The Clinical Study of the Safety and Efficacy of Istaroxime in Treatment of Acute Decompensated Heart Failure - A Multicenter, Randomized, Double-blind, Placebo Controlled, Parallel Group Clinical Study
Phase: PHASE2
Conditions: Acute Decompensated Heart Failure
Sponsor: Windtree Therapeutics

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
1
CVie Therapeutics Company Limited.  
Unit110-111, Bio-informatics center, 
No2. Science Park West Avenue, 
Hong Kong Science Park, Shatin, N.T. HK –  
 
 
 
Project N0. CVT-CV-002 
General Protocol Amendment N 2 
Final Version with SC and DSMB Members  
 
CFDA Drug Clinical Trial  
Original number: 20130181 
Admission number: JXHL1300197国       
Approval document no.: 2015L00219 
  
EUDRACT No.: 2013-000540-26 
 
 
CLINICAL STUDY PROTOCOL OF ISTAROXIME 
 
The clinical study of the safety and efficacy 
of Istaroxime in Treatment of Acute Decompensated Heart 
Failure 
- A multicenter, randomized, double-blind, placebo 
controlled, parallel group clinical study 
CONFIDENTIAL 
Final Version, 7 February, 2013 
Amended Final Version 08 April 2013 
Amended Final Version 23 July 2015 
Amended Final Version 30 November 2017 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
3
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PROTOCOL APPROVAL BY THE PRINCIPAL INVESTIGATOR  
 
I, the undersigned ____________________________________________________ 
Last Name   
 
 
 
 
 
First Name  
 
Principal Investigator at the following site: 
SITE _______________________________________________________________ 
 
 
declares:  
 
to have read carefully and thoroughly the Study Protocol:  
 
CODE: CVT- CV-002  
 
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Approved by the Sponsor: CVie Therapeutic Company Limited, and 
by the Chair of Steering Committee: Dr. JUN Huang 
  
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to agree to the above-mentioned Protocol  
x
to conduct the Study in accordance with Study Protocol and in agreement with the 
Sponsor’s and the Study Chairman’s requests.  

Signing this document I declare to have read the paragraph relevant to Study 
acknowledgement and confidentiality and authorise CVie Therapeutics Company 
Limited, to record my data on a computerised archive containing all the data pertinent 
to the Study.  
 
 
Signature _________________________________    Date: ___/____/___ 
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Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
4
GENERAL INFORMATION 
Protocol No.: 
 
 
CVT-CV-002  
Title:   
 
 
 
The clinical study of the safety and efficacy 
of Istaroxime in Treatment of Acute Decompensated Heart 
Failure—A multicenter, randomized, double-blind, placebo 
controlled, parallel group clinical study 
Date and Version:   
Amended Final Version 30 November 2017 
Study Sponsor: 
   
CVie Therapeutics Company Limited 
Unit110-111, Bio-informatics center, No2. Science 
ParkWestAvenue, Hong Kong Science Park, Shatin, N.T. HK  
 
Product: 
 
 
   
Istaroxime (PST2744) 
 
Pharmaceutical form:  lyophilized powder for intravenous infusion 
Dosage:  
 
 
   
0.5-1.0 µg/kg/min 
Control Drug:  
   
Placebo 
Pharmaceutical form:  lyophilized powder for intravenous infusion 
 
Chair of Steering Committee for China: Prof. JUN Huang, MD,  
Jiangsu Province Hospital, Nanjing 
 
Co-Chair of Steering Committee for Italy: Prof. METRA Marco, MD,  
Spedali Civili, Brescia 
 
Sponsor’s Medical Expert:           Prof. Giuseppe Bianchi, MD 
CVie Therapeutics Company Limited 
Phone +39 348 8545560 +39 02 745569 p.zza 
Adigrat 4, 20133 Milan, ITALY  
 
 
 
 
 
 
 
 
 
 
The information in this protocol is confidential. It is provided to every potential Investigator 
and his/her team, as well as to the Institutional Review Board, that must statute on the 
trial. No information can be provided to a third party without the written agreement of CVie 
Therapeutics, except for the medical personnel directly concerned by the trial and for the 
information needed by the patient and his/her family. 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
5
 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS 
 
 
 
 
 
 
 
 
 
 
 
 
8 
 
SYNOPSIS  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10 
 
1. INTRODUCTION 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
18 
 
1.1 Indications  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
18 
 
1.2 Pharmaceutical and background information  
 
 
 
 
 
 
19 
 
2. STUDY OBJECTIVE 
 
 
 
 
 
 
 
 
 
 
 
 
 
24 
 
3. STUDY DESIGN  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24 
 
4. STUDY POPULATION  
 
 
 
 
 
 
 
 
 
 
 
 
25 
 
4.1 Inclusion Criteria 
 
 
 
 
 
 
 
 
 
 
 
 
 
25 
 
4.2 Exclusion Criteria 
 
 
 
 
 
 
 
 
 
 
 
 
 
26 
 
5. STUDY PRODUCTS AND DOSE REGIMEN  
 
 
 
 
 
 
 
27 
 
5.1 Test Drug 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
27 
 
 
5.1.1 Istaroxime  
 
 
 
 
 
 
 
 
 
 
 
 
 
27 
 
 
5.1.2 Placebo 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
28 
 
5.2 Drug Blind Code 
 
 
 
 
 
 
 
 
 
 
 
 
 
28 
 
5.3 Drug Packaging  
 
 
 
 
 
 
 
 
 
 
 
 
 
29 
 
5.4 Emergency Envelope/Emergency Unblinding Card 
 
 
 
 
 
31 
 
5.5 Drug Distribution 
 
 
 
 
 
 
 
 
 
 
 
 
 
32 
 
5.6 Dosage Regimen  
 
 
 
 
 
 
 
 
 
 
 
 
 
32 
 
 
5.6.1 Method of administration and compliance  
 
 
 
 
 
33 
 
5.7 Concomitant Medication  
 
 
 
 
 
 
 
 
 
 
 
34 
 
6. CLINICAL ENDPOINTS  
 
 
 
 
 
 
 
 
 
 
 
 
35 
 
6.1 Efficacy Endpoints  
 
 
 
 
 
 
 
 
 
 
 
 
35 
 
6.2 Safety Endpoints 
 
 
 
 
 
 
 
 
 
 
 
 
 
36 
 
6.3 PK Parameters  
 
 
 
 
 
 
 
 
 
 
 
 
 
37 
 
7. STUDY PROCEDURE 
 
 
 
 
 
 
 
 
 
 
 
 
 
38 
 
8. EFFICACY ASSESSMENTS  
 
 
 
 
 
 
 
 
 
 
 
42 
 
9. SAFETY ASSESSMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
42 

 
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10. REPORTING OF ADVERSE EVENTS  
 
 
 
 
 
 
 
 
43 
 
10.1 Definition of adverse event (AE) 
 
 
 
 
 
 
 
 
 
43 
 
10.2 Definition of Serious Adverse Event (SAE)  
 
 
 
 
 
 
44 
 
10.3 Relativity assessment of AE and study drugs 
 
 
 
 
 
 
45 
 
10.4 Severity assessment of Adverse Event 
 
 
 
 
 
 
 
 
46 
 
10.5 Reporting of SAE  
 
 
 
 
 
 
 
 
 
 
 
 
46 
       10.6 Reporting of Pregnancy 
 
 
 
 
 
 
 
 
 
 
 
47 
 
11. ADVERSE DRUG REACTION (ADR) 
 
 
 
 
 
 
 
 
 
47 
 
11.1 Definition of ADR  
 
 
 
 
 
 
 
 
 
 
 
 
47 
 
11.2 Reporting and monitoring of ADR  
 
 
 
 
 
 
 
 
47 
 
12. PHARMACOKINETIC ASSESSMENT  
 
 
 
 
 
 
 
 
48 
 
13. DATA MANAGEMENT  
 
 
 
 
 
 
 
 
 
 
 
 
48 
 
13.1 Filling the CRFs and its handover  
 
 
 
 
 
 
 
 
48 
 
13.2 Data entry and amendment  
 
 
 
 
 
 
 
 
 
 
48 
 
13.3 Locking the Database  
 
 
 
 
 
 
 
 
 
 
 
49 
 
14. STATISTICAL ANALYSIS  
 
 
 
 
 
 
 
 
 
 
 
49 
 
14.1 Datasets 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
49 
 
14.2 Statistical Plan  
 
 
 
 
 
 
 
 
 
 
 
 
 
50 
 
15. QUALITY CONTROL AND QUALITY ASSURANCE 
 
 
 
 
 
52 
 
15.1 Drug Accountability 
 
 
 
 
 
 
 
 
 
 
 
 
53 
 
16. MONITORING AND STUDY COMMITTEES 
 
 
 
 
 
 
 
53 
 
16.1 Monitoring  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
53 
 
16.2 Steering Committee 
 
 
 
 
 
 
 
 
 
 
 
 
53 
 
16.3 Data Safety Monitoring Board  
 
 
 
 
 
 
 
 
 
54 
 
17. ETHICS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
55 
 
18. RESPONSABILITY AND PUBLICATION  
 
 
 
 
 
 
 
55 
 
19. ANTICIPATED PROGRESS AND COMPLETATION  
DATE OF THE STUDY 
 
 
 
 
 
 
 
 
 
 
 
 
56 
 
20. REFERENCES  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
57 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
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21. DECLARATION SIGNATURE PAGE 
 
 
 
 
 
 
 
 
 
59 
 
22. APPENDICES  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
61 
 
22.1 Appendix A: Istaroxime Infusion Rate  
 
 
 
 
 
 
 
61 
 
 
 
 
 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
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LIST OF ABBREVIATIONS 
 
 
A 
Pick mitral flow velocity during atrial contraction 
Aa 
Atrial myocardial contraction velocity 
ADHF 
Acute decompensated heart failure 
AE 
Adverse event 
Ae 
Amount excreted unchanged into urine 
Ae% 
Percent of the dose excreted unchanged into urine 
ALAT 
Alanine aminotransferase 
ANOVA 
Analysis of variance  
ASAT 
Aspartate aminotransferase 
AUC 
Area under the curve 
AUC0-∞ 
Area under the concentration-time curve extrapolated to infinity 
AUC0-t 
Area under the curve over the time interval 0 to the time of last quantifiable 
concentration 
AUMC 
Area under the first moment versus time curve 
BNP 
Brain natriuretic peptide 
BP 
Blood pressure 
Bpm 
Beats per minute 
cTnT 
Cardiac Troponin T  
CHF 
Chronic Heart Failure 
CI 
Confidence interval 
Cl 
Clearance 
ClR 
Renal clearance 
Cmax 
Maximum concentration 
CMH 
Cochran-Mantel-Haenszel 
CRF 
Case Report Form 
CRO 
Contract Research Organization  
CRT 
Cardiac Resynchronization Therapy 
Ct 
Last quantifiable concentration  
CV 
Coefficient of variation 
Da 
Diastolic myocardial velocity 
DBP 
Diastolic blood pressure 
DSMB 
Data Safety Monitoring Board 
DRQ 
Data request Query  
E 
Pick mitral flow velocity during early rapid filling 
Ea 
Early diastolic myocardial relaxation velocity 
E/Ea 
ratio for estimation of LV filling pressure 
E/A 
ratio of the early over late filling velocity 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
e-CRF         
Electronic case report form  
EF             
Ejection fraction 
eGFR 
Estimated glomerular filtration rate  
GCP 
Good Clinical Practice 
Hb 
Hemoglobin 
HF 
Heart failure  
HR 
Heart rate 
ICD 
Implantable Cardioverter Defibrillator 
IUPAC 
International Union of Pure and Applied Chemistry 

 
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Amended Final Version (30 November 2017)  
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i.v. 
Intravenous 
ITT 
Intention-to-treat 
λz 
Terminal rate constant 
LC/MS/MS 
Liquid chromatography coupled to mass spectrometry 
LVEF 
Left ventricular ejection fraction 
MBP 
Mean blood pressure 
MedDRA 
Medical Dictionary for Regulatory Activities  
MRT 
Mean residence time 
NT-proBNP 
N-terminal brain natriuretic peptide precursor 
PCWP 
Pulmonary capillary wedge pressure  
PD 
Pharmacodynamics 
PK 
Pharmacokinetics  
PP 
Per protocol 
PR 
Interval between P and R waves  
QRS 
Interval between the beginning of Q wave and the end of S wave  
QT 
Interval between the beginning of Q wave and the end of T wave 
RR 
Interval between R and R waves  
Sa 
Systolic myocardial velocity 
SAE 
Serious adverse event 
SAS 
Safety analysis set 
SBP 
Systolic blood pressure 
SD 
Standard deviation 
SE 
Standard error 
SERCA2a 
Sarcoplasmic reticulum calcium ATPase isoform 2a 
SV 
Stroke Volume  
SVR 
Systemic vascular resistance 
t1/2 
Terminal half-life 
Tinf 
Infusion duration 
tlast 
Time of last quantifiable concentration 
tmax 
Time to maximum concentration 
TSH 
Tyroid Stimulating Hormone 
URL 
Upper Reference Limit 
Vss 
Volume of distribution at steady-state 
VAS 
Visual Analog Scale 
Vz 
Volume of distribution during the terminal phase 
WHO 
World Health Organization 
WHO-DD 
World Health Organization Drug Dictionary 
 
 
 
 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
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SYNOPSIS  
Title 
The clinical study of the safety and efficacy of Istaroxime in Treatment of 
Acute Decompensated Heart Failure - A multicenter, randomized, double-
blind, placebo controlled, parallel group clinical study. 
 
Indication 
Acute Decompensated Heart Failure (ADHF) 
Objective 
To Assess the safety, tolerability and efficacy of two different doses of 
istaroxime (0.5 and 1.0 µg/kg/min), a new agent with lusitropic and 
inotropic activities that improves the cardiac contraction -
relaxation cycle. The 2 doses of istaroxime (0.5 and 1.0 µg/kg/min) will 
be infused i. v. for 24 hours in comparison with placebo, in treatment of 
Chinese and Italian patients with Acute Decompensated Heart Failure. In a 
subset of Italian patients and in a subset of Chinese patients pharmacokinetics 
and metabolism of istaroxime shall also be studied. 
 
