistent terminology will be used to identify a population. Common nomenclature will include (a) Intent-to-Treat Set, (b) Safety or Safety Set, and (c) Full Analysis Set.

• Sub-population(s) or special population(s) descriptions will provide sufficient detail to ensure comprehension of the population (e.g., ITT Females) used for analysis in a table, figure or a data listing.

• Population sizes will be presented for each treatment and overall as totals in the column header as (N=xxx), where appropriate.

• Population sizes shown with summary statistics are the samples sizes (n) of subjects with non-missing values.

• All population summaries for categorical variables will include categories that the subjects had a response in these categories. Percentages corresponding to null categories (cells) will be suppressed.

• All calculated percentages between 1% and 99% will be rounded to one decimal place. A percentage of 100% will be reported as 100%. Any computation of percent that results in 0% is to be reported as 0 (no % sign).

• Population summaries that include p-values will report the p-value to five decimal places (e.g., 0.00001) for comparisons involving the primary and secondary efficacy endpoints and to three decimal places with a leading zero (0.001) for all other comparisons. All p-values reported on default output from statistical software (i.e., SAS® Software) may be reported at the default level of precision.

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15. CHANGES FROM THE PROTOCOL-SPECIFIED ANALYSES

Changes to the analysis plan subsequent to Protocol Amendment 2 were incorporated in Protocol Amendment 3. (Protocol Amendment 4 introduced an ECG substudy but no changes to the planned analysis were made.) SAP version 2.0 was updated to incorporate all planned analyses described in Protocol Amendments 3 and 4. Protocol Amendment 5 incorporated additional efficacy outcomes and refinements to the SAP; this SAP is consistent with the analyses described in Protocol Amendment 5. This SAP incorporates SAPs detailing the analyses of the pharmacoeconomic, pharmacokinetic, and biomarker data and the ECG substudy.

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16. REFERENCES

• ASA. (1999) Ethical Guidelines for Statistical Practice. Prepared by the Committee

on Professional Ethics, August 7, 1999.

• Decker, C. Calculating a Nonparametric Estimate and Confidence Interval Using SAS® Software.

• Fleiss, J.L., Levin, B., and Paik, M.C. (2003), Statistical Methods for Rates and Proportions. Third Edition, New York: John Wiley & Sons.

• Hochberg, Y., and A.C. Tamhane. 1987. Multiple Comparison Procedures. New York: John Wiley and Sons.

• Hodges, J.L. and Lehmann, E.L. (1963). Estimates of location based on rank tests, The Annals of Mathematical Statistics, 34: 598:611.

• SAS Institute Inc., Logistic Regression Examples Using the SAS System, Version 6, First Edition, Cary, NC: SAS Institute Inc., 1995. 163 pp.

• US Federal Register. (1998) International Conference on Harmonization; Guidance on Statistical Principles for Clinical Trials. Department of Health and Human Services: Food and Drug Administration [Docket No. 97D-0174]. Federal Register Volume 63, Number 179, pages 49583-49598. September 16, 1998.

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17. APPENDIX A. STATISTICAL ANALYSIS PLAN FOR PHARMACOECONOMIC EVALUATION

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18. APPENDIX B. STATISTICAL ANALYSIS PLAN FOR PHARMACOKINETIC ANALYSIS

Clinical Development

Relaxin/RLX030A

RLX.CHF.003: A Phase II/III, Multicenter, Randomized, Double-blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety

of Relaxin in Subjects With Acute Heart Failure RAP Module: Statistical Methodology for Pharmacokinetic and

Immunogenicity Analysis

Author(s): Elaine Unemori, Yinuo Pang

Document type: RAP Documentation

Document status: Final

Release date: 13 June 2012

Property of Novartis Confidential

May not be used, divulged, published or otherwise disclosed without the consent of Novartis

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Table of Contents List of Abbreviations .............................................................................................76

1 INTRODUCTION TO RAP................................................................................77

1.1 Scope ...............................................................................................................77

