Conditions: Acute Decompensated Heart Failure
Keywords: safety efficacy istaroxime
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 120
Healthy volunteers: no

Brief summary:
To assess the safety, tolerability and efficacy of two different doses of istaroxime, 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 via i. v. for 24 hours in comparison with placebo, in treatment of Chinese and Italian patients with Acute Decompensated Heart Failure.

Detailed description:
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). The study will be conducted in 96 Chinese and Italian patients with Acute Decompensated Heart Failure. This is a phase II, multicenter, randomized, double-blind, placebo-controlled, parallel group study. Patients were 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).

In all the Italian patients and in a subset of Chinese patients pharmacokinetics and metabolism of istaroxime shall also be studied.

Arms:
arm 1: IV infusion of placebo for 24 hours arm 2: The istaroxime treatment dosed at 0.5 µg/kg/min via IV infusion for 24 hours arm 3: The istaroxime treatment dosed at 1.0 µg/kg/min via IV infusion for 24 hours

Interventions: intervention 1: Placebo intervention 2: Istaroxime
Intervention descriptions: intervention 1: IV of matching saline solution intervention 2: IV infusion of 0.5 µg/kg/min or 1.0 µg/kg/min istaroxime

Protocol document:
ICF_002.pdf: 
CVie Therapeutics Company Limited 
Protocol No CVT-CV-002 
Patient Information Sheet and Informed Consent Form 
Jiangsu Province Hospital Version 18 April 2017 
Page 1 
 
 
 
 
Patient Information Sheet and Informed Consent Form 
 
"The clinical study of the safety and efficacy of Istaroxime in Treatment of Acute 
Decompensated Heart Failure - A multicentre, randomized, double-blind, placebo 
controlled, parallel group clinical study-" 
 
Protocol N° CVT-CV-002 (Final version, 23 July, 2015) - EUDRACT N°: 2013-000540-26 
 
 
Dear Mr./Mrs., 
 
 
The heart failure is a highly disabling condition that negatively impact the quality of life. You have 
been hospitalized for a further worsening of your symptoms (breathing difficulty, fatigue). The drugs 
usually administered to a patient like you (diuretics, traditional inotropes) are only partially 
effective and are associated in some cases to a worsening of the patient conditions during the 
following weeks and months. Therefore, it will be important to can use new drugs able to improve 
in the future both your symptoms and your conditions. Istaroxime is a new drug with favourable 
characteristics for the treatment of patients with the heart failure. Its effectiveness and tolerability 
have been demonstrated after a short-time infusion (6 hours), but data concerning the infusion of 
24 hours are lacking. This will be evaluated in this clinical study that is aimed at demonstrating the 
security, tolerability and efficacy of Istaroxime 24 hour infusion in comparison with placebo in 
patients with heart failure. 
With this document, you have been asked to participate in the above mentioned Research. 
This document is aimed at informing you about the purpose of the study. You are asked to carefully 
read this written document of informed consent and ask all your questions to the Physician who is 
responsible of this Research, and to the Physician who will follow you during the study in order to 
take a conscious decision on your participation. You are not obliged to participate to this study, and 
if you decide to not participate, this decision will not impact on your future medical assistance. If by 
reading this document, you will have any questions, you can ask at any time explanations about the 
terms or information that are not clear to you. In case whereby, after having read and understood all 
the information provided, you decide to take part in this clinical trial, you will be asked to 
personally sign and date the Informed Consent Form attached to this document and one copy will 
be delivered to you for keeping it. Before taking any decision, if you want, you can ask an opinion 
to your relatives or to your Physician. 
 
 


CVie Therapeutics Company Limited 
Protocol No CVT-CV-002 
Patient Information Sheet and Informed Consent Form 
Jiangsu Province Hospital Version 18 April 2017 
Page 2 
 
 
 
1. Basic Information 
 
a. Which is the purpose of this clinical study? 
The heart failure is a clinical condition that does not permit to the heart to pump blood in order to 
cope with the body need. It is a widespread pathology that affects more than 30 million of people 
worldwide. During acute heart failure episodes, the ability of the heart to pump blood from the 
pulmonary circle to the peripheral circulation is impaired and, among the various symptoms, the 
most serious ones are the difficulty to breathing and a serious fatigue. You have been hospitalized 
for a worsening of your symptoms. The therapy you are following has not avoided a worsening of 
your symptoms. In this case, it is necessary an intravenous therapy. The drugs that at the moment 
are used for the intravenous therapy are only partially effective and, in patients with low systolic 
blood pressure or at the lower normal limits, as you, can favour the development of other cardiac 
complications (coronary insufficiency, arrhythmias, long-term myocardial damage). 
Istaroxime is a drug developed for increasing the cardiac ability to pump blood in this kind of patients 
without inducing the above mentioned complications. Istaroxime is an experimental product, that 
means that its use has not been yet approved by a special monitoring Agency like the Food and 
Drug Administration (FDA) from USA and the European Medicinal Agency (EMA). 
The aim of this clinical trial is to confirm the safety and efficacy of an intravenous (iv) infusion of 
Istaroxime for the treatment of the acute heart failure. Other objectives of this study are the 
evaluation of the Istaroxime and its by-product blood levels after its administration. 
 
b. Who is sponsoring this clinical study? 
This clinical study is financed by the CVie Therapeutics Company Limited and by Dr. JUN Huang, 
who represents the Study Sponsor, thus meaning that they are responsible for the starting, 
managing and /or financing of this clinical study. 
 
c. Why have you asked me to participate to this study? 
You have been asked to this study because you have been hospitalized for the treatment of the 
acute heart failure and because the Study Physician have considered that you could have 
benefit from this drug. The inclusion criteria to the study include a physical examination and 
laboratory analysis that will be evaluated before you can be enrolled in this study. The decision to 
participate to this study is exclusively yours. If you decide to not participate this will not negatively 
impact on your medical care. 
 
d. How many other subjects like me will participate to this study? 
About 120 subjects will participate to this study, among which 24 cases coming from Italy, 96 cases 
from China. 
 
 
e. Which kind of experimental product/s will be investigated in his study? 