Study Design 
A multicenter, randomized, double-blind, placebo-controlled, parallel group 
study. 
Study Period 
This study includes a screening period (Days -1), a treatment period (Day 1), a 
post-treatment period (Days 2-4), and a follow-up period (which includes one 
patient visit on Day 30).  
Subject 
Selection 
Criteria 
Inclusion criteria 
Patients who fulfill the following inclusion criteria at screening will be 
considered for the study: 
1. Signed informed consent; 
2. Male or female patients 18-85 years (inclusive); 
3. Admission for a recurrent ADHF episode with dyspnea at rest or minimal 
exertion and need of intravenous diuretic therapy (≥20 mg iv. furosemide); 
4. Systolic blood pressure between 90 and 125 mmHg (limits included) without 
signs or symptoms of hypoperfusion including cardiogenic shock, cold 
extremities and peripheral vasoconstriction, oliguria/anuria, signs of cerebral 
hypo perfusion such as confusion; 
5. Left ventricular (LV) Ejection fraction (EF) ≤ 40 % measured by 2D-
Echocardiography 
6. E/Ea ratio >10 
7. BNP ≥ 350pg/mL or NT-pro-BNP ≥1400 pg/mL   
8. Adequate echocardiography window (defined as visualization of at least 13/16 
segment of the left ventricle); 
 
Exclusion Criteria 
Any of the following criteria established at screening would render a patient 
ineligible for the study: 
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)  

 
Protocol N°: CVT-CV-002 
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2. Current (within 6 hours prior to screening), during the screening or planned 
(if the patients was treated with dopamine or nitrates iv infusion) (through 
the completion of study drug infusion) treatment or within 12 hours prior to 
screening if the patient was treated with with other iv. vasodilators or other  
inotropic agents and vasopressors  
3. Current or need of mechanical support (intra-aortic balloon pump, 
endotracheal intubation, mechanical ventilation, or any ventricular assist 
device),  
4. Ongoing treatment with oral digoxin. Patient treated with digoxin cannot 
be randomized. (However, if digoxin treatment has been stopped during the 
last week before signing informed consent form and the digoxin plasma 
level is < 0.5 ng / ml, patient may be randomized; if the patients have 
stopped digoxin for more than one week before signing informed consent 
form, it is not necessary to test the plasma level of digoxin.) 
5. History of hypersensitivity to the study medication or any related 
medication 
6. Diagnosis of cardiogenic shock within the past month; 
7. Acute coronary syndrome or stroke within the past 3 months; 
8. Coronary artery bypass graft or percutaneous coronary intervention within 
the past month or planned in the next month; 
9. Primary hypertrophic or restrictive cardiomyopathy or systemic illness 
known to be associated with infiltrative heart disease; 
10. Cor pulmonale or other causes of right-sided HF not related to left 
ventricular dysfunction; 
11. Pericardial constriction or active pericarditis; 
12. Atrial fibrillation with marked irregularities of heart rhythm (Inclusion 
criteria 8 can not be met); 
13. Life threatening ventricular arrhythmia or ICD (implantable cardioverter 
defibrillator) shock within the past month; 
14. CRT (cardiac resynchronization therapy), ICD or pacemaker implantation 
within the past month; 
15. Valvular disease as primary cause of HF (except those corrected by surgery 
without obstruction in left ventricle output); 
16. Heart rate >120 bpm or < 50 bpm 
17. Acute respiratory distress syndrome or ongoing sepsis;  
18. Fever >38° 
19. History of bronchial asthma or porphyria; 
20. Donation or loss of blood equal to or exceeding 500 mL, during the 8 
weeks before administration of study medication; 
21. Positive testing for HIV, Hepatitis B and/or Hepatitis C with abnormal 
liver functions; 
22. Participation in another interventional study within the past 30 days; 
23. The following laboratory exclusion criteria, verified based on results 
obtained within the last 24 hours of hospitalization:  
a. Serum creatinine > 3.0 mg/dl (> 265 µmol/L); 
b. Aspartate aminotransferase (ASAT) or alanine aminotransferase 
(ALAT) > 3 x upper limit of normal, 
c. Hemoglobin (Hb) < 10 g/dL, 
d. Platelet count < 100,000/µL, 
e. Serum potassium > 5.3 mmol/L or < 3.8 mmol/L, 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
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(Note:  
1. All the hematology and biochemistry results obtained within the past 24 hours 
before signing ICF can be used as baseline without retesting the patients. 
2. For HIV、HBV、HCV、T3、T4、TSH, results obtained within one week 
before the patient signed informed consent form can be accepted.) 
Study Drugs 
Test drug: Istaroxime（10 mg per vial） 
Mode of 
administration 
Intravenous infusion via a syringe pump. 
Treatment 
duration 
Treatment by i.v. infusion will last 24 hours. 
Dosing 
scheme 
Istaroxime 0.5 – 1.0 μg/kg/min since the beginning. A continuous i.v. infusion 
for 24 hours not exceeding 144 mg for 24 hours of istaroxime for patients with 
body weight > 100 kg shall be carried out. 
Sample Size 
96 Chinese patients and 24 Italian patients  
Study 
Procedures 
 Screening period (between Hours -24 to -1)   
Within a maximum of 24 hours before administration of study medication 
(istaroxime), a medical screening will be performed on all prospective patients 
to assess suitability for the study. Prior to conducting any study specific 
procedures, the investigator or his/her designee will explain the study fully to 
the patient and provide him/her with a copy of the Patient Information Sheet 
and Informed Consent Document. If the patient is willing to participate in 
the study, s/he and the investigator or his/her designee will both sign the 
Informed Consent Document and a copy of the signed document will be kept 
by the patient.  
 
 Treatment period (Day 1) 
1) Confirm eligibility; 
2) Randomization of patients (after eligibility has been confirmed)  
3) Insertion of multiple lumen intravenous catheter (or administration via 
peripheral vein) 
4) Start istaroxime or placebo infusion (date and time of infusion start must 
be recorded in the CRF) 
5) cTnT (at pre-dose: two samples, then at 3 and 6, 12, 24, 48 and 72 hours 
after start of infusion)  
6) NT pro-BNP at baseline and at the end of 24 hours infusion 
7) Blood samples collection for metabolites and PK (at pre-dose, 0.5-3-6-12-
24 after start of infusion and at 0.25, 0.5, 1, 4 12, 24, 48 hours after the end 
of infusion) in a subset of Italian patients and in a subset of Chinese 
patients pharmacokinetics and metabolism of istaroxime shall also be 
studied. 
8) Vital signs (including arterial oxygen saturation and dyspnoea at pre-dose,3, 
6, 12 and 24 after the start of infusion) 
9) 12-lead ECG profile (pre-dose, 3, 6, 12 hours and between 23 hours and 23 
hours 55 minutes after infusion, totally 5 times, each time 3 measurements)  
10) Stop Day -1 Holter 
11) Start 24-hour Holter ECG (Day 1 recording; to be started immediately 
before initiation of the study drug infusion) 

 
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12) Echocardiography at baseline and 6 and 24 hours after infusion start  
13) 24-hours urine collections for measurement of istaroxime and its 
metabolites and urinary creatinine for the calculation of the creatinine 
clearance; 
14) Blood collection for K+ and eGFR between 23 hours and 30 minutes and 
23 hours and 55 minutes since infusion start; 
15) Concomitant medication monitoring (including chronic medication; dose, 
date and time must be recorded on CRF) 
16) Adverse events monitoring 
 
 Post-treatment period (Day 2 to Day 4) 
Evaluations at 24 hours (day 2) from randomization include: 
1) Vital signs (including arterial oxygen saturation and dyspnea); 
2) 12-lead ECG (triple ECGs) (between 23 hours and 23 hours 55 minutes since 
infusion start); 
3) Stop 24-hour Holter ECG; 
4) Start 24-hour Holter ECG (Day 2 recording); 
5) Stop istaroxime infusion (date and time of infusion end must be  
recorded in the CRF); 
6) Serum potassium level and 24-hour urine collection for measurement of 
istaroxime metabolites and urinary creatinine for calculation of the creatinine 
clearance; 
7) Serum creatinine clearance and calculation of eGFR; 
8) cTnT (50 % or 20 % relative increase over the basal cTnT levels, 
respectively for patients with cTnT basal levels < or > of the 99 % URL (upper 
reference levels, as defined for the Roche hs test, in patients with normal renal 
function, eGFR≥85 ml/min); in patients with eGFR below this value, the renal 
function variations must be considered in evaluating the significance of the 
cTnT changes); 
9) NT pro-BNP; 
10) Metabolites;  
11) Echocardiography;  
12) Concomitant medication monitoring (including chronic medication  
must be recorded in the CRF); 
13) Adverse Events monitoring. 
Evaluations at 48 hours (day 3) include: 
1) Vital signs (including arterial oxygen saturation and dyspnea); 
2) 12-Lead ECG (single ECGs); 
3) Stop 24-hour Holter ECG; 
4) Standard hematology; 
5) Standard blood chemistry; 
6) Serum potassium level; 
7) 24-h urine collection for measurement of istaroxime metabolites and 
urinary creatinine for the calculation of creatinine clearance ; 
8) Calculation of eGFR; 
9) NT-proBNP; 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
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10) cTnT; 
11) Blood samples for istaroxime metabolites; 
12) Echocardiography 
13) Adverse events monitoring; 
14) Concomitant medication monitoring (including chronic medication must 
be recorded in the CRF); 
 
Evaluations at 72 hours (day 4) include: 
1) 
cTnT and NTproBNP (at 72 hours after start of infusion)  
2) 
Vital signs (including arterial oxygen saturation and dyspnoea) 
3) 
Physical examination (HF signs included) 
4) 
12-lead ECG 
5) 
Adverse events monitoring 
6) 
Concomitant medication monitoring (including chronic medication) 
7) 
Istaroxime metabolites 
8) 
Serum potassium and creatinine levels for calculation of eGFR 
9) 
Creatinine clearance  
 Follow-up period and visit (Day 5 to Day 30) 
During the follow-up period the Investigator/designee will make every 
effort to establish patient outcomes. 
Evaluations on Day 30 (follow-up visit) include: 
 
1) Vital signs (including arterial oxygen saturation and dyspnoea); 
2) 12-lead ECG in triplicate; 
3) Calculation of eGFR; 
4) Standard hematology; 
5) Standard blood chemistry; 
6) NT-proBNP  
7)  cTnT; 
8) Urine pregnancy test (β-HCG) for females of childbearing potential 
9) Urinalysis; 
10) Physical examination (HF signs included); 
11) Adverse events monitoring; 
12) Concomitant medication monitoring (including chronic medication) 
Efficacy 
Parameters 
Efficacy endpoints  
1. Primary efficacy end-point: 
 
Change from baseline to 24 hours after infusion start (treatment period 
Day 1) in the E/Ea ratio assessed by tissue Doppler. 
 
2. Secondary efficacy end-points: 
 
Change from baseline to 24 hours in the treatment period Day1 
(addressing the differences between the changes at 6 and 24 hours from 
baseline) of the following Echo-Doppler parameters: 
- 
LV Ejection fraction (EF) 

 
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Amended Final Version (30 November 2017)  
15
- 
LV end systolic and end diastolic volumes 
- 
Stroke volume index (SVI) 
- 
E, A and E/A ratio  
 Difference between the changes at 6 and 24 hours from baseline of the 
Tissue Doppler parameter E/Ea 
 Others Tissue Doppler parameters such as Sa, Da and Aa 
 
Changes in dyspnoea assessed at 3, 6, 12, 24, 48 hours after infusion 
start by Visual Analog Scale (VAS) (including only patients presenting 
dyspnoea at baseline); 
 Area under the curve (AUC) on changes in dyspnoea assessed at 3, 6, 
12, 24, 48 hours after infusion start by VAS (including only patients 
presenting dyspnea at baseline); 
 Changes in BNP from baseline at 24 hours; 
 
Proportion of patients with hospital readmissions or emergency visits 
for cardiovascular reasons by Day 30; 
 
Proportion of patients with episodes of worsening HF defined by the 
need to increase the dose or reinitiate i.v. therapy with diuretics and/ or 
other inotropic agents during the hospitalization; 
 
Length of the hospitalization; 
Safety 
Parameters 
Safety endpoints: 
The following safety endpoints will be assessed during treatment and the 
post-treatment/follow-up periods: 
 
Incidence of adverse events;  
 
Change in vital signs (including body temperature and dyspnoea); 
 
Change in 12-lead ECG parameters; 
 
Incidence of clinically or hemodynamically significant episodes 
of supraventricular or ventricular arrhythmias detected by 
continuous ECG dynamic monitoring;  
 
Change in laboratory parameters (hematology, blood chemistry and 
urinalysis);  
 
Change in renal function; 
 
Change in in cTnT; 
 
Incidence of cTnT elevation (>50% or > 20% relative increase over 
the basal cTnT levels at baseline, for patients with cTnT levels at 
baseline < or  ≥ of the 99% URL (upper reference levels, as 
defined for the Roche hs test, in patients with normal renal 
function, eGFR ≥85 ml/min); in patients with eGFR below this 
value, the renal function variations must be considered in 
evaluating the significance of the cTnT changes); 
 
Mortality at Day 30; 
PK 
parameters 
Full plasma and urine PK profile: 
 The following PK metrics will be computed for E and Z isomers (when 
applicable) 
of 
istaroxime 
plasma 
concentrations 
using 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
16
non-compartmental analysis: Cmax, tmax, AUC0-t, AUC0-∞, z, t½, ClT, 
MRT, Vss, Vz; 
 the following PK metrics will be computed for E and Z isomers (when 
applicable) of istaroxime urine concentrations: Ae, Ae%, ClR; 
 In addition, the following PK metrics will be computed as above for 
plasma and urine concentrations of the E and Z isomers (when 
applicable) of istaroxime metabolites PST2915, PST2922, and PST3093: 
Cmax, tmax, AUC0-t, AUC0-∞, z, t½ and, if possible, Ae and Ae%; 
This endpoint will be addressed in all the Italian patients and in a subset of 
Chinese patients. 
 