2 STATISTICAL ANALYSIS PLAN ...................................................................78

2.1 Introduction .....................................................................................................78 2.2 Study objectives ..............................................................................................78 2.3 Study design and treatment .............................................................................79 2.4 Statistical methods ..........................................................................................80

2.4.1 Analysis sets...................................................................................80 2.4.2 Statistical methods for analysis of RLX030 PK parameters ..........80 2.4.3 Statistical methods for anti-RLX030 antibody analysis ................83

2.5 References .......................................................................................................84

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List of Abbreviations Acronym BAPK Bio-Analytics & Pharmacokinetics BAPKDBL BAPK Database Lock BA Bioanalytics BLQ Below level of quantitation CDBL Clinical Database Lock CIS Clinical Information Sciences CP Clinical Pharmacology CPE Clinical Pharmacology Expert CPL Clinical Program Leader CPS Clinical Pharmacology Scientist CSE Clinical Safety and Epidemiology CSP Clinical Study Protocol CSR Clinical Study Report ED-CTL Clinical Trial Leader eGFR Estimated Glomerular Filtration Rate CTT Clinical Trial Team DBL Database lock D5W 5% Dextrose in Water IIS Integrated Information Sciences FIR First Interpretable results FPFV First Patient First Visit KPI Key performance indicator LDG Last Data Generated LLOQ Lower Limit of Quantitation LPLV Last Patient Last Visit LPK Lead Pharmacokineticist MDRD Modification of Diet in Renal Disease MW Medical Writer PD Pharmacodynamics PK Pharmacokinetics RAP Report and Analysis Preparation SBP Systolic Blood Pressure TDM Trial Data Manager TLG Tables, listings, graphs TMF Trial Master File TS Translational Science

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1 INTRODUCTION TO RAP

1.1 Scope

This document contains detailed information to aid in the production of Statistics & Programming input into the PK Appendix of the Clinical Study Report (CSR), including summary tables, figures and listings for PK analysis for trial “RLX.CHF.003 RELAX-AHF.”

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2 STATISTICAL ANALYSIS PLAN

2.1 Introduction

The pharmacokinetics (PK) of RLX030 have been evaluated over a wide range of doses from 6 to 960 µg/kg/day following multiple routes of administration in previous human studies. RLX030 elimination was shown to be fairly rapid, with most of the RLX030 being cleared within 1-2 hours of intravenous (i.v.) bolus injection or after the end of the infusion. An effective half-life of approximately 1.1 hours following i.v. bolus administration (based on the mean residence time) has been observed. RLX030 serum clearance was estimated to fall into the range of 89-229 mL/kg/hr. In general, a dose-independent PK was observed across the wide range of doses tested.

Antibodies against RLX030 have not been observed in subjects treated with RLX030 via the i.v. route or via the subcutaneous (s.c.) route for less than 2 weeks. Anti-RLX030 antibodies were only observed in subjects, mostly scleroderma patients, following long-term continuous s.c. administration for up to 6 months, and were found to affect RLX030 PK but to be non-neutralizing. Although increased serum concentrations of RLX030 were observed in the presence of anti-RLX030 antibodies, no toxicity or altered pharmacology was observed with increased RLX030 exposure.

In the RELAX-AHF study, RLX030 is administered via continuous i.v. infusion for 48 hr. Steady state concentrations (Css) and clearance of RLX030 will be calculated and are intended to provide confirmation that the appropriate dose of RLX030 has been administered and the PK is generally consistent with those observed in the other RLX030 clinical studies in heart failure patients. The potential effect of renal impairment, concomitant medication use, and baseline (intrinsic) patient characteristics on RLX030 clearance will be assessed. Finally, RLX030 Css and clearance will be summarized in the four weight ranges, specified in the Pharmacy Manual (Appendix I), that were used for study drug compounding.

Although it is not anticipated that RLX030 administered for 48 hr via the i.v. route will elicit anti-RLX030 antibodies, the presence of these antibodies will be assessed in these patients following completion of dosing.

This analysis plan describes the statistical analyses and the presentation of data related to RLX030 PK and anti-RLX030 antibodies in the RELAX-AHF study.