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Protocol No CVT-CV-002 
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The only experimental product that will be investigated in this study is Istaroxime, administered at 
two different doses 0.5 and 1.0 μg/kg/min and compared with placebo. Placebo is an inactive drug 
that resembles from outside to the true drug, but does not contain any active drug; it is therefore 
completely inactive and not dangerous. In this document istaroxime is defined as "experimental 
product". The experimental product will be administered to you by intravenous route (i.v.) for 24 
hours. 
 
f. Which probability I have to receive the experimental product? 
If you meet the enrolment criteria and you decide to participate to the study, you will be assigned at 
random (as for tossing a coin) to one of the two treatment groups (istaroxime or placebo). Istaroxime 
will be administered a low or high dose. If you will be assigned to receive istaroxime and you will be 
included within the first 60 subjects enrolled in the study, you will receive the low dose; if you will 
be within the second group of 60 subjects enrolled in the study you will receive istaroxime at high 
dose. The assignment to istaroxime or placebo is based on a list created by a computer. The 
probability to receive istaroxime will be of the 66.6% (2 over 1). 
 
g. Can I know which product (istaroxime or placebo) I'm receiving? 
Since this is a blind study, neither you nor your Study Physician or the study personnel will know 
the group you belong (istaroxime or placebo). The reason of this is to be sure to be not influenced 
when judging the safety and efficacy of the experimental product. If needed, in emergency 
situation, the information on the treatment will be available. 
 
h. How long does the study last? 
The length of your participation to this study will last about 1 month (30 days). A Screening period, 
not longer than approximately 24 hours, the i.v. infusion for 24 hours, daily evaluations during your 
hospitalization up and not after day 4, and finally, one final medical evaluation at day 30 are planned. 
Your Study Physician or the Sponsor can decide to exclude you from the study at any time. 
 
 
2. Study Procedures 
 
 
a. Which are my responsibilities if I participate to the study? 
If you participate to this clinical study, you will be asked to sign this form of informed consent. To 
establish your eligibility to the study, you will be subjected to a series of screening procedures (see 
below) including questions about your cardiac failure and the therapy received for your pathology, 
and questions concerning other clinical conditions affecting you. The screening procedures will be 
performed within 24 hours from the signature of this form of informed consent for the treatment of 
your cardiac failure. 


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If you will be eligible to this study and accepts to participate, you will receive the experimental 
product or placebo by iv infusion for 24 hours as global. Due toyour clinical conditions, your 
hospitalization will last al least 4 days, which corresponds to the minimum hospitalization time for 
the acute heart failure. The final evaluations are planned about 1 month after your enrollment in to 
the study and you will be asked to come back to our Centre for a medical visit. 
 
b. Which kind of analysis or procedures is expected in this study? 
Screening phase 
 
 
After signing the informed consent form, you will be subjected to a screening to establish if 
you are eligible for this study. The following evaluations and procedures will be performed: 
1. Evaluation of all the inclusion or exclusion criteria of the study; 
2. Evaluation of your medical and pharmacological assessment, collection of your 
demographic data; 
3. Physical inspection 
4. ECG; 
5. Holter ECG; 
6. Blood and urine sampling for laboratory analysis; 
7. Pregnancy test ( only for child-bearing women); 
8. Echocardiography; 
9. Recording of any medical event that can happen during the screening phase 
 
 
  
Treatment and follow-up: 
 
 
After having concluded the screening phase and you will be considered elegible for the enrolment, 
the i.v. treatment with the experimental product will start. The day of starting of the treatment is 
defined as day 1 of the study. The i.v. infusion of the experimental product will be performed within 
24 hours. During this period and for further 48 hours after the experimental drug administration, 
you must be hospitalized (up to day 4 of the study). Beside the procedures listed below, during all 
the time of your participation to the study, the Study Physician or the Study personnel will record 
the other medical treatments you will receive and any medical or hospitalized event that you could 
receive. 
 
Day 1 of the study 
 
 
After starting the experimental product administration, the following evaluations will be performed: 
1. Blood sampling for laboratory analysis; 
2. Vital signs; 


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3. ECG; 
4. Holter ECG; 
5. Echocardiogram; 
6. Recording of any medical event 
 
 
Day 2 of the study: 
1. Physical examination; 
2. ECG; 
3. Holter ECG (only if needed); 
4. End of the experimental product infusion; 
5. Echocardiogram; 
6. Blood sampling for laboratory analysis; 
7. Recording of any medical event 
 
 
Day 3 of the study: 
 
 
1. Physical examination; 
2. ECG; 
3. Blood sampling for laboratory analysis; 
4. Recording of any medical event 
5. Echocardiogram; 
 
 
Day 4 of the study: 
1. Physical examination; 
2. ECG; 
3. Blood sampling for laboratory analysis; 
4. Recording of any medical event 
 
 
Day 30 of the study: 
 
 
The final evaluations will be performed about 1 month after the start of your participation into the 
study. Unless you is not yet hospitalized, you will be asked to come back to the experimental 
Centre. The following procedures and evaluations will be performed: 
1. Physical examination; 
2. ECG; 
3. Blood sampling for laboratory analysis; 
4. Pregnancy test only for child-bearing women; 
5. Recording of any medical event 


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Further information on the study procedures 
During the study, due to the need of related laboratory examinations, the global volume of collected 
blood will be about 165 ml, which means about one third of a typical blood donation (500 ml). The blood 
sample will be collected within 4 days. During all the study period, all your side effects will be 
monitored. If a relevant side effect or abnormal laboratory data will happen, you could be asked 
to have further medical visits at the experimental Centre up to when the situation is solved or is 
considered stable by the Study Physician. 
 
c.   What does it happen at the end of the clinical study? 
 
 
It is planned that your involvement in the study will end about 1 month after the starting of the i.v. 
infusion of the experimental product. However, since you are a volunteer participation, you can leave 
the study when you desire. If after the end of the study you think to have had a side effect 
related to the experimental product or to the study procedures, you must contact your Study Physician: 
Dr._____________________by the telephone number:_______________________. 
 
 
3. Safety: POTENTIAL RISKS AND DISCOMFORTS 
 
a. Which are the general risks of participating to this research study? 
 
 
168 adult subjects have been treated with different doses of istaroxime in clinical experimentations, 
without showing evidence of severe dangers for the heath and the welfare of the patients. However, 
the administration of any experimental product requires a careful evaluation of possible side 
effects. Since istaroxime is an experimental product, not all the potential side effects in human being 
are known. The side effects are medical unfavorable conditions or consist in a worsening of a pre-
existing medical condition and they can happen during the participation to a study or, generally 
speaking, when undergoing to a therapy. One of the aims of this study is to know the possible 
side effects of istaroxime. The Study Physician will keep you under strict observation during the study 
and will discuss with you about any possible problems related to the risks, discomforts and side 
effects, If you participate to the study, during the study you or one of your relatives must 
immediately warn the nurses or the Study Physician in case of unusual health condition, lesions or 
side effects. If you have any concern about the study, contact your Study Physician.  
 
 
b. Which are the known side effects of the experimental product? 