Statisical 
Analysis 
Primary efficacy endpoint: 
The primary efficacy endpoint (change from baseline in E/Ea ratio) will be 
analyzed using a linear mixed model for repeated measures including 
treatment, centre, timepoint, gender, baseline cTnT (normal <URL, abnormal 
≥URL), atrial fibrillation (Yes/No) and treatment*timepoint interaction as 
fixed effects and baseline and baseline*timepoint interaction as covariates. 
The primary comparison will be 0.5 μg/kg/min dose of istaroxime versus 
placebo at 24 hours. Highest dose of istaroxime (1.0 μg/kg/min) versus placebo 
will be tested as a secondary comparison. 
Additional analyses separated by cohort will be implemented for sensitivity 
purpose.  
Secondary efficacy endpoints 
The following secondary endpoints: 
 
Change from baseline to 24 hours (addressing the differences between 
the changes at 6 and 24 hours from baseline) of the following Echo-
Doppler parameters: 
- 
LV Ejection fraction (EF) 
- 
LV end systolic and end diastolic volumes 
- 
Stroke volume index (SVI) 
- 
E, A and E/A ratio  
 Change from baseline to 24 hours in the E/Ea ratio assessed by tissue 
Doppler (difference between the changes at 6 and 24 hours from 
baseline) 
 
Others Tissue Doppler parameters such as Sa, Da and Aa 
 
Changes in dyspnoea using VAS score will be analysed using a mixed 
model for repeated measures similar to the one used for the primary 
efficacy endpoint. 
AUC on changes in dyspnoea by VAS and changes in BNP will be analyzed 
using an ANCOVA model with treatment, centre, gender, baseline cTnT (normal 
<URL, abnormal ≥URL) and atrial fibrillation (Yes/No) as fixed effects and 
baseline dyspnea as covariate. 
Number and proportion of patients with: 
 
hospital readmissions or emergency visits for cardiovascular reasons 
within Day 30 
 
episodes of worsening HF defined by the need to increase the dose or 
reinitiate i.v. therapy with diuretics and/ or other inotropic agents during 
the hospitalization 
will be summarized by treatment groups using descriptive statistics.  

 
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Length of hospitalization will be summarized by treatment group using 
descriptive statistics. 
 
Safety endpoints 
The number and the percentage of patients experiencing adverse events, adverse 
drug reactions, serious adverse events and adverse events leading to study 
withdrawal will be summarized by treatment group. Adverse events will also be 
summarized by treatment group by means of System Organ Class and Preferred 
Term using the MedDRA dictionary. 
Vital signs (including body temperature and dyspnoea), 12-lead ECG 
parameters, incidence of clinically or hemodynamically significant episodes of 
supraventricular or ventricular arrhythmias, laboratory parameters, renal 
function, cTNT, increase of cTNT and mortality will be summarized by 
treatment group using descriptive statistics. 

 
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1. INTRODUCTION 
1.1 Indications 
Congestive Heart Failure and Acute Decompensation 
 
Congestive heart failure (CHF) is a complex pathophysiologic syndrome associated with 
significant morbidity and mortality. Figures from the USA and Europe suggest that 
respectively some 5 and 10 million adults have CHF. The incidence and prevalence continue 
to rise as the proportion of elderly in the population increases [1].  In patients with CHF, 
acute decompensation is a major cause of hospitalization and of cost associated with 
treatment.  Acute decompensated heart failure (ADHF) is characterized by a rapid or gradual 
onset of symptoms and signs secondary to abnormal cardiac function. As said, it often results 
in unplanned hospitalization and a need for urgent therapy [2]. 
 
Hospitalizations for ADHF are increasing with more than 1 million per year in the USA and 
as many in Europe.  In-hospital mortality ranges from 4 to 7% in large ADHF registries 
[2, 3]. Post-discharge mortality is approximately 10% during the next 1-2 months, and 
hospital readmission common, with 25 to 30% of patients being readmitted within the first 3 
months after discharge [2- 4]. 
 
Signs and symptoms of heart failure (HF) result primarily from severe pulmonary 
congestion due to elevated left ventricular pressures, with or without low cardiac output. 
Cardiac dysfunction, renal impairment and neurohormonal activation play a significant role 
in the development of congestion by creating a vicious circle: systolic and/or diastolic 
dysfunctions lead to increased left ventricular diastolic pressure and blood volume resulting 
in increased pulmonary capillary wedge pressure (PCWP) [5]. Increased PCWP might 
further lead to increased pulmonary arterial pressure, increased right ventricular and atrial 
pressures, inducing pulmonary and/or systemic congestion with dyspnoea and edema. 
Activation of neurohormonal systems and renal dysfunction also contribute to the 
development of congestion through impaired sodium and water handling [5]. 
 
Approach to Treatment 
 
In chronic congestive heart failure, treatment involves both non-pharmacological and 
pharmacological interventions. Stabilization and improvement of cardiac hemodynamics is 
an important goal of therapy and diuretics, ACE inhibitors, Beta Blockers and vasodilators 
are the most commonly utilized agents. Cardiac glycosides such as digoxin, which is mainly 
used as adjunctive therapy, have an extremely long history in the management of heart failure 
[6]. Little controversy exists as to the benefit of digoxin in patients with symptomatic left 
ventricular systolic dysfunction (LVEF ≤ 40%) and concomitant atrial fibrillation [7]. 
The therapeutic goals in patients hospitalized with ADHF are to reverse acute hemodynamic 
abnormalities, relieve symptoms, and preserve myocardial tissue and renal function without 

 
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inducing arrhythmia or hypotension [8]. Commonly-used agents in the treatment of ADHF 
with normal to high systolic blood pressure (SBP) include diuretics and vasodilators (e.g. 
nitroglycerin, nitroprusside, and nesiritide). Patients with low blood pressure (BP) may 
require intravenous (i.v.) inotropic support for their low output state, essentially adrenergic 
stimulating agents such as dobutamine or dopamine, or phosphodiesterase inhibitors such 
as milrinone. However, none of the currently available agents encompasses all the properties 
desired for an “ideal” agent for treatment of ADHF: diuretics may worsen renal function; 
vasodilators and nesiritide are associated with symptomatic hypotension; conventional 
inotropes induce myocardial cell calcium accumulation and may cause atrial and ventricular 
arrhythmias, myocardial ischemia due to increase of oxygen consumption, cell apoptosis and 
possibly increase post-discharge  mortality [9, 10]. Thus, conventional inotropes such as 
dobutamine, dopamine or milrinone are indicated in the presence of peripheral 
hypoperfusion with or without congestion or in pulmonary edema refractory to diuretics and 
vasodilators [11]. 
Development of drugs that improve contractility without producing calcium overload, 
therefore represents an important goal in the treatment of ADHF. 
1.2 Pharmaceutical and background information 
Istaroxime  
Background Information 
Istaroxime is a first-in-class luso-inotropic agent under development for the treatment of acute 
decompensated heart failure.  It possesses a dual mode of action, combining inotropic 
(myocyte contraction) and lusitropic (myocyte relaxation) effects. Istaroxime is a potent 
positive inotropic agent that increases myocardiac contractility through inhibition of Na+/K+-
ATPase, subsequently augmenting cytoplasmic calcium levels. In addition, istaroxime exerts 
positive lusitropic effects through activation of sarcoplasmic reticulum calcium ATPase 
isoform (SERCA2) promoting reuptake of cytoplasmic calcium with subsequent myocardial 
relaxation, thus decreasing left ventricular diastolic pressure and congestion. Based on its 
mechanism of action, preclinical studies and early clinical findings, it is anticipated that 
istaroxime at the doses proposed in this trial will be devoid of the deleterious effects of 
conventional inotropes. Istaroxime does not increase heart rate (HR), minimizes the increase 
in oxygen consumption, is less arrhythmogenic and does not cause hypotension [12, 13, 14]. 
 
 
Chemical Structure, Formula and Molecular Weight 
 
Istaroxime is an original derivative of androstanedione, a lipophilic steroid based compound, 
chemically unrelated to cardiac glycosides or to phosophodiesterase inhibitors. 
 

 
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IUPAC name: 
Androstane-3,6,17-trione 
(E,Z)-3-[O-(2-
aminoethyl)]oxime hydrochloride 
 
 
  
Other names:      (E,Z)-3-[O-(2-aminoethyl)oxime]-5α-androstane-3,6,17-trione 
monohydrochloride 
 
(E,Z)-3-(2-Aminoethoxyimino)androstane-6,17-dione   
hydrochloride 
 
 
Molecular weight:  
 
 
396.95 
 
Molecular formula: 
C21 H33 ClN2 O3 
 
 
Molecular structure: 
 
Stereoisomerism: 
The active substance is a 1:1 mixture of E and Z isomers 
at the oximic C(3)=N double bound. 
 
International Non-proprietary Name, Brand Name and Code Name 
International non-proprietary name: 
Istaroxime 
     
Brand name: 
Not yet designated 
 
Code names: 
PST2744  
 
 
Mechanism of Action 
Istaroxime is a dual-action Na+/K+-ATPase inhibitor/sarcoplasmic reticulum calcium pump 
(SERCA2) activator that improves myocardial efficiency by modulating calcium cycling in 
cardiac myocytes. Na+/K+-ATPase   inhibition   augments   cytoplasmic   calcium   levels   
and   subsequently   promotes myocardial contractility, enhancing inotropy. SERCA2 

 
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activation promotes the reuptake of cytoplasmic calcium with subsequent myocardial 
relaxation and improved filling of the heart. As a result of the combination of these two 
mechanisms, istaroxime improves cardiac function [13, 14, 15]. Compared to inotropes such 
as dobutamine or milrinone, istaroxime, at therapeutic doses, does not induce myocardial 
“calcium intoxication”, does not increase HR, oxygen consumption, and proarrhythmic effect. 
Moreover, being devoid of vasodilating effects, istaroxime does not induce arterial hypotension 
[14]. 
  
Brief Summary of Non-clinical Pharmacology 
Hemodynamic in vivo studies showed enhanced cardiac systolic and diastolic function in 
anesthetized guinea pigs, pigs and dogs both with and without HF. In guinea pigs with 3-
month aortic banding, echocardiographic results showed that i.v. istaroxime infusion 
significantly increased both contraction and relaxation indices [16]. In pigs without HF, the 
effects of istaroxime on contractility and HR were evaluated in comparison with dobutamine 
as the reference inotropic agent: istaroxime showed a positive inotropic effect on cardiac output 
and stroke volume similar to dobutamine, however without increasing HR or oxygen 
consumption as seen with the latter [17]. 
 
Pharmacological studies carried out in the dog model of advanced HF, escalating doses  of  i.v.  
istaroxime from  0.5  µg/kg/min to 5.0 µg/kg/min were administered, each dose being 
maintained for one hour. Istaroxime substantially improved systolic and diastolic left 
ventricular function [18]. Furthermore, istaroxime was administered i.v. for 24 hours at 1, 3, 
and 4 μg/kg/min, compared to saline 50 mL/24h infusion in conscious male Beagle dogs  
with  HF   after  repeated  coronary  microembolization  [19].  Istaroxime induced a dose-
dependent increase in cardiac inotropy (+dP/dtmax), which peaked after 6-8 hours and 
remained constant throughout the experiment. Cardiac lusitropy (-dP/dtmax) improved 
although not statistically significantly during infusion rates of 1 and 3 μg/kg/min. 
Administration of istaroxime at 3 μg/kg/min, induced vomiting after 4-5 hours of continuous 
infusion. At 4 μg/kg/min, vomiting episodes increased and persisted for the first 10-12 hours 
of infusion. The maximum tolerated dose of istaroxime in dogs was 3 μg/kg/min for 24 hours. 
 
Noteworthy, unlike classic cyclic adenosine monophosphate-dependent positive inotropic 
agents such as milrinone or β-adrenergic compounds like dobutamine, istaroxime improves 
both inotropic and lusitropic states with much less increase of oxygen consumption. These 
improvements were not associated with any apparent increase in positive chronotropic and 
proarrhythmic activity. 
 
The toxicological profile of 24 hour infusion of istaroxime was investigated in rats and 
dogs under GLP conditions. The maximum tolerated dose (MTD) was 10 μg/kg/min in rats 
and 3 μg/kg/min in dogs. 
 
In conclusion, istaroxime is a powerful luso-inotropic agent that, in animal models of HF in 

 
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doses up to 4 µg/kg/min, does not increase the risk of arrhythmia. From the pharmacology data, 
istaroxime appears to be a promising and safe new drug for improving cardiac performance in 
HF. 
 
More detailed pre-clinical information can be found in the Investigator’s Drug 
Brochure [20]. 
 
Brief Summary of Human Pharmacology  
Istaroxime efficacy was first evaluated in two phase I-II safety and tolerability studies 
performed in patients with chronic HF and left ventricular dysfunction. In the first study, 
istaroxime was administered as 1-hour infusions of ascending doses from 0.005 to a 
maximum of 5.0 µg/kg/min in an escalating dose, placebo-controlled, double-blind design in 
6 patients for each dose. No increases in HR or arrhythmias were detected at any of the doses 
administered including the highest dose (5.0 μg/kg/min).  Reported AEs were gastrointestinal 
symptoms and injection site pain starting at the dose of 3.33 μg/kg/min. Hemodynamic 
measurements within 2 hours prior to dosing and during the 1-hour infusion were carried 
out using bio-impedance methodology. Istaroxime infusions up to a dose of 1.0 μg/kg/min did 
not appear to have an effect on any of the measured hemodynamic parameters. At the 
subsequent dose levels of 1.67, 3.33, and 5.0 μg/kg/min, dose-dependent increases from 
baseline were observed at the end of the infusion period in cardiac index, acceleration index, 
velocity index, and left cardiac work index, with no clinically significant decrease from 
baseline in systemic vascular resistance (SVR) [20].  
 
In a second double-blind study, patients received 1-hour istaroxime infusions at doses 
escalating from 0.005μg/kg/min to 3.0µg/kg/min. Swan Ganz catheterization was used to 
measure hemodynamic parameters. No clinically relevant inotropic and lusitropic effects 
were observed at the lowest doses of 0.005, 0.0167 and 0.5 µg/kg/min. At the 1.0, 2.0, and 
3.0 µg/kg/min infusions clinically relevant lusitropic and inotropic effects were observed. The 
echocardiographic sub-study results, performed in a subset of patients, confirmed a strong and 
consistent improvement in left ventricle systolic function with a significant decrease in left 
ventricle systolic volume and increases in ejection fraction (EF) and stroke volume (SV).  In 
addition the patients evaluated had severe left ventricle diastolic impairment at baseline, and 
demonstrated a significant improvement in diastolic function as measured by E/E’ (ratio of 
transmitral peak early filling velocity to early diastolic velocity of the mitral annulus) assessed 
10 minutes prior to completion of the istaroxime infusion. Cardiac tolerability was good, 
without any significant arrhythmia even at the highest dose of 3.0µg/kg/min. Nausea and 
vomiting were observed at the dose of 3.0 µg/kg/min. 
 