2.2 Study objectives

Primary objectives: • To characterize the Css and clearance of RLX030 based on serum concentrations of

RLX030 determined at 24 hr following the start of study drug infusion in patients with AHF enrolled in RLX.CHF.003 RELAX-AHF

• To summarize the frequency of anti-RLX030 antibody-positive patients at Day 14 (and potentially Day 60) following the start of study drug infusion in patients with AHF enrolled in RLX.CHF.003 RELAX-AHF

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Secondary objectives:

• To quantify the effect of renal impairment, measured by eGFR, on RLX030 Css and clearance

• To summarize RLX030 Css and clearance in subgroups defined by use of concomitant medications

• To summarize RLX030 Css and clearance according to baseline (intrinsic) characteristics, including age, gender, and race

• To summarize RLX030 Css and clearance at 24 hr following the start of study drug infusion in each of the 4 weight ranges specified for dosing in the Pharmacy Manual

2.3 Study design and treatment

The study for which this PK analysis plan is written is a Phase III multicenter, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of RLX030 in subjects with acute heart failure. Approximately 1160 subjects are to be enrolled in the study, with 2 parallel groups (placebo and RLX030 at a dose of 30 µg/kg/day) of approximately 580 subjects each. RLX030 or placebo is administered via continuous i.v. infusion for 48 hr. Patients randomized to the placebo arm are to receive standard therapy for AHF plus placebo. Patients randomized to the active RLX030 study drug arm are to receive standard AHF therapy plus active RLX030 study drug. Subjects are required to have an estimated glomerular filtration rate (eGFR) (calculated according to the MDRD formula) of 30-75 mL/min/1.73m2. Compounding of study drug is performed according to four subject weight ranges specified in the Pharmacy Manual (Appendix I): 40-59 kg, 60-74 kg, 75-114 kg, and 115-160 kg. All subjects are administered a constant volume of study drug compounded in D5W per unit of time, i.e. 10 mL/hr.

Study drug decrease or discontinuation rules based on decreases in systolic blood pressure (SBP) are included in the protocol. If at any time during dosing, the subject’s SBP is decreased by > 40 mm Hg from baseline but is > 100 mm Hg in 2 consecutive measurements 15 min apart, the study drug infusion rate will be decreased by 50% for the remainder of the study drug administration. Study drug administration will continue for 48 hours unless the subject’s SBP at any time during dosing in 2 consecutive measurements, 15 min apart, is <100 mm Hg, at which time the study drug infusion will be permanently terminated.

Although no formal PK analyses are to be performed, RLX030 concentrations will be quantified using serum samples drawn at baseline and at 24 hr after initiation of study drug dosing to estimate Css and to confirm that the appropriate dose was administered. Serum samples collected at 48 hours after the start of study drug infusion and at Days 3, 4, 5, 14 and 60 may also be used for analysis of RLX030 or factors that have the potential to affect RLX030 PK, such as anti-RLX030 antibodies. As such, samples from baseline, Day 14 and if needed, Day 60, time points will be used for antibody analysis.

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2.4 Statistical methods

2.4.1 Analysis sets

Full Analysis Set (FAS) (PK): For analysis of RLX030 PK, all subjects who received any study drug and have had a RLX030 measurement performed will be included in the evaluation. In subjects for whom the actual treatment received does not match the treatment assignment, the actual treatment received will be used for the analysis.

Efficacy Analysis Set: RLX030 PK will also be summarized in all subjects who received at least 24 hr of study drug and have had a RLX030 measurement performed. In subjects for whom the actual treatment received does not match the treatment assignment, the actual treatment received will be used for the analysis.

FAS (Ab): For analysis of anti-RLX030 antibodies, all subjects who received any study drug and have had an anti-RLX030 antibody measurement performed will be included in the evaluation. In subjects for whom the actual treatment received does not match the treatment assignment, the actual treatment will be used for the analysis.

2.4.2 Statistical methods for analysis of RLX030 PK parameters

To account for endogenous circulating relaxin that may exist in AHF patients (Dschietzig et al., 2001, Fisher et al., 2001), RLX030 will be measured at baseline, as well as at the 24 hr time point. RLX030 concentrations below the LLOQ (lower limit of quantitation) of the assay will be replaced by the value representing BLQ (below the limit of quantification).