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No serious adverse events (such as death or serious cardiovascular events) have been 
happened during the previous 3 studies on patients with acute heart failure. The only 
observed side effects were: gastroenteric discomforts, emesis or diarrhea, and pain at the 
drug injection site at doses higher than those tested in this study. At the doses used in this 
study, this kind of side effects was observed in 2 patients over 60. 
As for any other drug, you can have an allergic reaction to Istaroxime. The possible 
symptoms include headache, skin rushes, flushing, swelling, breath shortness, nausea and 
sometime vomits. The allergic reactions can be also serious or endangering of life, such as 
dizziness, difficulties in breathing or swallowing or a reduction of blood pressure. If you 
should experience any of these symptoms during the study, you must immediately warn 
you Study Physician. 
 
c. Which are the risks associated to the experimental product in association with other 
drugs? 
The risks due to the use of istaroxime in association with other drugs are not known. At 
screening, you must inform the Study Physician about all the drugs you are taking and, 
during the study, you must inform the Study Physician before taking any other new drug 
and before changing the drugs you are currently taking. 
 
d. Which are the risks associated to the procedures of this research study? 
Your Study Physician will discuss with you the possible risks associated to the analysis and 
procedures planned for this study (for instance, blood sampling, diagnostic imaging, 
echocardiogram, intravenous administration or other medical procedures). 
 
Blood sampling 
During the study, blood will be periodically taken. The possible side effects of blood 
sampling are soreness, pain, bleeding, bruising and /or infection at the injection site. Blood 
sampling may cause nausea and/or dizziness. 
 
Electrocardiogram (ECG) 
ECG is an examination that measures the electric activity of the cardiac beating by using 
electrodes (small adhesive disposable disks put on the skin). Neither pain nor risks are 
associated to the ECG. When the electrodes will be removed, you could feel a light 
discomfort or skin irritation. 
 
Continuous intravenous administration 
The experimental product will be administered by continuous intravenous infusion for 24 
hours through a venous catheter inserted in a high blood flow vein. The rare complications


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are associated to this procedure are: vessel lesion, of the catheter, phlebitis, infections. 
 
 
e. What does it happen if I /my partner became pregnant during the study? 
Since this is an intravenous administration lasting 24 hours, performed in the hospital and 
followed by 3-4 days of hospitalization, this case is highly improbable. 
However, if you are a woman and became pregnant or suspect to be pregnant during this 
study, you must immediately inform the Study Physician. The treatment with the experimental 
product will be immediately stopped and your safety (related to the pregnancy) will be 
monitored up to the study conclusion. Your Study Physician will ask you your consent for 
furnishing to the Sponsor of the study information about the pregnancy and the child. 
If you are a man and your partner is or became pregnant during the study, you must 
immediately inform the Study Physician. Your Study Physician will ask you contact 
information and your consent for furnishing to the Sponsor of the study information about 
the pregnancy of the mother and the child. The men of the study whose partners are or 
became pregnant should practice abstinence or use the condoms for at least 5 days from 
the end of the treatment with the experimental product to guarantee that the unborn baby 
cannot be exposed to istaroxime through the seminal fluid. 
 
4. POTENTIAL BENEFITS 
 
 
a. Which are the therapeutic benefits foreseen for this experimental product? 
Istaroxime is currently under development for the treatment of the cardiac failure. The 
potential benefits, listed above have been demonstrated after 6 hour infusion. However, it is 
not known if these benefits are also present after 24 hours of infusion. This study has been 
planned to ask this question. 
 
b. Will I get benefit by participating to this study? Will other subjects get benefit? 
For the reasons listed at paragraph n°1, you could get a direct benefit by participating to this 
study due to the favorable effects of the drug in patients with low or at the lower normal limits 
of arterial blood pressure. Of course, you will not get this benefit if you will take the placebo. 
Other subjects, in the future, could get benefits from the knowledge about this study. This 
information could allow the Physicians to better know the experimental product use for the 
acute heart failure in patients like you for whom the present therapy is not satisfactory. 
 
5. ALTERNATIVE THERAPY 
a. If I decide not participating to this study,  are there other alternative available treatments 
or drugs to this treatment? 


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Jiangsu Province Hospital Version 6 April 2016 
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Your alternative at the treatment with this experimental product, it is not to participate to this 
study. You should discuss with your Study Physician to know other treatment options that 
may be available for you. These options can include an increase of the dose of the drug you 
are currently taking for curing your cardiac failure or adding new treatment. 
 
b. Are there any benefits with these new treatments? Are there any risks? 
All type of treatments deserves benefits and potential risks. Your Study Physician can explain 
you all the treatments available for you and their benefits and risks. 
 
 
 
 
6. POTENTIAL  COSTS/REMBOURSES 
 
 
a. How much does this study cost to me? 
The sponsor of this study will provide experimental product and related examinations for free. 
 
b. Can I be reimbursed for any expense I can have as consequence of the study? 
To participate to the Study you will not receive any compensation. If you have a private 
health insurance it is better if you verify with your insurer that your participation in the study  
does not impact on your insurance, before consent to participate. 
 
c. Will I receive any compensation for the use of my biological sample? 
You should know that, by the use of your samples in the Study, a commercial pharmaceutical 
product and/or a procedure could be developed. The Sponsor, other researchers or research 
company can patent or sell discoveries produced by this research. If this happens, neither 
the Sponsor nor the Study Physician will give you any compensation. 
 
7. CONFIDENTIALITY 
 
 
a. a. How the confidentiality of my clinical documents will be protected? 
The management of the medical information obtained by the clinical research is governed 
by national and international rules on the data protection and medical confidentiality ( Good 
Clinical Practice (GCP) ). The medical information collected during this study will be firstly 
verified for their accurateness. Then, it will be transferred in the study data bank and 
processed for analyzing, disclosing and publishing the study results for scientific aims 
The confidentiality of your clinical documents will be protected according to the currently 
regulations. If the results of the experiments will be published, your identity will be kept 


CVie Therapeutics Company Limited 
Protocol No CVT-CV-002 
Patient Information Sheet and Informed Consent Form 
 
Jiangsu Province Hospital Version 6 April 2016 
Page 10 
 
 
confidential. The results and other information of the study could be transmitted to the 
Regulatory Agencies of the Countries where the experimental product is under evaluation 
for the approval. Neither your results nor your samples will be identified by your name. The 
data related to your participation in the study will be associated to you by a number that 
identifies the person, the birth date and the sex. 
 
b. Who will have access to my medical information if I sign this Informed Consent Form? 
Your participation to the study must be annotated in your clinical folders. If the Physician who 
will follow you in this study is not your family doctor, the latter shall be informed of your 
participation to the study. The representatives of the Sponsor can ask to have a direct access 
to your clinical documents to verify the information collected for the study. Your clinical 
folders can also be examined and photocopied by the members of the Ethical Committee of the 
experimental Centre, of a Regulatory Agency or by an authorized Sponsor representative. 
This people can know your name, other personal information such as the birth date and the 
sex, your medical information, but they do not disclose your name to others. By signing this 
Informed Consent Form, you (or your legal representative) authorize the access to these 
confidential information. The paragraph "information of the personal data management", at 
this I n f o r m e d  C o n s e n t  Form, will give you further information on the management of 
your personal data related to the study. Some of your data, including the ECG, that can 
contain confidential information, can be send to a central office for the analysis and 
interpretation and then send to the Sponsor and/or the Study Physician. The members of 
the personnel of the central office will keep the confidentiality of your information. 
 