A further phase I placebo-controlled dose escalating study was performed to evaluate the 
safety and tolerability of istaroxime 6-hour infusion in healthy male volunteers [20]. Four 
different escalating istaroxime doses (0.67, 1.33, 1.67, and 2.0 µg/kg/min) were infused i.v. 
for 6 hours. Each dose, except for the 2.0µg/kg/min dose administered in 1 cohort only, was 
infused in two different cohorts of 4 subjects (3 subjects receiving istaroxime and 1 receiving 

 
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placebo in each cohort). No clinically relevant abnormalities were observed on vital signs and 
ECGs. While the 0.67 and 1.33 µg/kg/min doses were well tolerated, the highest dose group 
(2.0 µg/kg/min) could not be completed due to non-serious adverse events (AEs), mainly 
gastrointestinal disorders (nausea and vomiting) and pain at the injection site. The 1.67 
µg/kg/min dose was relatively well tolerated by 2 of the 6 subjects. In this treatment group, 
the infusion was terminated prior to 6 hours in 4 subjects due to severe AEs (nausea and 
vomiting). All AEs resolved shortly after cessation of the infusion. 
 
Finally, to determine the minimum effective dose, a phase IIa, double-blind, randomized, 
placebo-controlled, dose escalating (0.5-1.0-1.5 µg/kg/min) efficacy study (HORIZON-HF) 
was conducted assessing the hemodynamic effects of istaroxime in 120 patients 
hospitalized with worsening HF and reduced left ventricle systolic function [14]. The 
primary end point of the study was the change from baseline in PCWP after 6 hours of 
infusion. Dose-dependent improvements in PWCP were observed with a statistically 
significant effects of all the 3 istaroxime doses versus placebo (– 3.21 mmHg with the 
0.5 μg/kg/min dose, – 3.33 mmHg with the 1.0 μg/kg/min dose, and –4.73 mmHg with the 
1.5 μg/kg/min dose). HR tended to decrease during the 6-hour infusion but no significant 
differences were observed at infusion end between treatment groups. Systolic blood pressure 
(SBP) slightly increased dose-dependently in all istaroxime-treated groups but not in the 
placebo group, reaching a statistically significant difference from the dose of 1.0 µg/kg/min. 
A trend towards an increase in cardiac output was also observed in the treated groups. A 
significant increase in cardiac index (CI) was observed only in patients with a CI<2.5 at 
baseline. Statistically significant difference between istaroxime and placebo were found for 
LV end systolic volume at 1.0 µg/kg/min and for LV end diastolic volume at 1.5 
µg/kg/min. Doppler echocardiography and tissue Doppler imaging showed 
changes in the E/Ea parameter that resulted statistically significant at 0.5 
µg/kg/min (-3.8, p= 0.03) and close to significance at 1.0 µg/kg/min (-1.12, p= 
0.059). In conclusion, echocardiographic data showed istaroxime to improve diastolic and 
systolic volumes, as well as Doppler and tissue Doppler parameters of diastolic function. As 
far as safety concerns, this study showed an increase in gastrointestinal TEAEs in the 
highest dose group (1.5 µg/kg/min) in which 10% and 17% of patients experienced nausea 
and vomiting, respectively. No patients experienced serious or clinically relevant episodes 
of arrhythmia during the infusion and post-infusion period. A mild increase in the frequency 
of isolated ventricular extrasystoles was found in the 1.5 µg/kg/min group in 8 out of 30 
patients. It is difficult to ascertain whether this increase was related to drug infusion or to 
spontaneous variability of patients, because of insufficient baseline recordings. One patient 
out of 30 in the istaroxime 1.5 µg/kg/min group experienced tachycardia with angina and 
ST-change during infusion. The QTc interval measured at 6 hours after infusion was shortened 
by -25, -38 and -49 ms at the three Istaroxime doses, respectively 
Globally, istaroxime was well tolerated and safe up to 1.5 µg/kg/min in patients suffering 
from acute HF. Doses above 1.5 µg/kg/min were not completely tolerated due to 

 
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gastrointestinal disorders and infusion site pain. 
In conclusion, in this study the lowest tested dose of 0.5 µg/kg/min of istaroxime infused for 
6 hours produces a significant PWCP improvement over placebo, being the istaroxime safety 
profile satisfactory even at higher dose, in this setting. 
Pharmacokinetic results showed that istaroxime levels increased proportionally in plasma 
reaching steady state concentration within the 1hour infusion. Estimated half-life for istaroxime 
was about 10 to 20 minutes at infusion rates higher than 1.66 µg/kg/min. For rates of infusion 
lower than 1.66 µg/kg/min, concentration dropped below the limit of quantification (10 ng/ml) 
at 5 min after stopping of the infusion. 
The systemic exposure to istaroxime resulted to be linearly related to the administered dose 
[20]. A very high clearance was shown, resulting around 4500 ml/min for a 70 kg body 
weight man. The concentration of PST 2915, the main metabolite (6-alcholol) of the parent 
compound, was also measured, showing that its concentration slowly increased during 
infusion period, reaching a maximum concentration within 10 min after the stop of the 
intravenous infusion of istaroxime [20]. The metabolite maximum concentration resulted to 
be about one half of the corresponding Cmax of istaroxime. Half-life of PST 2915 resulted 
to be around 3 to 4.5 hours [20]. PST 2915 resulted to be an active metabolite showing the 
same stimulatory activity on SERCA2a of istaroxime but about 10 time less inhibitory activity 
on Na-KATPase and potency in in vivo animal models than istaroxime [20]. 
For infusion longer than 1 hour, PST 2915 plasma concentrations increase with time, while 
those of istaroxime remain at steady state, being constant the rate of infusion. 
To evaluate the risk of a prolongation of istaroxime intravenous infusion over six hours, 
pharmacokinetic, efficacy and tolerability clinical studies have to be carried out.  
The above clinical evidence indicates that istaroxime could be a future treatment option for 
patients with ADHF and a low cardiac output state. 
 
More detailed clinical information can be found in the Investigator’s Drug Brochure [20]. 
 
2. STUDY OBJECTIVE  
To assess the safety, tolerability and efficacy of two different doses of istaroxime (0.5 and 1.0 
µg/kg/min) in comparison with placebo, including cardiovascular and renal tolerability, as well 
as changes in biological markers such as N-terminal prohormone brain natriuretic peptide (NT-
proBNP) and troponin T (cTnT), in two cohorts, Chinese and Italian /Caucasian patients with 
Acute Decompensated Heart failure. 
In all the Italian patients and in a subset of Chinese patients pharmacokinetics and metabolism 
of istaroxime shall also be studied. 
 
3. STUDY DESIGN 
This is a phase II, multicenter, randomized, double-blind, placebo-controlled, parallel group 
study.  

 
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96 Chinese patients and 24 Italian patients will be randomly assigned to one of two doses of 
istaroxime or placebo in a 2:1 ratio within two sequential cohorts of 60 patients each. 
This 31-day study includes a screening period (Days -1), a treatment period (Day 1), a post-
treatment period (Days 2-4), and a follow-up period (which includes one patient visit on Day 
30).  
 
When a patient is considered fully eligible (i.e. s/he fulfills all the inclusion criteria and does 
not meet any exclusion criteria as stipulated), the patient will be randomized. 
Treatment details are presented below: 
 
Cohort I: 0.5 μg/kg/min istaroxime or placebo (40 patients randomized to istaroxime 0.5 
μg/kg/min group and 20 patients randomized to placebo group). 
Istaroxime 0.5 μg/kg/min (30 μg/kg/h) continuous i.v. infusion for 24 hours not exceeding 72 
mg per 24 hours for patients with body weight > 100 kg 
Cohort II: 1.0 μg/kg/min istaroxime or placebo (40 patients randomized to istaroxime 1.0 
μg/kg/min group and 20 patients randomized to placebo group). 
Istaroxime 1.0 μg/kg/min (60 μg/kg/h) continuous i.v. infusion for 24 hours not exceeding 144 
mg per 24 hours for patients with body weight > 100 kg 
 
Escalation to the next dose (second cohort) will occur after completion of the first cohort 
and determined by the evaluation of the safety data performed by the Data Safety 
Monitoring Board. 
 
4. STUDY POPULATION 
A total of 96 Chinese and 24 Italian patients hospitalized with ADHF not requiring inotropic 
therapy at inclusion into the study (according to the opinion of the Investigator) and not 
presenting signs and/or symptoms of severe low output state or peripheral hypo perfusion will 
be randomized. 
 
To be included into the study, patients have to fulfill eligibility criteria both at their 
admission to the hospital (screening criteria) and just prior to randomization (randomization 
criteria). 
4.1 Inclusion criteria 
Patients who fulfill the following inclusion criteria at screening will be considered for 
the study: 
1. Signed informed consent; 
2. Male or female patients 18-85 years (inclusive); 

 
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3.Admission for a recurrent ADHF episode with dyspnea at rest or minimal exertion and 
need of intravenous diuretic therapy (≥ 40 mg iv. furosemide); 
4. Systolic blood pressure between 90 and 125 mmHg (limits included) without signs or 
symptoms of hypoperfusion including cardiogenic shock, cold extremities and peripheral 
vasoconstriction, oliguria/anuria, signs of cerebral hypo-perfusion such as confusion; 
5. Left ventricular (LV) Ejection fraction (EF) ≤ 40 % measured by 2D-Echocardiography  
6. E/Ea ratio > 10 
7. BNP ≥ 350pg/mL or NT-pro-BNP ≥1400 pg//mL; 
8. Adequate echocardiography window (defined as visualization of at least 13/16 segment 
of the left ventricle); 
 
4.2 Exclusion criteria 
 
Any of the following criteria established at screening would render a patient ineligible for the 
study: 
 
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. Current (within 6 hours prior to screening), during the screening or planned (if the 
patients was treated with dopamine or nitrates iv infusion) (through the completion of study 
drug infusion) treatment or within 12 hours prior to screening if the patient was treated with 
other iv. vasodilators or other inotropic agents and vasopressors,  
3) Current or need of mechanical support (intra-aortic balloon pump, endotracheal 
intubation, mechanical ventilation, or any ventricular assist device),  
4. Ongoing treatment with oral digoxin. Patient treated with digoxin cannot be 
randomized. (However, if digoxin treatment has been stopped during the last week before 
signing informed consent form and the digoxin plasma level is < 0.5 ng / ml, patient may 
be randomized; if the patients have stopped digoxin for more than one week before signing 
informed consent form, it is not necessary to test the plasma level of digoxin.) 
 5. History of hypersensitivity to the study medication or any related medication; 
6. Diagnosis of cardiogenic shock within the past month; 
7. Acute coronary syndrome or stroke within the past 3 months; 
8. Coronary artery bypass graft or percutaneous coronary intervention within the past month 
or planned in the next month; 
9. Primary hypertrophic or restrictive cardiomyopathy or systemic illness known to be 
associated with infiltrative heart disease; 
10. Cor pulmonale or other causes of right-sided HF not related to left ventricular 
dysfunction; 
11. Pericardial constriction or active pericarditis; 
12. Atrial fibrillation with marked irregularities of heart rhythm (Inclusion criteria 8 can 
not be met); 

 
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13. Life threatening ventricular arrhythmia or ICD (implantable cardioverter defibrillator) 
shock within the past month; 
14. Presence of a CRT (cardiac resynchronization therapy), ICD or pacemaker devices 
implanted within the past month; 
15. Valvular disease as the primary cause of HF (except those corrected by surgery without 
obstruction in left ventricle output); 
 
16. Heart rate > 120 bpm or < 50 bpm 
17. Acute respiratory distress syndrome or ongoing sepsis; 
18. Fever >38° 
19. History of bronchial asthma or porphyria; 
 
20. Donation or loss of blood equal to or exceeding 500 mL, during the 8 weeks before 
administration of study medication; 
21. Positive testing for HIV, Hepatitis B and/or Hepatitis C with abnormal liver functions; 
22. Participation in another interventional study within the past 30 days; 
23. The following laboratory exclusion criteria, verified based on results obtained within 
the last 24 hours of hospitalization:  
a. Serum creatinine > 3.0 mg/dl (> 265 µmol/L); 
b. Aspartate aminotransferase (ASAT) or alanine aminotransferase (ALAT) > 3 x upper 
limit of normal, 
c. Hemoglobin (Hb) < 10 g/dL, 
d. Platelet count < 100,000/µL, 
e. Serum potassium > 5.3 mmol/L or < 3.8 mmol/L, 
(Note:  
1. All the hematology and biochemistry results obtained within the past 24 hours before signing 
ICF can be used as baseline without retesting the patients. 
2. For HIV、HBV、HCV、T3、T4、TSH, results obtained within one week before the 
patient signed informed consent form can be accepted.) 
5. STUDY PRODUCTS AND DOSE REGIMEN 
5.1 TEST DRUG 
5.1.1 
ISTAROXIME 
Name:   
 
  Istaroxime 
Composition: 
Istaroxime 10 mg, lactose 50 mg, and if needed sodium hydroxide as pH 
adjuvant. 
Dosage form:      Powder for solution for infusion. 
Presentation:      10 mg istaroxime glass vial to be reconstituted with 5 ml saline for injection, 
according to instructions in leaflet. 
Storage Condition: Istaroxime should be stored at room temperature below or equal to 25°C 
(77°F) in a secure location. Do not freeze. 
Source: provided by CVie Therapeutics Company Limited. 

 
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The certificate of analyses will be provided together with the product. 
 
5.1.2  PLACEBO  
Composition:  
Lactose 50 mg, and if needed sodium hydroxide as pH adjuvant. 
Dosage form:   
Powder for solution for infusion. 
Presentation:  
Glass vial to be reconstituted with 5 ml saline for injection, according to 
instructions in leaflet. 
Storage condition: Placebo should be stored at room temperature below or equal to 25°C (77°F) 
in a secure location. Do not freeze 
Source: provided by CVie Therapeutics Company Limited. 
The certificate of analyses will be provided together with the product. 
5.2 Drug Blind Code  
The computer generated randomization list stratified by centre and atrial fibrillation (Yes/No) 
will be prepared via a computerised method. Starting from the lowest number provided, in each 
center and and atrial fibrillation, at Day 1, patients will be sequentially assigned to the next 
randomization number, following the order in which they present themselves for the study.  
The investigator will keep the code break envelopes in a locked, secure storage facility.  
A code break envelope can only be opened in an emergency situation (mainly SUSAR 
occurrence) where the investigator considers it essential to know which treatment the subject 
was taking. If possible, it is recommended that the investigator contacts the Sponsor before 
opening an envelope. The monitor shall be promptly notified when a treatment code envelope 
is opened. The investigator shall provide a certified explanation of why the treatment code was 
opened in the Case Report Form for the subject or directly on the opened treatment code 
envelope. 
 
IMPORTANT: The randomization codes will only be disclosed to the laboratory in charge of 
the bioanalytical assay (plasma and urine PK assessment) of istaroxime and its metabolites, in 
order to prevent analyses of samples from patients who have received placebo. This laboratory 
will re-code identifiers of patients, allowing only to differentiate but not to identify the patients, 
in order to maintain blinding integrity for the other assessments of the study, until database 
lock. PK results will be maintained separate from the electronic CRF data until database lock 
at study completion. 
 