Descriptive statistics for relaxin/RLX030 concentrations at baseline and at the 24 hr time point will include arithmetic mean, geometric mean with its corresponding 90% confidence interval , median, SD, and CV, min and max. A value of “0” will be used for samples that are BLQ in these calculations. RLX030 Css will be estimated using the serum concentration determined at the 24 hr time point adjusted with the baseline value. It is anticipated that baseline concentrations of endogenous relaxin will be at or near the LLOQ of the assay. Css will be used to calculate RLX030 clearance for each subject as follows:

Clearance (mL/hr/kg) =1x10^3 * Rate of infusion (µg/kg/hr)/Css (ng/mL)

RLX030 will be assayed in Day 14 and if needed, Day 60 samples, in order to evaluate the possibility that there is interference from RLX030 in assays for anti-RLX030 antibodies (refer to Section 2.4.3).

Effect of renal impairment on RLX030 PK This study is designed to include only patients with mild to moderate renal impairment at screening, i.e. those with an eGFR in the range of 30-75 mL/min/1.73 m2. The effect of eGFR

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on RLX030 clearance will be determined, based on eGFR calculated according to the modified MDRD formula as follows:

eGFR (mL/min/1.73 m2) = 175 x (Scr) ^-1.154 x (Age) ^-0.203 x (0.742 if female) x (1.212 if black)

where Scr is the serum creatinine in mg/dL and age is in years.

Age at screening and serum creatinine measured at baseline will be used for this calculation. eGFR will be used as both categorical and continuous variables in the analyses to determine its potential effect on RLX030 clearance.

Patients will be classified as having mild or moderate renal impairment at baseline based on the following criteria:

18.1.1.1.1.1 Table 2-1 Definitions of renal impairment Group Description EMA Definition1

GFR (mL/min/1.73m2) FDA Definition2

GFR (mL/min/1.73m2)

1 Normal renal function >80 >90

2 Mild renal impairment 50 - 80 60 - 89

3 Moderate renal impairment 30 - <50 30 - 59

4 Severe renal impairment <30 15 - 29

5 End stage renal disease (ESRD) Requiring dialysis <15 not on dialysis; Requriing dialysis

Sources: 1CHMP/EWP/225/02 Note for Guidance on the Evaluation of the Pharmacokinetics of Medicinal Products in Patients with Impaired Renal Function, December 2004; 2FDA Guidance for Industry, Pharmacokinetics in Patients with Impaired Renal Function, 2010.

Descriptive statistics of RLX030 concentration and clearance will be summarized in the mild and moderate renal impairment groups according to both EMA and FDA definitions (Table 2-1). In addition, 90% confidence intervals about the geometric mean ratio of RLX030 clearance (moderate to mild renal impairment) will be presented (CHMP/EWP/225/02, 2004). Summary data of the other renal impairment subgroups will not be presented as it is anticipated that the number of subjects with renal function at baseline outside of these two categories will be very small, consistent with their renal function at screening.

Results will also include a graphical presentation of the relationship between eGFR and RLX030 clearance as a continuous variable (CHMP/EWP/225/02, 2004).

Effect of concomitant medications on RLX030 PK Although the likelihood of drug-drug interactions is lower for therapeutic proteins than for conventional drugs, potential effects of concomitant medications commonly administered to patients with AHF on RLX030 clearance will be examined. Drugs are reported according to

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pharmacology groups pre-specified on the Case Report Forms (e.g. ACE inhibitors, IV loop diuretics, etc.) and collection of concomitant medication usage occurs twice in the relevant time period: at the baseline time point, which includes the prior 24 hr, and at the 24 hr time point, which includes the prior 24 hr. Thus, usage of medications administered from 24 hr prior to the baseline assessment, which occurs immediately before start of study drug infusion, to the 24 hr time point at which the PK measurement is being made, will be captured.