8. DAMAGES 
 
 
a. What can I do if I presume to have suffered a damage as consequence to the 
participation at this study? 
If you think to have suffered a damage that are connected to the study, you must alert 
your Study Physician. The Study Physician and the study personnel will give you the 
needed treatment. 
 
 
b.  If I will suffer a damage because of the participation to the study will I be 
reimbursed? 
In case of any damage caused directly to your participation to the study you will be covered 
by a special insurance entered into by the Sponsor with the Insurance Company.  
Insurance companies will be based on relevant laws and regulations and insurance policy, your 
insurance compensation accordingly. 
 


CVie Therapeutics Company Limited 
Protocol No CVT-CV-002 
Patient Information Sheet and Informed Consent Form 
 
Jiangsu Province Hospital Version 6 April 2016 
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9. INFORMATION ON THE PROCESSING OF THE PERSONAL DATA 
 
 
a. Use of the data and related objectives 
The Centre and the Sponsor of the study, each one according to their competence and 
responsibility as required by the Good Clinical Practice will utilize, analyze and manage 
your personal data, with particular reference to your health and other data related to your 
origin, your life style etc., only for the conductance of the study as indicated below and for 
pharmacovigilance purposes, with the final objective to develop commercial pharmaceutical 
products. To this end the data will be collected by the Study Physician and his/her 
collaborators and, after their coding, they will be transmitted to the Sponsor and to the 
people and external Societies working by their self or making assistance to the clinical 
experiments or who will know the data as responsible of the data management. Some 
of these external Societies can be located also in foreign Countries outside Italy and 
China. The whole list of the external societies will be stored at the Experimental Centre. 
Your personal data, including information of your health condition, will be collected, used and 
managed by the above cited societies and will be analyzed by the Sponsor and his delegates 
to discover whether Istaroxime is beneficial for subjects suffering of acute cardiac failure. This 
is indispensable for the study development: if you If you have suffered a damage/developed a 
disease related to the study, the Study refuse to furnish your personal data, you could not 
participate into the study. 


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b. Data Characteristics 
The Physician who will follow you during the study will identify you by a personal code 
number (code): your data collected during the study, except your name, will be 
transmitted to the Sponsor or his designated persons, and will be recorded, analyzed, 
used, managed and stored with your code, your birth date, sex, weight and height. Only 
the Study Physician and the authorized persons can link this code to your name. 
 
c. Management of the data 
Neither the results nor your samples (if collected) will be identified with your name. If the 
results of the clinical study will be published your identity will be kept anonymous. Your 
participation into the study implies that, according to the laws on the clinical 
experimentations of drugs, the Sponsor personnel or the external societies that monitor and 
verify the study on behalf of the Sponsor, the Ethical Committee and the health authorities 
can have access to your data, including those contained in your original clinical folder, in a 
manner such as to ensure the confidentiality of your identity. 
 
d. Exercise of the Rights 
You can exert the Rights on the Confidentiality of your data according to law that controls such 
matter. 
However, you cannot know which drug you will receive and/or the results obtained during 
the development of the study. You can stop at any moment and without give any 
explanation your participation into the study: in this case, your biological sample (if 
collected), when no more needed for the study, will be destroyed and no other data on 
your person will be collected. However, the data already collected will be used and 
disclosed according to this form. 
You should inform your family Doctor or other Physicians of your participation into the 
clinical study. Furthermore, you must authorize the Study Physician to contact other 
Physicians who take care of you. If you want, the Study Physician can inform you about 
the general results of the study and, in case, of data useful for your disease, if produced. 
 
10. Rights and Obligations 
 
 
If I will accept to participate into this study, which warranties can I have? 
You are a a voluntary participation and you are free to withdraw from the clinical study at any time 
without problem for your future medical care. If you decide to withdraw, you must warn the Study 
Physician in order to interrupt your participation in a correct way and discuss your future medical 
care. Furthermore, your Study Physicians or the Sponsor can decide towithdraw you from this clinical 
study. 
 
You, or your legal representative will be immediately updated about any information 
on your will to continue to participate into the study. According to your Study 


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Physician and the Sponsor, it is possible that you or your legal representative can be 
asked to sign a revision or an appendix of the Informed Consent Form giving this 
information. 
 
You can ask about this study at any time. If you think to have suffered from an adverse 
side effect to the experimental product or to the procedures, or you feel always bad 
during the study, you must contact your Study Physicians. 
 
If you have any questions about the Informed Consent procedure or your rights as subject involved 
into the clinical study, you can contact your Study Physicians. 
If you want to file a complaint or consult questions concerning your rights as a study participant, please 
contact the Ethics Committee of Jiangsu Province Hospital by  025-68136360  (phone number). 
 
11. SIGNATURES 
By signing this Form, I undersigned (add name and surname) ……………………………….. 
 
 
 
I confirm that I have had adequate opportunity to discuss all the available treatments 
and all my doubts and my questions on this study have been satisfactory clarified. 
 
I agree that the research carried out by the Sponsor of the study, by using my clinical 
data collected during this study, could lead to the development of commercial 
pharmaceutical products. The Sponsor and other researchers could use these data to 
patent and sell the discoveries coming from the results of this research. In this case, 
neither the Sponsor nor other participants to this research will give me any reward. 
 
I agree that my personal data can be confidentially treated and can be transferred out 
of China, to subjects or societies indicated in the Information at paragraph 9 of this 
document and in particular to the Sponsor for the research objectives within the limits 
and the mode indicated in the present Information form. I undertake to inform my family 
doctor and any other physicians who will cure me about my participation in this study. 
 
I authorize the Study Physician to directly contact any other physicians who will cure 
me. 
 
I undertake to avoid to became pregnant (to make pregnant) up to 5 days after the 
study product administration. 
 
By signing this Form, I confirm to agree to participate to this study and to have receive a copy of 
this Informed Consent Form. 
 


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__________________________________ 
____________________ 
Subject signature 
date * 
Contact information: __________________ ID No.: ______________________ 
OR 
__________________________________ 
____________________ 
Legal Representative signature 
date  
(The document must be signed by legal representative when the subject age 
more than 65 years old.) 
Contact information: __________________ ID No.: ______________________ 
Relationship with the subject 
 
* Who is signing must write by him/herself the date. 
 