Emergency Un-blinding: 1) Un-blinding condition: any emergencies of unknown origin that 
occur to the subject during the trial, should find out the medication type, or the subject asks to 
quit and wants to know the medication type; 2) Un-blinding register: the principle investigator 
opens and reads the emergency letter of corresponding number, recording the reading date and 
reason in detail, and signs his/her name; 3) Un-blinding disposal: once the emergency letter is 

 
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read, the corresponding case is regarded as drop-out. 
 
IMPORTANT: An emergency code break by the Investigator may be authorized only in case 
of an unexpected serious adverse event (SUSAR), suspected to be related to an investigational 
product and requiring, according to the Investigator, the knowledge of the study treatment 
administered. In such a case, the following information must be documented in the e-CRF: 
reason for code break, date, name, qualification, patient initials and birth date (as recorded in the 
eCRF) and electronic signature of the person who requested the code break. At completion of 
the study, all emergency code breaks tracked by the e-CRF will be checked for verification of 
the blinding integrity. 
5.3 Drug packaging  
The test drugs and placebo are packaged in the glass vials of identical size.  
The test drug Istaroxime (10 mg test drug + 50 mg lactose) and the placebo (50 mg of lactose) 
are contained in glass vials of 7 ml each as lyophilized powder. 
 
Dummy: Packaging dummy technique will be adopted as follows: 
The investigational drug and placebo will be provided as kits containing 4 study boxes. Each 
study box for each patient will be used for 6 hours iv. infusion, for a total of 24 hours infusion 
for the 4 boxes. Each study box for the 6 hour infusion will contain 4 glass vials of 7 ml. 
According to the treatment (test drug-istaroxime or placebo) and to the dose (test drug-
istaroxime: 0.5 or 1.0 mcg/kg/min) the 6-hour box will contain: 
 
1. Istaroxime (0.5 mcg/kg/min): 
 2 glass vials of 7 ml containing lyophilized powder of test drug (10 mg ) + 50 mg of 
lactose 
 2 glass vials of 7 ml containing lyophilized powder of placebo (50 mg of lactose) 
 
2. Istaroxime (1.0 mcg/kg/min): 
 4 glass vials of 7 ml containing lyophilized powder of test drug (10 mg ) + 50 mg of 
lactose 
 
3. Placebo 
 4 glass vials of 7 ml containing lyophilized powder of placebo of istaroxime (50 mg 
of lactose) 
 
All these boxes will be uniformly packaged and the vials will be tightly covered by study special 
label. The clinical study approval document number, drug serial numbers, expiry date, usage 
method and attentions should be stated on the labels. 

 
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Packaging: External packing and inner packing, inner packing is 4 glass vials (see above) while 
external packing is a white box. Outside the box and vials are pasted by study special label (refer 
the contents to the label drafts only for Chinese sites). 
 
1). External packing box label of test drugs and control drug       (OK) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2). Inner packing box label of test drugs and control drug  
 
 
 
 
 
 
 
 
 
 
 
Istaroxime injection for ADHF clinical study 
 
Drug No.： ________ 
 
 
 
   
  Drug batch.：_______       
Study No.： ________  
 
 
 
 
  EUDRACT No.: ______ 
 
Strength：10 mg /vial，Lyophilized powder               Expiry date：_________ 
Quantity：infusion for 24h, contain 4 small boxes, each with 4 vials  
Storage condition：below 25℃(77°F) room temperature，don’t freeze 
Dose regimen：adjust infusion rate according to body weight, prepare drug every 6h 
Storage temperature: XXX°C. 
Sponsor: CVie Therapeutics Company Limited 
To be used only for clinical trials. Keep out of children’s reach. Please return the 
remaining. drugs  in timely 
Istaroxime injection for ADHF clinical study 
 
Drug No.： ________ 
 
 
 
   
  Drug batch.：_______       
Study No.： ________  
 
 
 
 
  EUDRACT No.: ______ 
 
Strength：10 mg /vial，Lyophilized powder               Expiry date：_________ 
Quantity：infusion for 6h, 4 vials  per box 
Storage condition：below 25℃(77°F) room temperature，don’t freeze 
Dose regimen：adjust infusion rate according to body weight, prepare drug every 6h 
 
Sponsor: CVie Therapeutics Company Limited 
To be used only for clinical trials. Keep out of children’s reach. Please return the 
remaining. drugs  in timely 
Approve No.：2015L00219 
Approve No.：2015L00219 

 
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3). Vial label of test drugs and control drug  
 
 
 
 
 
 
 
 
 
 
 
5.4 Emergency Envelope/Emergency Unblinding Card 
Emergency envelope is prepared for SAE happening, one opens the envelope to rescue in time, 
and avoid overall un-blinding. “A randomized, double-blind clinical study of Istaroxime”, and 
the randomization number are printed on the envelopes. The emergency envelope draft is as 
follow: 
 
Double-blind clinical study emergency  
Un-blinding record            Drug Randomization No.: 
Study project:  A randomized, double-blind clinical study of Istaroxime                        
Sponsor:  CVie Therapeutics Company Limited 
Clinical Study Responsible Sites:  XX 
The subject of this Randomization No. is randomly divided into: 
 
□ 
Test group: Istaroxime 0.5 mcg/kg 
□ 
Test group: Istaroxime 1.0 mcg/kg 
□ 
Control group: Placebo 
Reason for emergency unblinding: _____________________________  
Study center: _______________ 
 
Signature of investigator: _________  Date: ____________ 
 
Signature of principal investigator : __________  Date: ___________ 
Note: the case should withdraw from the study after emergency un-blinding 
Istaroxime injection for ADHF clinical study 
 
Drug No.： ________ 
 
 
 
   
  Drug batch.：_______       
Study No.： ________  
 
 
 
 
  EUDRACT No.: ______ 
 
Strength：10 mg /vial，Lyophilized powder               Expiry date：_________ 
Storage condition：below 25℃(77°F) room temperature，don’t freeze 
Dose regimen：adjust infusion rate according to body weight, prepare drug every 6h 
 
Sponsor: CVie Therapeutics Company Limited 
To be used only for clinical trials. Keep out of children’s reach. Please return the 
remaining. drugs  in timely 
Approve No.：2015L00219 

 
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5.5 Drug Distribution 
Registration: All drugs of each site will be stored in one place and managed by a specially-
assigned person. The distribution and reclaiming should be registered. 
 
Sequence: the investigator obtain the random no. and drug no. of the subject according to the 
visiting sequence of the qualified subjects in each study center, and the drug administration 
office dispense the study drugs of corresponding drug no.  
 
Distribution: The subjects are randomized to the test group (istaroxime) and control group 
(placebo) in a ratio of 2:1. All study drugs are accompanied by emergency envelopes of 
corresponding numbers and the emergency envelopes are kept by the principal investigator in 
attending site. 
The drugs should be given to the subjects according to their drug numbers, and shouldn’t be 
chosen. The random numbers should remain unchanged during the study. Each subject will be 
provided with a random number and sufficient study drugs. The investigator should fill in the 
Drug Distribution Registration Form exactly in time. 
 
Storage: The study drugs are stored, administrated and distributed by each site. The drugs 
should be stored hermetically in the shade. 
 
Remaining Drugs: If the drugs remain because patients do not take them on time, or quit 
midway, or the treatment changes, the drugs should be reclaimed and recorded in detail. 
 
5.6 Dosage Regimen 
Istaroxime, in powder form, will be provided in glass vials. NaCl 0.9% bags for preparation of 
the 4 x 50ml infusion over 24 hours, will be provided by the Center. The infusion pumps, sets, 
filters and connections will be provided by the research center. 
 
Preparation of the solution for infusion will be performed by a p h y s i c i a n  o r  a  
pharmacist of the Pharmaceutical Services Division, in the presence of a suitably qualified 
witness according to the randomization code. A record for the preparation of the infusion 
will be kept for each patient according to the research center's standard operating procedure 
and will be approved by the Sponsor before first dosing.  Stability data available support the 
infusion of the mixed preparation over 6 hours. 
 

 
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The istaroxime vials will be handled according to the manufacturer’s specifications to 
ensure that the products are appropriately mixed prior to infusion. 
 
In order to calculate the amount of istaroxime given to each patient in each cohort, the body 
weight will be determined to one decimal place. The body weight recorded on Day 1 at pre-
dose will be used for calculation of doses of istaroxime for each cohort. 
 
 
5.6.1 Method of administration and compliance 
 
Istaroxime and placebo will be provided as kits containing 4 study drug boxes. Each study 
drug box will contain 4 glass vials (see section 5.3). The infusion solution will be 
administered via a syringe pump. The exact istaroxime dose to be administered will be 
calculated according to patient body weight and will be modulated by the infusion rate (see 
Appendix A: infusion rates) 
 
On the treatment day, a solution for infusion will be prepared every 6 hours (with 4 vials) 
and will be administered to the patient via the syringe pump. 
 
The procedure for preparation of a 6-hour infusion is the following: 
 
1. Check that the Study Drug Box # displayed on the kit is the one that has been 
allocated to the patient; 
 
2. Select one of the 4 study drug boxes; 
 
3. Remove the flip offs from the containers contained in the box; 
 
4. Reconstitute each of the 4 vials with 5 mL of saline, taken from the NaCl 0.9% bag 
(supplied by the local center) that will be utilized for the preparation of the infusion 
solution, according to instructions in leaflet; 
 
5. Draw completely the clear solution from the 4 reconstituted vials into a single 50 mL 
syringe; 
 
6. Remove 30 ml of saline from the NaCl 0.9% bag with the same syringe used for the 
reconstitution of the 4 vials in order to obtain a total volume of 50 ml (20 ml of the 4 
vials+30 ml of saline from the NaCl 0.9 % bag) 
 
7. Mix the whole content of the syringe  
 

 
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8. Invert the syringe carefully 4 times and connect the syringe to the infusion line; 
 
9. Prime the infusion line to the most proximal point nearest to the catheter; 
 
10. Start the infusion pump at the pre-determined dosing time for the patients; Calculate 
the catheter volume according to the indications reported in the Section 14 of 
Operation Manual 
 
11. Modulate the infusion rate according to patient weight (see Appendix A: Infusion 
rates). Syringe infusion time is 6 hours. 
 
- 
The infusion syringe must be clearly labeled with the investigational product 
name, the study number, and the patient number. 
 
- 
A pre-printed label is available inside the Study Drug Box. In case of use, the 
Patient Screening or Randomization # has to be added by the person preparing 
the investigational product. 
 
Under close supervision, the investigational product will be continuously infused 
intravenously in a vein at high blood flow. 
 
In case of clotting of a line, the infusion tube has to be disconnected from the PAC 
and immediately   connected   with   the   infusion   line   available   on   the   introducer   
to   continue investigational product infusion. 
 
Treatment compliance will be verified by direct visual supervision at dosing by the 
Investigator or his/her designee. Drug accountability records will be kept as outlined in 
section 15.1. 
 
In case of a damaged study drug box, a broken vial, or for any need for replacement of 
one of the 4 study drug boxes within the kit, the Investigator must discard the entire study 
drug box, and obtain a replacement box using the study drug box number retrieved from the 
replacement list. 
 
5.7 Concomitant Medication 
All oral treatments, are permitted during the study with the exception of positive inotropic 
agents. 
Dosage of oral HF medications must remain stable for at least 6 hours before study entry, 
during the entire treatment period (24-hour infusion), until 72 hours (on Day 4) after the start 

 
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of infusion on Day 1. Concomitant medications should be administered in the pre-
randomization phase and after at least 6 hours from the initiation of study drug infusion, if 
possible.  
Intravenous diuretics administered during the screening period to stabilize the patient’s 
condition will be permitted during the treatment period at stable doses, unless changes are 
required due to clinically relevant variations of the patient’s condition.  
 
Any introduction of new drugs or increase of the dosage of oral or i.v. medications for the 
treatment of heart failure, during treatment and till the 72 hours after the start of infusion 
(on day 4), will be considered as "rescue medication". 
 
Patient supplementation of magnesium or potassium, prior to randomization, will not be 
considered as a change of the standardized therapy but just as a supplementation to maintain 
serum electrolytes within the normal ranges. 
 
Administration of prior medications (i.e. drug treatments administered during the last 
month) and concomitant medications/therapies (i.e. medications ongoing at admission time 
and with a start date between admission to the research center and Day 4 or discharge) must 
be reported on the appropriate medication pages of the CRF, including dosage information, 
dates and time of administration and reasons for use. The international non-proprietary name 
and the trade name in brackets should be used for concomitant medications.  The total daily 
dose or the stat dose, if appropriate, should also be reported. Rescue therapies will be 
recorded on the specific rescue medication page, recording the same information as for 
concomitant therapies, plus the time of administration of each single dose. 
 
In case an inotrope should be necessary as rescue medication during the investigational product 
infusion period, the investigational product infusion should be terminated. 
6. CLINICAL ENDPOINTS 
6.1 Efficacy Endpoints 
1. Primary efficacy end-points:  
 
Change from baseline to 24 hours after infusion start (treatment period Day 1) in the 
E/Ea ratio assessed by tissue Doppler. 
 
2. Secondary efficacy end-points: 
 Change from baseline to 24 hours in the treatment period Day1 (addressing the 
differences between the changes at 6 and 24 hours from baseline) of the following 
Echo-Doppler parameters: 
- 
LV Ejection Fraction (EF) 
- 
LV end systolic and end diastolic volumes 
- 
Stroke volume index (SVI)  

 
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- 
E, A and E/A ratio 
 Change from baseline to 24 hours in the E/Ea ratio assessed by tissue Doppler 
(difference between the changes at 6 and 24 hours from baseline) 
 Others Tissue Doppler parameters such as Sa, Da and Aa 
 
Changes in dyspnea assessed at 3, 6, 12, 24, 48 hours after infusion start by Visual 
Analog Scale (VAS) (including only patients presenting dyspnea at baseline);  
 Area under the curve (AUC) on changes in dyspnea assessed at 3, 6, 12, 24, 48 hours 
after infusion start by VAS (including only patients presenting dyspnea at baseline); 
 Changes in BNP from baseline at 24 hours; 
 
Proportion of patients with hospital readmissions or emergency visits for 
cardiovascular reasons by Day 30; 
 
Proportion of patients with episodes of worsening HF defined by the need to increase 
the dose or reinitiate i.v. therapy with diuretics and/ or other inotropic agents during 
the hospitalization; 
 
Length of hospitalization. 
 