Commonly used concomitant medication groups, whose constituent medications have been administered from 24 hr prior to the pre-treatment baseline assessment and 24 hr following study drug initiation, will be included in the analysis. Subjects will be separated into subgroups based on use or lack of use of drugs within these groups during this time frame. RLX030 clearance will be summarized using descriptive statistics for these subgroups.

Medication groups to be included in these analyses may include the following, depending on frequency of use:

1. IV vasodilators/inodilators: nitroglycerin, nitroprusside, nesiritide, levosimendan, milrinone, enoximone, norepinephrine, epinephrine, dopamine, dobutamine

2. IV loop diuretics: furosemide, bumetanide, torsemide, ethacrynic acid

3. Aldosterone inhibitors: spironolactone

4. β-blockers: atenolol, betaxolol, bisoprolol, metoprolol, carvedilol, labetalol

5. ACE inhibitors/ARBs: Benazepril, captopril, enalapril, fosinopril, lisinopril, zofenopril, moexipril, quinapril, ramipril, perindopril, candesartan, eprosartan, irbesartan, losartan, olmesartan, valsartan

6. Inotrope: Digoxin

7. Calcium channel blockers: felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nitrendipine, diltiazem, verapamil, amlodipine

Results will be summarized for each subgroup and reported as 90% confidence intervals about the geometric mean ratio of RLX030 clearance in subgroups with and without the concomitant medications for each of the seven concomitant medication categories listed above. Comparisons indicating a twofold or greater increment in systemic exposure in subgroups receiving the concomitant medication would indicate the presence of a potential drug-drug interaction (FDA guidance, In vivo drug metabolism/drug interaction studies—study design, data analysis, and recommendations for dosing and labeling, 1999).

Effect of baseline (intrinsic) characteristics on RLX030 PK In order to assess the potential effects of baseline patient characteristics on RLX030 clearance, the influence of age, gender and race on RLX030 Css and clearance will be evaluated (ICH M4 Common Technical Document for the Registration of Pharmaceuticals for Human Use – Efficacy). Descriptive statistics of RLX030 Css and clearance will be provided for subgroups

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of age, gender and race. Additionally 90% confidence intervals for the geometric mean ratio of RLX030 clearance may be constructed as appropriate to evaluate the effect of baseline characteristics.

Effect of compounding study drug within specified weight ranges on RLX030 PK In order to enable study drug compounding at study sites, patient body weights were grouped into 4 weight ranges and compounding standardized within these groups: 40-59 kg, 60-74 kg, 75-114 kg, and 115-160 kg. Descriptive statistics of RLX030 Css and clearance in patients grouped according to these weight ranges, as well as overall, will be summarized. Additionally 90% confidence intervals for the geometric mean ratio of RLX030 clearance may be constructed as appropriate to evaluate the effect of drug compounding.

2.4.3 Statistical methods for anti-RLX030 antibody analysis

Anti-RLX030 antibodies will be measured in serum samples collected at the Day 14 time point. To account for individual variability in background of each subject and to check for endogenous interference and/or pre-existing anti-RLX030 antibodies, anti-RLX030 antibodies will also be measured at baseline. Any subject with a positive antibody result at Day 14 will have their Day 60 sample assayed for anti-RLX030 antibodies, as well. Frequencies and percentages will be computed for occurrence of anti-RLX030 antibodies at each time point. Listings for subjects with a positive screening result at any time point will include subject number, time point, screening result, immunodepletion result, titer, and neutralizing antibody result.

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2.5 References

Dschietzig T, C. Richter, C. Bartsch, M. Laulel, F.P. Armbruster, G. Baumann, K. Stangl. The pregnancy hormone relaxin is a player in human heart failure. FASEB J. 2001; 15:2187-2195.

Fisher, C., S. Al-Benna, A. Kirk, J.J. Marin, J.J.V. McMurray. Transcardiac and transpulmonary gradients in the putative new cardiovascular hormone relaxin. Heart 2003; 89:789-790.

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19. APPENDIX C. STATISTICAL ANALYSIS PLAN FOR BIOMARKER EVALUATION

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20. APPENDIX D. STATISTICAL ANALYSIS PLAN FOR ELECTROCARDIOGRAM SUBSTUDY

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