 
I undersigned (add name and surname)………………………………………………………… 
as Study Physician, confirm to have furnished to the subject comprehensive explanations about 
the nature, the aim, the length and the potential risks associated to this study, and I believe in 
conscience that they have been understood. 
 
 
 
__________________________________ 
____________________ 
Study Physician signature 
 
date * 
Contact information: ______________________ 
*   Who is signing must write by him/herself the date. 
 
 
 

Prot_000.pdf: 
 
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

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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VERSION: $PHQGHG)LQDO9HUVLRQ1RYHPEHU 
 
Approved by the Sponsor: CVie Therapeutic Company Limited, and 
by the Chair of Steering Committee: Dr. JUN Huang 
  
x
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 


 
Protocol N°: CVT-CV-002 
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6
 
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)  
7
 
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)  
8
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 


 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
9
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)  
10
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 
Amended Final Version (30 November 2017)  
11
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)  
12
(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) 


 
Protocol N°: CVT-CV-002 
Amended Final Version (30 November 2017)  
13
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)  
14
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) 


 
Protocol N°: CVT-CV-002 
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, 
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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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Istaroxime, a novel intravenous inotropic and lusitropic agent: a randomized controlled 
trial in patients hospitalized with heart failure. J Am Coll Cardiol. 2008; 51(23): 2276-85. 


 
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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 
 
 

SAP_001.pdf: 
CONFIDENTIAL 
 
 
 
 
  
 
 
 
 
 
 
STATISTICAL ANALYSIS PLAN 
 
 
 
 
 
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 
 
 
 
Protocol CVT-CV-002 
 
 
 
 
 
Author: Lisa Comarella  
             CROS NT S.r.l. 
               
 
 
 
Version: Final 1.0 
Date of Production: 30 October 2018 
Identification Code: SAP_CVT-CV-002_1.0_181030  
 
 
 
 
Sponsor                                 CVie Therapeutics Company Limited  
Unit110-111, Bio-informatics center, 
No2. Science Park West Avenue, 
Hong Kong Science Park, Shatin, N.T. HK 
 
 




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DISTRIBUTION LIST 
 
 
Lisa Comarella, Director, Biostatistics (CROS NT S.r.l.) 
Marta Zanus, Director, Project Management (CROS NT S.r.l.) 
Giuseppe Bianchi, Appointed Medical Expert (CVie Therapeutics Company Limited) 
 


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TABLE OF CONTENTS 
1.0 
INTRODUCTION ........................................................................................................................................... 7 
1.1 
CHANGES FROM PROTOCOL ................................................................................................................... 7 
2.0 
VERSION HISTORY ...................................................................................................................................... 7 
3.0 
STUDY OBJECTIVES AND ENDPOINTS .................................................................................................. 8 
3.1 
STUDY OBJECTIVES .................................................................................................................................. 8 
3.2 
STUDY ENDPOINTS ................................................................................................................................... 8 
4.0 
STUDY DESIGN .............................................................................................................................................. 9 
4.1 
SAMPLE SIZE ............................................................................................................................................ 10 
4.2 
RANDOMIZATION.................................................................................................................................... 10 
5.0 
ANALYSIS SETS .......................................................................................................................................... 11 
5.1 
INTENT-TO-TREAT POPULATION (ITT) .............................................................................................. 11 
5.2 
PER-PROTOCOL POPULATION (PP) ...................................................................................................... 11 
5.3 
SAFETY POPULATION ............................................................................................................................ 11 
5.4 
OTHER ANALYSIS SET ............................................................................................................................ 11 
5.5 
TREATMENT MISALLOCATIONS .......................................................................................................... 11 
5.6 
PROTOCOL DEVIATIONS ....................................................................................................................... 11 
6.0 
DEFINITIONS AND DATA CONVENTIONS ........................................................................................... 11 
6.1 
DEMOGRAPHIC AND SCREENING/BASELINE CHARACTERISTICS VARIABLES ........................ 12 
6.2 
EXTENT OF EXPOSURE .......................................................................................................................... 13 
6.3 
EFFICACY VARIABLES ........................................................................................................................... 13 
6.4 
SAFETY VARIABLES ............................................................................................................................... 14 
6.5 
BASELINE .................................................................................................................................................. 16 
7.0 
STATISTICAL METHODOLOGY ............................................................................................................. 16 
7.1 
HANDLING OF MISSING DATA .............................................................................................................. 16 
7.2 
COVARIATES AND SUBGROUPS ........................................................................................................... 16 
7.3 
INTERIM ANALYSIS AND SEQUENTIALITY OF ANALYSES ............................................................ 17 
7.4 
GENERAL METHODOLOGY ................................................................................................................... 17 
7.5 
PATIENTS DISPOSITION ......................................................................................................................... 17 
7.6 
DEMOGRAPHIC AND SCREENING/BASELINE CHARACTERISTICS ............................................... 18 
7.7 
ANALYSIS OF EFFICACY ........................................................................................................................ 19 
7.8 
ANALYSIS OF SAFETY ............................................................................................................................ 22 
7.9 
OTHER PLANNED ANALYSIS ................................................................................................................ 23 
8.0 
GENERAL CONSIDERATIONS ................................................................................................................. 23 
8.1 
SOFTWARE TO BE USED ......................................................................................................................... 23 
8.2 
PROGRAMS AND TABLES QUALITY CONTROL ................................................................................ 23 
9.0 
PROGRAMMING SPECIFICATIONS ...................................................................................................... 24 
10.0 
ARCHIVING .................................................................................................................................................. 27 
11.0 
REFERENCES............................................................................................................................................... 27 
APPENDIX 1: MOCK-UPS OF TABLES, LISTINGS AND FIGURES .............................................................. 28 
 


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LIST OF ABBREVIATIONS 
 
A  
 
Pick mitral flow velocity during atrial contraction  
ADR   
Adverse Drug Reaction 
AE  
 
Adverse Event  
Ae  
 
Amount excreted unchanged into urine  
Ae%   
Percent of the dose excreted unchanged into urine  
ANCOVA  
Analysis of Covariance  
ANOVA 
Analysis of Variance 
ATC  
Anatomical Therapeutic Chemical 
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  
BNP   
Brain Natriuretic Peptide  
CI  
 
Confidence interval  
ClR  
 
Renal clearance  
Cmax   
Maximum concentration  
CRF 
 
Case Report Form  
CS 
 
Clinically Significant 
cTnT   
Cardiac Troponin T  
DMC   
Data Monitoring Committee  
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  
ECG   
Electrocardiogram  
eGFR   
Estimated glomerular filtration rate  
EMEA  
European Medicines Agency  
ICH  
 