6.2 Safety Endpoints 
The following safety endpoints will be assessed during treatment and the post-
treatment/follow-up periods: 
 Incidence of adverse events;  
 Change in vital signs (including body temperature and dyspnoea); 
 Change in 12-lead ECG parameters; 
 Incidence of clinically or hemodynamically significant episodes of supraventricular or 
ventricular arrhythmias detected by continuous ECG dynamic monitoring; 
 Change in laboratory parameters (hematology, blood chemistry and urinalysis);  
 Change in renal function; 
 
Change in cTnT; 
 
Incidence of cTnT elevation relative (>50% or >20% relative increase over the cTnT 
levels at baseline, for patients with cTnT basal levels at baseline < or  ≥ of the 99% 
URL (upper reference levels, as defined for the Roche hs test in patients with normal 
renal function, eGFR ≥ 85 ml/min); in patients with eGFR below this value, the renal 
function variations must be considered in evaluating the significance of the cTnT 
changes); 

 
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 
Mortality at Day 30. 
 
 
6.3 PK Parameters 
 
After infusion, the following PK parameters will be measured and/or calculated for plasma E 
and Z isomers (when applicable) of istaroxime and its metabolites PST2915, PST2922, and 
PST3093, using the validated software WinNonLin® 5.2 or higher (Pharsight Corporation): 
 
Cmax: 
Maximum plasma concentration achieved after single dose 
 
tmax: 
Time to achieve Cmax 
 
AUC0-t: 
Area under the concentration curve from administration to the last observed 
concentration time t, calculated after single dose with the linear trapezoidal 
method 
 
AUC0-: 
Area under the concentration/time curve extrapolated to infinity, calculated, if 
feasible, as AUC0-t + Ct/λz, where Ct is the last measurable drug concentration 
 
λz: 
Terminal elimination rate constant, calculated after single dose, if feasible, by 
log-linear regression using at least 3 points 
 
t1/2: 
Half-life, calculated, if feasible, as ln2/λz 
 
MRT: 
Mean Residence time, calculated, if feasible, as AUMC0- / AUC0- - ti/2, 
where AUMC0- is the area under the moment curve extrapolated to infinity 
and ti is the infusion duration 
 
ClT: 
Total Body Clearance, calculated, if feasible, as Dose / AUC0- 
 
Vz: 
Volume of distribution of the terminal phase calculated, if feasible as  
Dose / (AUC0- x λz) 
 
Vss: 
Volume of distribution at steady state calculated, if feasible as  
(Dose x AUMC0-) / AUC0-2 
 
 
The quality of log-linear regression (and, consequently, the reliability of the extrapolated PK 
parameters) should be demonstrated by a correlation coefficient R2 > 0.8. 
 
After infusion, the following PK parameters will be measured and/or calculated for urine E and 
Z isomers (when applicable) of istaroxime and its metabolites PST2915, PST2922, and 
PST3093, using the validated software WinNonLin® 5.2 or higher (Pharsight Corporation): 
 
Ae: 
Total amount of analyte excreted in urine 

 
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Ae%: 
Percentage of analyte excreted in urine  
 
ClR: 
Renal Clearance calculated, if feasible as Ae / AUC0- (for istaroxime only) 
 
 
These endpoints will be addressed in all the Italian patients and in a subset of Chinese patients. 
Data below the lower quantification limit (LQL) will be considered as 0 in the calculations and 
presented as BLQL in listings and tables. As a consequence of BLQL (i.e. 0) values, calculated 
geometric means (if requested) could be null. For this reason, in the presence of any null value, 
the geometric mean will be reported as not calculated (NC).  
7. STUDY PROCEDURES 
 Screening period (between Hours -24 to -1)   
 
Within a maximum of 24 hours before administration of study medication (istaroxime or 
placebo), a medical screening will be performed on all prospective patients to assess suitability 
for the study. Prior to conducting any study specific procedures, the investigator or his/her 
designee will explain the study fully to the patient and provide him/her with a copy of the 
Patient Information Sheet and Informed Consent Document. If the patient is willing to 
participate in the study, s/he and the investigator or his/her designee will both sign the 
Informed Consent Document and a copy of the signed document will be kept by the patient.  
1) Obtaining of ICF; 
2) Evaluation of screening inclusion and exclusion criteria; 
3) Stratification: Atrial fibrillation YES/NO; 
4) 
Medical history, demographic/anthropometric data; 
5) Alcohol and tobacco consumption patterns; 
6) Medication history (previous and concomitant), including a list of the patient’s previous 
and current intake of vitamins, over-the-counter drugs and herbal products for the last 3 
months. Chronic oral treatment with digoxin and other medications (e.g. diuretic and/or 
vasodilator) will also be recorded; 
7) Vital signs (including body temperature and dyspnea); 
8) 12-lead ECG; (Data obtained within 3 hours before the patient signed informed consent 
form can be accepted.) 
9) Holter ECG; 

 
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10) Standard blood chemistry and Standard haematology; 
11) TSH, T3 and T4; 
12) Estimated glomerular filtration rate (eGFR); 
13) Digoxin assay; 
14) Urinalysis; 
15) Alcohol breath test (at random, if deemed necessary by the Investigator/designee); 
16) Serum pregnancy test (beta human chorionic gonadotropin [β-hCG]) for females of 
childbearing potential; 
17) Echocardiography; 
18) Serum creatinine clearance; 
19) Human Immunodeficiency Virus, Hepatitis B and Hepatitis C tests; 
20) Adverse events monitoring (after obtaining informed consent); 
21) BNP, NT-proBNP and cTnT; 
 
 Treatment period (Day 1)  
1) Confirm eligibility; 
2) Randomization of patients (after eligibility has been confirmed);  
3) Insertion of multiple lumen intravenous catheter (or administration via peripheral vein); 
4) Start istaroxime or placebo infusion (date and time of infusion start must be recorded in 
the CRF); 
5) cTnT (at pre-dose in two samples, 3, 6, 12, 24, 48 and 72 hours after start of infusion); 
6) NT pro-BNP at baseline and at the end of 24 hours infusion 
7) Blood sample collection for metabolites and PK  (at pre-dose, 0.5, 3, 6, 12, 24 hours after 
start of infusion and 0.25, 0.5, 1, 4, 12, 24, 48 hours after the end of infusion) in a subset of 
Italian patients and in a subset of Chinese patients pharmacokinetics and metabolism of 
istaroxime shall also be studied; 
8) Vital signs (including arterial oxygen saturation and dyspnoea at pre-dose, 3, 6, 12 and 24 
hours after the start of infusion); 
9) 12-lead ECG profile (pre-dose, 3, 6, 12 hours and between 23 hours and 23 hours 55 
minutes after infusion, totally 5 times, each time 3 measurements); 
10) Stop 24-hour Holter ECG (Day -1 recording; Holter ECG monitor stops automatically 24 
hours after the recording has been started; 
11) Start 24-hour Holter ECG (Day 1 recording; to be started immediately before initiation of 
the study drug infusion); 
12) Echocardiography at baseline and 6 and 24 hours after infusion start; 
13) 24-hours urine collections for measurement of istaroxime and its metabolites and urinary 

 
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creatinine for the calculation of the creatinine clearance 
14) Blood collection for K+ and eGFR between 23 hours and 30 minutes and 23 hours and 55 
minutes since infusion start 
15) Concomitant medication monitoring (including chronic medication; dose, date and time 
must be recorded in the CRF); 
16) Adverse events monitoring. 
 
 Post-treatment period (Day 2 to Day 4) 
Evaluations on Day 2 (end of the 24 hour infusion) include: 
1) Vital signs (including arterial oxygen saturation and dyspnoea); 
2) 12-lead ECG (triple ECGs) (between 23 hours and 23 hours 55 minutes since infusion 
start); 
3) Stop 24-hour Holter ECG (Day 1 recording; to be stopped immediately after the end 
of study drug infusion;   
4) Start 24-hour Holter ECG (Day 2 recording); 
5) Stop istaroxime or placebo infusion (date and time of infusion end must be recorded in 
the CRF); 
6) Serum potassium levels and 24-hour urine collection for measurement of istaroxime 
metabolites and urinary creatinine for calculation of the creatinine clearance; 
7) Serum creatine clearance and calculation of eGFR; 
8) cTnT (50% or 20% relative increase over the basal cTnT levels, respectively for 
patients with cTnT basal levels < or ≥ of the 99% URL (upper reference levels, as 
defined for the Roche hs test, in patients with normal renal function, eGFR≥85 ml/min); 
in patients with eGFR below this value, the renal function variations must be considered 
in evaluating the significance of the cTnT changes); 
9) NT pro-BNP  
10) Metabolites 
11) Echocardiography; 
12) Concomitant medication monitoring (including chronic medication must be recorded in 
the CRF); 
13) Adverse Events monitoring 
 
Evaluations on Day 3 include: 
1) Vital signs (including arterial oxygen saturation and dyspnea) 
2) 12-Lead ECG (single ECGs) 
3) Stop 24-hour Holter ECG   
4) Standard hematology; 
5) Standard blood chemistry; 
6) Serum Potassium levels; 

 
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7) 24-h urine collection for measurement of istaroxime metabolites and urinary creatinine 
for the calculation of creatinine clearance; 
8) Calculation of eGFR; 
9) NT-proBNP 
10) cTnT; 
11) Metabolites; 
12) Echocardiography; 
13) Adverse events monitoring; 
14) Concomitant medication monitoring (including chronic medication must be recorded in 
the CRF) 
 
Evaluations on Day 4 include: 
1) cTnT and NT-proBNP (at 72 hours after start of infusion)  
2) Vital signs (including arterial oxygen saturation and dyspnoea) 
3) Physical examination (HF signs included) 
4) 12-lead ECG 
5) Adverse events monitoring 
6) Concomitant medication monitoring (including chronic medication) 
7) Metabolites (at 72 hours after start of infusion) 
8) Serum potassium and creatinine levels for calculation of eGFR 
9) Creatinine clearance 
 Follow-up period and visit (Day 5 to Day 30) 
During the follow-up period the Investigator/designee will make every effort to establish 
patient outcomes. 
Evaluations on Day 30 (follow-up visit) include: 
 
1) Vital signs (including arterial oxygen saturation and dyspnoea); 
2) 12-lead ECG in triplicate; 
3) Calculation of eGFR; 
4) Standard hematology; 
5) Standard blood chemistry; 
6) NT-proBNP ; 
7) cTnT; 
8) Urine pregnancy test (β-HCG) for females of childbearing potential; 
9) Urinalysis; 

 
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10) Physical examination (HF signs included); 
11) Adverse events monitoring; 
12) Concomitant medication monitoring (including chronic medication). 
 
8. EFFICACY ASSESSMENTS 
 
Change from baseline to 24 hours after infusion start in the E/Ea ratio assessed by tissue Doppler 
will be assessed as primary end-point. Other echocardiographic parameters (LVEF, LV end 
diastolic and end systolic volumes, SVI, E/A ratio, other Tissue Doppler parameters such as Sa, 
Da and Aa) will be assessed as secondary end-points. These parameters will be assessed also at 
48 hours as further exploratory analyses. 
 
Dyspnoea will be assessed at baseline using a 100 mm VAS scale. Further assessments will be 
repeated at 3, 6, 12, 24 and 48 hours after the start of drug infusion only in patients presenting 
dyspnoea at baseline.  
NT-proBNP will be measured by the local laboratory only for evaluation and screening inclusion. 
While at baseline, at 24 hours after infusion start (day 2), on Day 3, at day 4 or at discharge) and 
at day 30, NT-proBNP will be measured by the central laboratory 
Episodes of worsening HF, defined by the need to increase the dose or reinitiate i.v. therapy with 
diuretics and/ or other inotropic agents during the hospitalization, will be recorded.  
 
9. SAFETY ASSESSMENTS 
1. AEs will be monitored throughout the study (i.e. from the time of signature of informed 
consent to treatment end + 30 days); 
2. Physical examination (HF signs included) at screening, baseline, on study Day 4 or at 
discharge, and on study Day 30 during the follow up visit; 
3. Vital signs (HR, BP, respiratory rate, body temperature and dyspnoea)) will be 
measured at screening,  (concomitantly to hemodynamic measurements) during the pre-
treatment period, at 3, 6, 12, and 24 hours after infusion start, and then on study Day 3 
and Day 4 or at discharge, and on study Day 30 during the follow up visit; 
4. 12-lead ECG (HR, RR and PR intervals, QRS duration, QTc, rhythm, ectopy, 
ischemic changes) will be recorded and locally analyzed at screening and on study 
Day1, Day2, Day 3 and Day 4 or at discharge, and on study Day 30 during the follow 
up visit; 
5. Continuous Holter monitoring will take place during screening, treatment (Day 1), and 
Day 2. 
6. Blood samples will be collected and sent to the local laboratory during screening, 48 
hours after infusion starts and on Day 30 to measure the following safety parameters: 

 
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 a) Hematology: red blood cell (RBC) count, Hb, hematocrit (Hct), white 
blood cell (WBC) count, differential WBC count, platelet count; 
 b) Blood chemistry: electrolytes (Na+, K+, Ca++, Cl-), albumin, total 
protein, glucose, creatinine, eGFR, BUN (blood urea nitrogen), alkaline 
phosphatase, ALAT, ASAT, total and conjugated bilirubin, gamma 
glutamyl transferase (γ-GT), total cholesterol, triglycerides; 
 
7. Blood samples for creatinine will be collected by the local laboratory at each time after 
infusion start 
8. cTnT or cTnI will be measured locally during screening. While cTnT only will be 
measured by the central laboratory , at pre-dose in two samples, at 3, 6, 12, 24, 48,  72 
hours after infusion start or at discharge and at 30days by using the hs test from Roche 
cobas. Incidence of cTnT elevation (>50% or > 20% relative increase over the basal 
cTnT levels at baseline, for patients with cTnT levels at baseline < or  ≥ of the 99% 
URL (upper reference levels, as defined for the Roche hs test, in patients with normal 
renal function, eGFR ≥85 ml/min); in patients with eGFR below this value, the renal 
function variations must be considered in evaluating the significance of the cTnT 
changes). 
 
 
10. REPORTING OF ADVERSE EVENTS 
 10.1 Definition of Adverse Event (AE) 
Adverse event (AE) is an adverse medical event that occurs following informed consent 
signature, the event is not necessarily related to the treatment. Therefore, adverse events 
may be any discomfort and unconscious signs (such as abnormal laboratory examination 
results), symptoms or illnesses. 
● Any adverse event, whether serious or not, whether related to the treatment or not, should 
be recorded on the CRF. 
● Investigator should follow up the patients that dropped out because of any adverse event 
until recovery, and record the date of AE occurring and recovering. 
 If during adverse event follow-up, the adverse event progresses to an "SAE", or if a subject 
experiences a new SAE whose relationship to the study drug(s) cannot be ruled out, the 
investigator must immediately report the information to the sponsor.  