International Conference on Harmonisation 
ITT  
 
Intention-to-treat  
LV 
 
Left Ventricular 
LVEF  
Left Ventricular Ejection Fraction  
λz  
 
Terminal rate constant  
MedDRA  
Medical Dictionary for Regulatory Activities  
MMRM 
Mixed Models for Repeated Measures 
NCS 
 
Not Clinically Significant 
NR 
 
No result 
NT-proBNP  N-terminal prohormone brain natriuretic peptide 
PK  
 
Pharmacokinetics  
PP  
 
Per protocol  
PR  
 
Interval between P and R waves  
PT 
 
Preferred Term 
QRS   
Interval between the beginning of Q wave and the end of S wave  


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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  
SAP 
 
Statistical Analysis Plan 
SAS 
 
Statistical Analysis Systems 
SD  
 
Standard deviation  
SOC 
 
System Organ Class 
SVI  
 
Stroke Volume Index 
t1/2  
 
Terminal half-life  
Tmax   
Time to maximum concentration  
TEAE  
Treatment-emergent adverse event 
URL   
Upper Reference Limit  
Vss  
 
Volume of distribution at steady-state  
VAS   
Visual Analog Scale  
Vz  
 
Volume of distribution during the terminal phase  
WHO-DD  
World Health Organization Drug Dictionary 
 


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1.0 
INTRODUCTION 
This document outlines the statistical methods to be implemented in the analysis of the data 
of CVT-CV-002 Clinical Trial. The purpose of this plan is to provide general guidelines from 
which the analysis will proceed, containing a more technical and detailed elaboration of the 
principal features of the analysis described in the protocol. Any changes to the protocol or 
Case Report Form (CRF) may necessitate updates to the Statistical Analysis Plan (SAP). In 
case of deviations from this updated SAP, explanations will be provided in the clinical study 
report. 
Relevant applicable CROS NT quality document will be followed also according to the 
contract with the Client. 
 
1.1 
CHANGES FROM PROTOCOL 
The analysis of echocardiographic parameters is changed from the protocol:  
 The analysis of echocardiographic parameters will be performed separately for 
each cohort, instead of performed on the combined cohorts.  
 An analysis of variance (ANOVA) model with treatment as covariate is now 
the primary analysis, instead of a mixed model for repeated measures 
(MMRM) including treatment and additional covariates. 
 Missing response data will not be imputed in the ANOVA analysis.  
The justification for these changes are documented in the Memo to File prepared by 
Tri Tat, dated 24Oct2018, entitled: Change in analysis of echocardiographic parameters. 
 
2.0 
VERSION HISTORY 
 
Version 
Number 
Summary/Reason for changes 
Date Issued 
to Client 
Draft 1 
Draft specification based on: 
 Study protocol (general protocol amendment no.1, 08 
April 2013) 
 CRF (Version Final 2, 23 July 2013) 
02 September 
2013 
Draft 2 
The following  change has been implemented after sponsor’s 
revision: 
 The echocardiographic parameters to be considered in the 
global test have been defined. 
15 October 
2013 
Final 1.0 
The following  change has been implemented after sponsor’s 
revision: 
The analysis of echocardiographic parameters is changed to 
an analysis of variance using only treatment as covariate.  
30 October 
2018 
 
 


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3.0 
STUDY OBJECTIVES AND ENDPOINTS 
 
3.1 
STUDY OBJECTIVES 
 
3.1.1 Primary Objective 
The primary objective of this study is to evaluate the efficacy of two different 
doses of istaroxime (0.5 and 1.0 µg/kg/min) in comparison with placebo on 
the E/Ea ratio in patients with Acute Decompensated Heart failure. 
 
3.1.2 Secondary Objectives 
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 
cardiac 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 
(PK) and metabolism of istaroxime shall also be studied. 
 
3.2 
STUDY ENDPOINTS  
 
3.2.1 Primary Endpoint  
 The primary efficacy endpoint is the change from baseline to 24 hours 
after infusion start (treatment period Day 1) in the E/Ea ratio assessed 
by tissue Doppler. 
 
3.2.2 Secondary Endpoints 
The secondary efficacy endpoints are the following:  
 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 Left Ventricular Ejection Fraction (LVEF) 
o LV end systolic and end diastolic volumes 
o Stroke volume index (SVI), and CI 
o E, A and E/A ratio 
o Ea and TAPSE 
 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; 
 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);  


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 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 dyspnoea at baseline); 
 Changes in Brain Natriuretic Peptide (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 heart failure 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. 
 
The safety endpoints are: 
 Incidence of Adverse Events (AEs);  
 Change in vital signs; 
 Change in 12-lead Electrocardiogram (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 (Estimated glomerular filtration rate 
(eGFR)); 
 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% Upper Reference Limit (URL, as defined 
for the Roche hs test to be 14 ng/L) respectively; 
 Mortality at Day 30. 
 
The PK parameters are the following: 
 Istaroxime plasma concentrations: Cmax, Tmax, AUC0-t, AUC0-∞, λz, t½, 
ClT, MRT, Vss, Vz; 
 Istaroxime urine concentrations: Ae, Ae%, ClR; 
 PST2915, PST2922, and PST3093: Cmax, Tmax, AUC0-t, AUC0-∞, λz, t½ 
and, if possible, Ae and Ae%. 
 
 
4.0 
STUDY DESIGN 
This is a phase II, multicenter, randomized, double-blind, placebo-controlled, parallel 
group study.  
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. 


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This 31-day study includes a screening period (Day -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. 
 
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. 
 
 
4.1 
SAMPLE SIZE 
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 (Gheorghiade M. et al. Haemodynamic, echocardiographic, and 
neurohormonal effects of Istaroxime, a novel intravenous inotropic and lusitropic 
agent: a randomized controlled trial in patients hospitalized with heart failure. J Am 
Coll Cardiol. 2008; 51(23): 2276-85), 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. 
 
4.2 
RANDOMIZATION 
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 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.  
 


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5.0 
ANALYSIS SETS 
 
5.1 
INTENT-TO-TREAT POPULATION (ITT) 
All randomized patients who will receive at least one administration of the study 
medication and with at least one available evaluation of efficacy after the baseline 
will be included in the ITT population. 
The ITT population is the primary population for the efficacy analysis. 
 
5.2 
PER-PROTOCOL POPULATION (PP) 
All randomized patients from the ITT population without any major protocol 
deviation will be included in the PP population.  
Exact definition of major protocol deviations will be discussed by the clinical team 
case by case during the blind review of the data. 
 
5.3 
SAFETY POPULATION 
All randomized patients who will receive at least one administration of the study 
medication will be included in the safety population. 
 
5.4 
OTHER ANALYSIS SET 
All randomized patients will be included in the randomized population. The 
randomized population will be considered for descriptive purposes. 
 
 
5.5 
TREATMENT MISALLOCATIONS 
In case an error occurs in treatment allocation, the following rule will be followed: if 
a patient was randomized but received the incorrect treatment, he/she will be reported 
under his/her randomized treatment group for all analyses performed on the 
randomized and ITT populations, but he/she will be reported under the treatment 
actually received for all analyses performed on the safety. The patient will be 
excluded from the PP population.  
 
5.6 
PROTOCOL DEVIATIONS 
All the protocol deviations will be discussed case by case before unblinding of the 
treatment code with the clinical team during the blind review of the data and 
described in the Data Review Report. 
 
 
6.0 
DEFINITIONS AND DATA CONVENTIONS 
This section contains definitions and conventions that will be used for the analysis. 
 


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6.1 
DEMOGRAPHIC AND SCREENING/BASELINE CHARACTERISTICS 
VARIABLES 
 
 
General, demographic and baseline characteristics 
Age 
If the date of birth is not missing, age will be calculated using the following rules: 
 If month of date of screening is greater than month of date of birth or (month of 
date of screening is equal to month of date of birth and day of date of screening is 
greater than or equal to day of date of birth), then: 
  
Age (years) = year (date of screening) – year (date of birth) 
 
 Otherwise:  
 
Age (years) = year (date of screening) – year (date of birth) - 1 
 
In order to determinate the age of patient, the following rules will be applied for the 
partial date of birth: 
 if only the day is missing, the 15th day of the month will be assumed; 
 if the day and the month are missing, June 30th will be assumed. 
If the date of birth is missing, the age recorded in the CRF will be used. 
 
Duration of smoking (years) 
Duration of smoking will be calculated using the following formula: 
 For ex-smoker:  
Duration of smoking (years) = (stop date – start date)/365.25  
 For smoker:  
Duration of smoking (years) = (date of screening – start date)/365.25  
 
In order to determinate the duration of smoking, the following rules will be applied 
for the partial dates of start/stop of smoking: 
 for start date and stop date, the first day of the month will be assumed; 
 if the month of start date or stop date is missing, January 1st will be assumed. 
 
Past disease and concomitant disease 
Past disease 
A disease is considered as past disease if it is not ongoing at screening visit 
(“ongoing” box is not ticked). 
 
Concomitant disease 
A disease is considered as concomitant disease if it is ongoing at screening visit 
(“ongoing” box is ticked). 
 


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Previous and Concomitant medications 
The following categories of medications will be identified: 
 previous medication (start date < date of first randomized study medication 
intake and stop date ≤ date of first randomized study medication intake); 
 medication maintained during the treatment period (start date < date of first 
randomized study medication intake and stop date > date of first randomized 
study medication intake or ongoing); 
 concomitant medication (start date ≥ date of first randomized study medication 
intake). 
 
In case of missing or incomplete dates not directly allowing allocation to any of the 
three categories of medications, a worst-case allocation will be done according to the 
available parts of the start/end dates. The medication will be allocated to the first 
category allowed by the available data, according to the following order: 
 concomitant medication; 
 medication maintained during the treatment period; 
 previous medication. 
 
Study medication intake 
Date/time of first randomized study medication intake is the date/time of first study 
medication intake recorded at Day 1. 
 
Date/time of last randomized study medication intake is the date/time of last study 
medication intake recorded in the Study Termination form. 
 
6.2 
EXTENT OF EXPOSURE 
Extent of exposure will be calculated in hours as difference between end date/time of 
infusion and start date/time of infusion: 
 
Extent of exposure (hours) = end date/time of infusion - start date/time of infusion 
 
6.3 
EFFICACY VARIABLES 
 
AUC0-48h on changes in dyspnoea 
AUC0-48h on changes in dyspnoea will be calculated using the following formula: 
 













h
h
i
i
i
i
i
h
VAS
VAS
t
t
AUC
48
3
1
1
48
0
2
 
 
with i = 3, 6, 12, 24, 48 hours. 
 


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In the above formula, ti represents the scheduled timepoints in hours for VAS score 
measurements (see table below) and ΔVASi represents the change from pre-dose of 
the value of the VAS score at the timepoint i. The change from pre-dose at the 
timepoint i = 0 will be zero.  
 
Time of evaluation (hours) 
T pre-
dose 
T3h 
T6h 
T12h 
T24h 
T48h 
t0 
t1 
t2 
t3 
t4 
t5 
Time point for calculation 
 
The scheduled timepoint will be used for the calculation of AUC0-48h.  
 
In the calculation of AUC0-48h, missing values will be replaced as follows: 
 single, isolated missing values (different from pre-dose and last value) will be 
replaced by linear interpolation using the adjacent values; 
 if the last value is missing, it will be replaced by the previous value; 
 in case of pre-dose value is missing and/or two or more consecutive values are 
missing and/or three or more values are missing, no replacement will be 
performed and the entire curve will be considered as missing. 
 
The AUC0-48h will be calculated after the replacement of missing values. 
 
AUC0-48h will be calculated only for patients with VAS score at screening different 
from 100 or missing. 
 
Length of hospitalization (days) 
Length of hospitalization will be calculated using the following formula: 
 
Length of hospitalization = 4 + Number of days of hospitalization after day 4  
 
If the variable “Number of days of hospitalization after day 4” is missing, the length 
of hospitalisation will be 4 and the number of days of hospitalization after day 4 
considered in the analysis will be zero. 
 
6.4 
SAFETY VARIABLES 
Pre-treatment adverse events 
An AE will be classified as pre-treatment AE if it starts before the date/time of first 
randomized study medication intake (AE onset date/time < date/time of first 
randomized study medication intake). 
 
 


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Treatment-emergent adverse event (TEAE) 
An AE will be classified as a TEAE if it starts on or after the date/time of first 
randomized study medication intake (AE onset date/time ≥ date/time of first 
randomized study medication intake). 
In case of missing or incomplete dates/times not allowing a direct allocation to any of 
the two categories of AEs, a worst-case allocation will be done according to the 
available parts of the onset dates/times and the end dates. The AE will be allocated to 
the first category allowed by the available data, according to the following order: 
 TEAE 
 Pre-treatment. 
 
Serious adverse event (SAE) 
A SAE is an AE judged as serious. 
 
Adverse drug reaction (ADR) 
An ADR is an AE judged as “Certainly related”, “Possibly related”, “Possibly 
unrelated”, “Uncertain”. 
 
Adverse event leading to discontinuation 
An AE leading to discontinuation is an AE with action taken equal to “Study drug 
withdrawn and/or patient withdrawn from the study”. 
 