 
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Even if the subject does not return to normal or to his or her previous state, the follow-up can 
be considered finished when the following procedures have been taken:  
 The investigator judges that the follow-up of the subject concerned is no longer necessary 
based on the progress made during the follow-up,  
 The reason for such a judgment is entered as a comment on the case report form for the 
follow-up. 
10.2 Definition of Serious Adverse Event (SAE) 
 ‘Serious adverse event’ is an event that induces hospitalization, prolonging the length of 
hospitalization, disability, affecting work ability, imperiling life or death, and congenital 
malformation. 
(1) SAE is one of the following situations: 
● That causes death 
● Teratogenesis, carcinogenesis or birth defect 
● That is dangerous to life and could induce permanent or remarkable disability 
● That induces permanent injury in organ function 
● That induces hospitalization or prolongs the length of hospitalization 
(2) Hospitalization 
According to the clinical study reports, the adverse events leading to hospitalization or 
prolongs the length of hospitalization will be regarded as serious adverse event (SAE). 
Including: hospitalized in a healthcare facility for the first time (even less than 24 hours) and 
the hospital transferred to the emergency/intensive care ward. 
The hospitalization does not include the following institutions: 
 
rehabilitation institutions; 
 
asylum; 
 
occasional care service ; 
 
nursing institution; 
 
sanatorium; 
 
routine hospitalization; 

 
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(3) Hospitalization or prolonging the length of hospitalization without emergency clinical 
adverse event does not belong to SAE. For example: 
 
hospitalization because of previous disease, which is unrelated to adverse event 
and with no worsening (e.g. examinations for the continuous abnormalities in 
laboratory tests prior to treatment); 
 
for social reasons (such as homelessness); 
 
for management reasons (such as routine health examinations per year or for 
convenience of drug administration); 
 
hospitalization assigned or permitted by the protocol during the trial; 
 
voluntary hospitalization unrelated to AE (such as selective plastic surgery); 
 
Scheduled treatment or surgery for the whole protocol and/or subject’s benefit, 
which is recorded at baseline; 
 
Just for infusion of blood products. 
10.3  Relativity assessment of AE and Study Drug 
 
Relativity between AE and test drugs is assessed by investigator according to the following: 
1) Certainly related 
Evidence of having taken test drugs; time sequence of AE happening and taking the drugs is 
credible; more rational to explain for AE with test drugs than other reasons; positive 
withdrawal reaction; positive repetition medication test; AE pattern accords with the past 
understanding of such drug or this kind of drugs. 
2) Possibly related 
Evidence of having taken test drugs; time sequence of AE happening and taking the drugs is 
credible; AE may be induced by the test drugs, or other reasons; positive withdrawal reaction. 
3) Possibly unrelated 
Evidence of having taken test drugs; AE is more likely to be induced by other reasons; 
negative or equivocal withdrawal reaction; negative or equivocal repetition medication test. 
4) Certainly unrelated 
The patient has not taken test drugs; or time sequence of AE happening and taking the drugs is 
incredible; or AE is induced by other notable reasons. 
5) Uncertain 
 

 
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10.4 Severity assessment of Adverse Event 
 
The strength or the serious degree of the adverse events can be divided into3 levels: 
Mild: mild discomfort, no significant impact on daily life (work and study); 
 Moderate: moderate discomfort and impact on daily life, patients self-conscious symptom 
obvious, but can stand, no need to stop medicine 
Severe: severe discomfort, significant impact on daily life, need for staying in bed, patients 
self-conscious symptom obvious is remarkable, and cannot stand, so need to stop medicine 
 
10.5 Reporting of SAE 
 
Any ‘serious adverse event’ happens during the trial (from the time of signature of informed 
consent to visit Day 30):  
Whether it is related to the drugs, the investigator should fill in ‘Serious Adverse Event 
Reporting Form and within 24 hours after learning it, informs, by fax or e-mail, CFDA, local 
drug supervising departments, NHFPC, the sponsor- CVie Therapeutics Company Limited, 
the site's principal investigator and the ethics committee. 
Department 
Name 
Contact Information 
CFDA 
China Food and Drug 
Administration 
Tel: 
010-88330724 
Fax: 
010-88363228 
NHFPC 
Health Care Authority - 
National Health and 
Family Planning 
Commission 
Tel: 
010-68792201 
Fax: 
010-68792734 
Local Food and 
Drug Administration 
 
 
 
Sponsor:  CVie 
Therapeutics 
Company Limited  
Eric Tseng 
Email: erictseng@cvie.com 
Giuseppe Bianchi 
Tel: +39-02745569; +39-3488545560 
Email: 
giuseppebianchi1935@cvie.com 

 
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10.6 Reporting of Pregnancy 
If a woman becomes pregnant during the study period until Day 30, the investigator should 
report the information to the sponsor as if it is an SAE. The expected date of delivery or expected 
date of the end of the pregnancy, last menstruation, estimated fertility date, pregnancy result and 
neonatal data etc., should be included in this information. 
The investigator will follow the medical status of the mother, as well as the fetus, as if the 
pregnancy is an SAE and will report the outcome to the sponsor. 
When the outcome of the pregnancy falls under the criteria for SAEs [spontaneous abortion, 
induced abortion, stillbirth, death of newborn, congenital anomaly (including anomaly in a mis-
carried fetus)], the investigator should respond in accordance with the report procedure for SAEs. 
Additional information regarding the outcome of a pregnancy (which is categorized as an SAE) 
is mentioned below. 
 
"Spontaneous abortion" includes abortion and missed abortion. 
 
Death of an infant within 1 month after birth should be reported as an SAE regardless of 
its relationship with the study drug.  
 
If an infant dies more than 1 month after the birth, it should be reported if a relationship 
between the death and intrauterine exposure to the study drug is judged as "possible" by 
the investigator. 
 
In the case of a delivery of a living newborn, the "normality" of the infant is evaluated at 
the birth. 
"Normality" of the miscarried fetus is evaluated by visual examination unless test results which 
indicate a congenital anomaly are obtained prior to miscarriage. 
11. ADVERSE DRUG REACTION (ADR) 
11.1 Definition of ADR 
 
Harm reaction which unrelated to the purpose of treatment with the use of given medications 
at a normal dosage. 
11.2 Reporting and monitoring of ADR 
 
Reporting and monitor ADR is the process of finding, reporting, evaluating and controlling the 
adverse drug reaction. 
12.  PHARMACOKINETIC ASSESSMENT  
Plasma concentrations of istaroxime and its metabolites PST2915, PST2922, and PST3093 will 

 
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be measured by liquid chromatography coupled to mass spectrometry (LC/MS/MS). When 
applicable, E and Z isomers will be measured separately. 
 
In a subset of patients, a blood sample will be collected during the study period. In addition, 
a full PK profile will be established in a subset of Italian patients and in a subset of Chinese 
patients (to have 16 patients with istaroxime 0.5 µg/kg/min and 8 patients with istaroxime 1.0 
µg/kg/min 24 total patients). 
Blood will be sampled at the following time-points: 0 hour (pre-dose); 0.5, 3, 6, 12, and 24 
hours after infusion start; 0.25, 0.5, 1, 4, 12, 24 hours after infusion end. 
 
In the same subset of patients, concentrations of istaroxime and its metabolites will be 
measured by LC/MS/MS in urine collected from pre-dose to 48 hours after infusion end (3 
collections will be scheduled: the first from pre-dose to 24 hours after investigational product 
infusion start, the second at 48 hours after infusion start, and the third at 72 hours after infusion 
start). 
 
13.  DATA MANAGEMENT 
13.1 Filling the CRFs and its handover 
 
Data Management will identify and implement the most effective data acquisition and 
management strategy for the clinical trial protocol and deliver datasets which support the 
protocol objectives. Subject data will be entered into an e-CRFs in a validated data system. 
 
13.2 Data entry and amendment 
 
The data manager will prepare a Data Management Plan (DMP) to describe the procedures and 
processes of data collection and data coding (including data entry, data validation, data 
clarification process and database closure). For data coding (e.g., AEs, medical and surgical 
history, concomitant diseases, prior and concomitant medication), internationally recognized 
and accepted dictionaries will be used. The coding of the medical history, concomitant diseases 
and adverse events will be performed using MedDRA and the coding of concomitant medication 
by using WHO drug dictionary (WHODD). The clinical data will be reviewed for legibility, 
completeness, and logical consistency. Automated validation programs identify missing data, 
out-of-range data, and other data inconsistencies. Requests for data clarification will be 
forwarded to the investigative site for resolution. All plausibility checks will be defined in the 
Data Validation section of the DMP to be approved by the sponsor. Clinical data management 
will be performed in accordance with applicable CRO standards and data cleaning procedures 
with the objective of removing errors and inconsistencies in the data which would otherwise 

 
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impact on the analysis and reporting objectives. 
 
13.3 Locking the Database 
 
At the completion of the data management activities and when the clinical data are clean, the 
clinical database will be locked by the Data Manager and the clinical data released to the 
statistician for the analysis. With database lock no further changes are allowed to the clinical 
data. 
14 STATISTICAL ANALYSIS 
14.1 Datasets 
 
Four populations will be defined: 
1) Intention-To-Treat population (ITT): all randomized patients who will take at least one dose 
of the study medication and with at least one available evaluation of efficacy after the baseline. 
2) Per-protocol population (PP): all randomized patients from the ITT population without any 
major protocol deviation; exact definition of major protocol deviations will be discussed by the 
clinical team case by case during the blind review of the data. 
3) Safety population: all randomized patients who will take at least one dose of the study 
medication. 
4) Pharmacokinetic population (PK): all randomized patients selected for the PK analysis (32 
total patients).  
The primary efficacy endpoint will be analysed both in the ITT and in the PP populations; the 
secondary efficacy variables will be analysed in the ITT population. The PK analysis will be 
performed in the Pharmacokinetic population. 
Analysis of safety variables will be performed in the safety population. In case of deviation 
between as-randomised treatment and treatment actually received, the treatment actually 
received will be used in the analysis of safety and PK variables (i.e. an as-treated analysis will 
be performed). 
 
14.2 Statistic Plan  
All statistical analyses will be performed using Statistical Analysis Systems (SAS®) Software 
(release 9.2) on a Windows 7 pro operating system. 
 
Sample size 

 
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No formal sample size calculation has been calculated since there is no preliminary study to 
evaluate these endpoints at 24 hours after the infusion start. 
In a previous study [14], there was a statistically significant decrease of the primary efficacy 
endpoint with istaroxime 0.5 μg/kg/min respect to placebo at 6 hours (twenty-nine patients in 
both groups). Since this study will investigate the primary efficacy endpoint at 24 hours and it 
is supposed that the profile at 24 hours should be similar to the one at 6 hours, it is expected that 
forty patients per group will be sufficient to evaluate a potential difference between istaroxime 
groups and placebo group on the primary efficacy endpoint at 24 hours. 
 
Descriptive statistics 
Descriptive statistics will be provided for each variable in summary tables by treatment group. 
Quantitative variables will be summarized by using n (sample size), mean, 95% confidence 
interval (CI) of the mean, median, standard deviation, minimum and maximum. Categorical 
variables will be summarized by using frequency count and percent distribution. 
 
Principles of statistical analysis 
For quantitative efficacy and safety variables, analysis within treatment groups will be presented. 
Mean changes from baseline and their 95% CIs will be calculated.  
For all inferential analyses, p-value will be rounded to three decimal places. Statistical 
significance will be declared if the rounded p-value will be less than or equal to 0.05. 
 
Subject’s accountability 
Disposition of patients, patient status and patients excluded from populations will be 
summarized by treatment group. 
 
Description of the population-description of baseline characteristics 
Descriptive statistics will be presented at baseline for demographic and baseline characteristics 
for the ITT population. 
 
Primary efficacy endpoint 
The primary efficacy endpoint (change from baseline in E/Ea ratio) will be analyzed using a 
linear mixed model for repeated measures including treatment, centre, timepoint, gender, 
baseline cTnT (normal <URL, abnormal ≥URL), atrial fibrillation (Yes/No) and 
treatment*timepoint interaction as fixed effects and baseline and baseline*timepoint 
interaction as covariates. 
The primary comparison will be 0.5 μg/kg/min dose of istaroxime versus placebo at 24 hours. 
Highest dose of istaroxime (1.0 μg/kg/min) versus placebo will be tested as a secondary 
comparison. 
Additional analyses separated by cohort will be implemented for sensitivity purpose.  
 

 
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Secondary efficacy endpoints 
The following secondary endpoints: 
 
Change from baseline to 24 hours in the treatment period Day 1 (addressing the 
differences between the changes at 6 and 24 hours from baseline) of the following 
Echo-Doppler parameters: 
o LV Ejection fraction (EF) 
o LV end systolic and end diastolic volumes 
o Stroke volume index (SVI) 
o E, A and E/A ratio  
 
Change from baseline to 24 hours in the E/Ea ratio assessed by tissue Doppler 
(difference between the changes at 6 and 24 hours from baseline) 
 Others Tissue Doppler parameters such as Sa, Da and Aa 
 
Changes in dyspnoea using VAS score will be analysed using a mixed model for repeated 
measures similar to the one used for the primary efficacy endpoint. 
A global test considering multiple echocardiographic parameters at 6 and 24 hours will also be 
performed. 
 
AUC on changes in dyspnea by VAS and changes in BNP will be analyzed using an ANCOVA 
model with treatment, centre, gender, baseline cTnT (normal <URL, abnormal ≥URL) and atrial 
fibrillation (Yes/No) as fixed effects and baseline dyspnea as covariate. 
 
Number and proportion of patients with: 
 
hospital readmissions or emergency visits for cardiovascular reasons within Day 30 
 
episodes of worsening HF defined by the need to increase the dose or reinitiate i.v. 
therapy with diuretics and/ or other inotropic agents during the hospitalization 
will be summarized by treatment groups using descriptive statistics.  
Length of hospitalization will be summarized by treatment group using descriptive statistics. 
 
Safety endpoints 
The number and the percentage of patients experiencing adverse events, adverse drug reactions, 
serious adverse events and adverse events leading to study withdrawal will be summarized by 
treatment group. Adverse events will also be summarized by treatment group by means of 
System Organ Class and Preferred Term using the MedDRA dictionary. 
Vital signs (including body temperature and dyspnoea), 12-lead ECG parameters, incidence of 
clinically or hemodynamically significant episodes of supraventricular or ventricular 
arrhythmias, laboratory parameters, renal function, cTNT, increase of cTNT and mortality will 
be summarized by treatment group using descriptive statistics. 
 
Interim Analysis 
A safety interim analysis on cTNT changes is planned after the completion of Cohort 1. 

 
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PK analysis of istaroxime and its metabolites 
PK parameters Cmax, AUC0-t, AUC0- (if feasible) and Ae for istaroxime and its metabolites will 
be analysed using analysis of variance (ANOVA). The data will be dose-normalised and 
transformed prior to analysis using a neperian logarithmic transformation.  
The statistical analysis will take into account gender, dose, centre, baseline cTnT (normal <URL, 
abnormal ≥URL) as fixed factors. The statistical method for testing linearity is based upon the 
90% confidence interval for the ratio of the population geometric means (Dose1/Dose2), for the 
parameters under consideration. This method is equivalent to the corresponding two one-sided 
test procedure with the null hypothesis of no linearity at the 5% significance level.  
Acceptance criterion for linearity will be a 90% confidence interval of the ratio of geometric 
means within the range 75.00 – 133.33%. 
tmax will be analyzed using a non-parametric model providing an estimate and 90% CI to the 
median difference of dose pairs. 
 
15. QUALITY CONTROL AND QUALITY ASSURANCE 
 
During the trial，monitor will be appointed by Sponsor to visit the study centers regularly, in 
order to ensure rigorous performance of all contents in the protocol and accuracy of study 
materials. 
Investigators must attend unified training to unify the record manner and the judgment 
standard. 
● the clinical trial process should be strictly conducted in a blind state.   
●Investigators should fill in the CRF accurately, detailed, and seriously according to the 
requirements, to insure that contents of the CRF are true and credible; 
●Judgment standard of laboratory examination abnormality should be subject to the 
normal range of the centers; 
●All observation results and findings of the trial should be verified to ensure the 
reliability of the data and that each conclusion comes from source data. Corresponding 
data management should be taken during the trial and data processing period; 
●For possible drop-out, active measures should be taken to keep the dropout rate within 
20%. 

 
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15.1 Drug Accountability 
The Investigator must ensure the maintenance of accurate records of the amounts and dates 
investigational products were received from the Sponsor, dispensed and unused supplies returned 
or destroyed. All clinical supplies must be accounted for at the termination of the study and a 
written explanation provided for discrepancies. All unused supplies must be returned promptly to 
the Sponsor or destroyed properly at the study site; written authorization from the Sponsor must be 
obtained prior to destruction. The Investigator will maintain a record of the amount and dates when 
unused supplies were returned to the Sponsor or destroyed locally. When the above tasks are 
delegated to a Pharmacist, the latter is under the supervision of the Investigator, who remains 
ultimately responsible for the maintenance of accurate drug dispensing records. 
 
16. MONITORING AND STUDY COMMITTEES 
 
16.1 Monitoring 
 
 
CVie Therapeutics Company Limited, Hong Kong, is the study Sponsor. CVie Therapeutics will 
delegate CROs (CROS NT, Verona and CRONOS Srl, Ravenna) for the operational aspects of 
the study (set up, e-CRF and data management) and for monitoring of the Italian centers. Local 
operational representatives will be determined in each participating country before the launch of the 
study. 
At mutually convenient times during the study, the Investigator will allow CVie Therapeutics or its 
representative to review all completed e-CRFs and related source documentation, i.e. patients’ 
office, hospital, clinic and laboratory records. At timely intervals, and at the closing of the 
study, all investigational products will be accounted for, and dispensing records will be made 
available to CVie Therapeutics or its representative. 
 
In case CRFs require support information, CVie Therapeutics may request copies of patient 
records or other source documents from the Investigator. All necessary steps will then be 
taken to protect patient identity. Adherence to local and national laws governing protection 
of patient identity will be ensured. 
 
16.2 Steering Committee 
 
The main tasks of the Steering Committee will be to provide input to the Sponsor on proto-
col content, follow study progress, and answer technical or medical questions on the study. 
This Committee will meet periodically (even by teleconference) during the planning, imple-
mentation and conduct of the Study and will be under the direction of the two Committee Re-
sponsible, one for each Country (China and Italy): 
 
 Prof. Jun  Huang - Chairmen and Responsible for China 
The First Hospital Affiliated to Nanjing Medical University 

 
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Prof. Marco Metra - Co-Chairmen and Responsible for Italy 
Head of the U.O. of Cardiology, Applied and Experimental Medicine  
Brescia University – Spedali Civili Brescia- Italy 
 
The Steering Committee Members will be:  
 
For China 
Prof. Jian Zhang  
 
 
 
 
 
 
 
 
 
 
 
 
 
Chairman of the Association of Heart Failure in China, Fuwai Hospital CAMS&PUMC - China 
 
Prof. LIU Lisheng 
Chinese Academy of Medical Sciences - Fu Wai Hospital and Cardiovascular Institute – China 
 
For Italy: 
Prof. Gianfranco Parati 
Internal Medicine Bicocca University, Milan – Head of the U.O. of Cardiology S. Luca Hospital – 
Centro Auxlogico Italiano – Milan, Italy 
 
 
 
16.3 Data Safety Monitoring Board  
 
An independent DSMB will be established prior to study initiation. The committee will consist 
of at least two physicians (cardiologists) and a statistician who are not otherwise associated 
with the study, and who are experienced in HF multicenter trials. 
The main tasks of the Board will be to provide recommendations to the Sponsor concerning 
safety aspects and study continuation/discontinuation within a cohort and from one cohort to 
the next one. If a cohort is stopped, the randomisation will be ended and the study treatment 
will be prematurely discontinued in patients on treatment. Escalation to the next dose level 
will not take place. 
After the completion of cohort I, a safety interim analysis will be performed and the DSMB 
members will evaluate the incidence of troponin (cTnT) elevation. At interim analysis, the 
DSMB may recommend to stop the cohort if the incidence of troponin elevation is found to be 
statistically significantly higher on the istaroxime dose than on placebo, or if this difference is 
deemed to be clinically relevant. Consequently, the clinical study will be prematurely terminated. 
Randomization data and datasets needed to conduct the interim analysis will be given to the 
DSMB statistician.  The DSMB will be unblinded  when  performing  the  interim  analysis. 
During the interim analysis, DSMB members will analyze and discuss the results in closed 

 
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sessions. The DSMB is required to keep confidential and undisclosed until study conclusion: i) 
these discussions, ii) the detailed minutes thereof, and iii) the interim analysis results. At study 
conclusion, after database lock and unblinding, the DSMB will make the minutes and interim 
analysis results available as part of the study documentation. 
 
Members of the DSMB will be:  
 
Prof. Aldo Maggioni – Chairman of the DSMB 
Head of Centro Ricerche Associazione Nazionale Medici Cardiologi (ANMCO) 
Firenze – Italy 
 
Prof. Luigi Tavazzi – Member of the DSMB as Physician  
Scientific Director GVM Care and Research Unit Maria Cecilia Hospital 
Ravenna- Italy 
 
Prof. Renato Urso – Member of the DSMB as Statistician  
Unità of Pharmacology "Giorgio Segre" – Siena University  
Siena - Italy 
17. ETHICS 
Prior to the starting of the trial, the study protocol, informed consent form and other materials 
for the patients should be submitted to the ethics committee of the responsible study center for 
approval. Sponsor must obtain the signed and approval document from the ethics committee 
before starting the study. Any amendments of the protocol and ICF should be approved by the 
EC. ICF should be signed again if necessary. 
Investigator should obtain patient’s informed consent and the signed ICF before 
implementation. Ensure patients’ rights and keep confidential to patients’ information 
throughout the study. 
 
18. RESPONSIBILITY AND PUBLICATION 
 
Investigator should keep confidential all information provided by sponsor. Other staff 
participating in the study and ethics committee should keep confidential the information as 
well. Without the written permission, the information cannot be provided to others. 
All data and results of the clinical study are possessed by sponsor and investigator. 
Investigator agrees not to submit the results of this study for publication or presentation before 
the sponsor review the accuracy and no leakage of confidential information in the manuscript. 

 
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19. ANTICIPATED PROGRESS AND COMPLETION DATE OF THE STUDY 
Procedure 
Time 
Working Time 
Final version of study protocol, ICF and 
CRF  
 
 
Approval document of ethics committee 
 
 
Initial meeting and articles delivery of each 
center 
 
 
The first subject into screening 
 
 
The last subject enrolled 
 
 
The last subject finishing efficacy 
observation 
 
 
The collection of CRF 
 
 
Statistics analysis report 
 
 
Clinical report 
 
 
Summing-up meeting 
 
 
Anticipated completion date 
 
 
 
 
 
 
 
 

 
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20. REFERENCES 
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Mozaffarian D, Mussolino M,  Nichol G, Roger VL, Rosamond W, Sacco R, Sorlie P, 
Roger VL, Thom T, Wasserthiel- Smoller S, Wong ND, Wylie-Rosett J; on behalf of the 
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[10].Gheorghiade M, Mebazaa A. The challenge of acute heart failure syndromes. Am J 
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[11].Nieminen MS et al. Executive summary of the guidelines on the diagnosis and treatment 
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[12]. Istaroxime–Agent 
for Congestive Heart Failure.http://www.drugdevelopment- 
technology.com/projects/istaroxime/ Accessed 18 August 2008.  
[13]. Gheorghiade M, Ambrosy AP, Ferrandi M, Ferrari P. Combining SERCA2a activation 
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[14]. Gheorghiade M. et al. Haemodynamic, echocardiographic, and neurohormonal effects of 
Istaroxime, a novel intravenous inotropic and lusitropic agent: a randomized controlled 
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[15].Rocchetti M et al. Modulation of sarcoplasmic reticulum function by Na+/K+  pump 
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aminoethoxy)imino)androstane-6,17-dione hydrochloride]. J Pharmacol Exp Ther 2005; 
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[16].Micheletti R et al. Istaroxime, a stimulator of sarcoplasmic reticulum calcium 
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[17].Recchia F. Haemodynamic profile of Istaroxime in normal farm pigs - Final report. 
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[18].Sabbah HN et al. Hemodynamic properties of a new-generation positive luso-inotropic 
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[19].Mattera GG et al.  Istaroxime: a new luso-inotropic agent for heart failure. Am J Cardiol. 
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[20].Investigator’s Drug Brochure: Istaroxime, Edition number 1, 21 January 2013. 

 
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21. DECLARATION SIGNATURE PAGE 
The clinical study of the safety and efficacy of Istaroxime in Treatment of Acute 
Decompensated Heart Failure - A multicenter, randomized, double-blind, placebo 
controlled, parallel group clinical study. 
 
Declaration of investigator: 
I have read the protocol carefully. I will conduct this clinical trial according to the Declaration 
of Helsinki, as well as to the moral, ethical and scientific principles required by GCP. I agree to 
conduct this clinical study in accordance with GCP, the protocol design and requirements, and 
fulfill the investigator’s responsibility. I have known the procedures and time requirements of 
reporting SAE, and I will record and report the SAE and give medical treatment to the subject 
in time. I ensure that all data will be recorded in CRF accurately, completely and timely. I will 
support and coordinate with the monitor. And I will accept the monitoring, auditing or 
inspection to ensure the quality of the clinical trial. 
 
Responsible study center: 
Principal Investigator (signature): ______________    Date: _____________ 
    
Study center: 
Principal Investigator (signature): ______________    Date: _____________ 
 
Statistics institute: 
Project Leader (signature): ____________________     
Date: _______________ 
 
 
 
 
 
 
 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
60
The clinical study of the safety and efficacy of Istaroxime in Treatment of Acute 
Decompensated Heart Failure - A multicenter, randomized, double-blind, placebo 
controlled, parallel group clinical study. 
 
Declaration of Sponsor: 
As the sponsor of the clinical trial, we will carry out the responsibility of sponsor defined in 
GCP. We are responsible for initiation, application, organization, financial support and 
management of the clinical trial. We will assign qualified monitors and auditors for quality 
control of the study. According to relevant laws and regulations, we will give active treatment 
and afford corresponding cost to the patients with AE related to the study drugs, and give 
advisable financial compensation to those with SAE related to the study drugs. And we will 
provide the investigators the legal and financial guarantee. 
 
 
Sponsor: CVie Therapeutics Company Limited 
 
Project Leader (signature): ____________________    Date: _______________ 
 
Head of the company (signature): _______________    Date: _______________ 
 
 

 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
61
 
22. APPENDICES 
 
22.1 Appendix A: Istaroxime Infusion Rate 
 
Reconstitute 4 vials with 5 mL of saline each every 6 hours, draw into one single 50 mL syringe, 
remove 30 ml of saline from the NaCl 0.9% bag with the same syringe used for the reconstitution of 
the 4 vials in order to obtain a total volume of 50 ml (20 ml of the 4 vials+30 ml of saline the NaCl 
0.9% bag) and infuse at the following rate (according to the precision of the electrical syringe of about 
0.1 mL per hour). 
 
 
PATIENT 
WEIGHT (kg) 
INFUSION RATE 
(ml/h) 
PATIENT 
WEIGHT (kg) 
INFUSION RATE 
(ml/h) 
PATIENT 
WEIGHT (kg) 
INFUSION RATE 
(ml/h) 
35 
2.6 
57 
4.3 
79 
5.9 
36 
2.7 
58 
4.3 
80 
6.0 
37 
2.8 
59 
4.4 
81 
6.0 
38 
2.8 
60 
4.5 
82 
6.1 
39 
2.9 
61 
4.6 
83 
6.2 
40 
3.0 
62 
4.7 
84 
6.3 
41 
3.0 
63 
4.7 
85 
6.3 
42 
3.2 
64 
4.8 
86 
6.4 
43 
3.2 
65 
4.9 
87 
6.5 
44 
3.3 
66 
5.0 
88 
6.6 
45 
3.3 
67 
5.1 
89 
6.7 
46 
3.5 
68 
5.1 
90 
6.7 
47 
3.5 
69 
5.2 
91 
6.8 
48 
3.6 
70 
5.2 
92 
6.9 
49 
3.6 
71 
5.3 
93 
7.0 
50 
3.7 
72 
5.4 
94 
7.0 
51 
3.8 
73 
5.5 
95 
7.1 
52 
3.9 
74 
5.5 
96 
7.2 
53 
4.0 
75 
5.6 
97 
7.2 
54 
4.0 
76 
5.7 
98 
7.3 
55 
4.1 
77 
5.8 
99 
7.4 
56 
4.2 
78 
5.8 
>100 
7.5