Adverse event leading to death 
An AE leading to discontinuation is an AE with outcome equal to “Fatal”. 
 
Count of adverse events 
Two AEs with the same Preferred Term (PT) and classified in the same category (pre-
treatment AE or TEAE) will be considered as two different events when calculating 
the “number of events” in the tables.  
 
Incidence of cTnT elevation relative 
Percent change from baseline in cTnT will be calculated using the following formula 
at all post-dose timepoints (3h, 6h, 12h, 24h, 48h, 72h): 
% change from baseline = 100 * (cTnt at timepoint – cTnT at screening) / cTnT at 
screening 
 
The following categories will be defined at each timepoint: 
 For patients with normal cTnT value (< URL) at screening: 
o A patient will be classified as a “patient with a percent change from baseline 
in cTnT >50%” if the percent change from baseline in cTnT is greater than 
50% (percent change from baseline in cTnT > 50%); 
 For patients with abnormal cTnT value (≥ URL) at screening: 


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o A patient will be classified as a “patient with a percent change from baseline 
in cTnT >20%” if the percent change from baseline in cTnT is greater than 
20% (percent change from baseline in cTnT > 20%); 
 
 
6.5 
BASELINE  
The baseline used in the analysis for each variable (for which comparison with 
baseline is requested) is reported in the table below: 
 
Endpoint 
Baseline 
Laboratory parameters, renal function 
Screening 
Echocardiographic parameters, VAS 
score for dyspnoea, vital signs 
Pre-dose Day 1 
BNP, cTnT 
Mean (pre-dose 1, pre-dose 2) at Day 1 
 
 
7.0 
STATISTICAL METHODOLOGY 
 
7.1 
HANDLING OF MISSING DATA 
The number of patients with missing data will be presented under the “Missing” 
category, if present. Missing values will be included in the denominator count when 
computing percentages. 
When continuous data are being summarised, only the non-missing values will be 
evaluated for computing summary statistics. 
Missing or incomplete data will be treated as described in sections 6.1, 6.3 and 6.4.  
There will be no imputation of missing echocardiographic data. 
Other critical missing data will be discussed prior to treatment unblinding, if any. 
Decisions will be taken during the blind review of the data and will be fully 
documented in the Data Review Report. 
 
7.2 
COVARIATES AND SUBGROUPS 
Linear Mixed Models for Repeated Measures (MMRM) will include 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. 
Analysis of Covariance (ANCOVA) models will include treatment, centre, gender, 
baseline cTnT (normal <URL, abnormal ≥URL) and atrial fibrillation (Yes/No) as 
fixed effects and baseline as covariate. 
ANOVA models for echocardiographic parameters will include treatment only. For 
analysis of Cohort 1, treatment is defined as (0.5 μg/kg/min dose, placebo). For analysis 
of Cohort 2, treatment is defined as (1.0 μg/kg/min dose, placebo). 
All baseline values considered in the statistical analyses have been defined in the 
paragraph 6.6. 


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7.3 
INTERIM ANALYSIS AND SEQUENTIALITY OF ANALYSES 
After the completion of cohort I, a safety interim analysis will be performed and the 
Data Monitoring Committee (DMC) members will evaluate the incidence of cTnT 
elevation. At interim analysis, the DMC may recommend to do not proceed with the 
next cohort if the incidence of cTnT 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 
by an independent statistician to the DMC statistician. The DMC will be unblinded 
when performing the interim analysis. 
During the interim analysis, DMC members will analyze and discuss the results in 
closed sessions. The DMC is required to keep confidential and undisclosed until 
study conclusion: i) these discussions, ii) the detailed minutes thereof, and iii) the 
interim analysis results. 
 
For the primary endpoint, 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. 
 
7.4 
GENERAL METHODOLOGY 
Descriptive statistics will be provided for all variables in the summary tables by 
treatment group according to the type of variable summarized.  
Quantitative variables will be summarised by using n, mean, standard deviation (SD), 
95% confidence interval (CI) of the mean, median and range (minimum and 
maximum). Categorical variables will be summarised by using frequency 
distributions and percentages. 
Hypothesis testing will be carried out at the alpha = 0.05 level (two-sided) when 
comparing treatments. 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. 
Statistical analyses will be carried-out pooling together the patients treated with 
placebo in the two groups with the exception of the echocardiographic parameters 
that will be analysed separately for each cohort.  
All data collected in the CRF will be presented in the listings.  
 
 
7.5 
PATIENTS DISPOSITION 
Disposition of patients will be presented by treatment group for the randomized 
population. 
The number of patients included in each of the randomized, safety, ITT, PP will be 
summarised for each treatment group and for placebo subjects by cohort and overall.  


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Randomized patients who discontinued from the study prematurely will also be 
presented with a breakdown of the reasons for discontinuation by treatment group for 
the randomized population. 
Major and minor protocol violations will also be summarised for each treatment 
group in the randomized population. 
 
7.6 
DEMOGRAPHIC AND SCREENING/BASELINE CHARACTERISTICS 
The baseline demographic characteristics will be summarised by treatment group, and 
for placebo subjects by cohort and overall, by means of descriptive statistics. 
The following characteristics will be provided for ITT population: 
 Age (years) 
 Gender (male/female) 
 Race 
 Smoking habits 
 Alcohol consumption – Alcohol Breath test 
 Viral serology 
 Digoxin assay 
 
Past disease and concomitant disease 
Past disease and concomitant diseases will be coded using Medical Dictionary for 
regulatory activities (MedDRA) dictionary (version 16.0) and frequency distributions 
and percentages will be summarised by treatment group for the ITT population by 
System Organ Class (SOC) and PT. 
 
Medications 
Medications will be coded using World Health Organization Drug Dictionary (WHO 
DD), 2012Q1 (1st Quarter of 2012).  
Previous medications, medications maintained during the treatment period and 
concomitant medications will be summarised by treatment group for the ITT 
population through frequency distributions and Anatomical Main Group (1st level of 
the Anatomical Therapeutic Chemical (ATC) classification), Chemical Subgroup (4th 
level of the ATC classification) and Preferred Name. 
Subjects experiencing more than one medication classified in the same category 
(previous medications, medications maintained during the treatment period, 
concomitant medications) within the same anatomical main group, chemical 
subgroup and preferred name will be counted only once. 
 
Echocardiographic parameters at baseline 
Descriptive statistics will be presented for the following echocardiographic 
parameters at baseline: EDD, ESD, LV end diastolic volume, LV end systolic 
volume, LVEF, 2D global longitudinal strain, Left atrium diameter, Left atrium 
area, Left atrium volume, ERO, CI, SVI, E, A, E/A ratio, Sa, Ea, E/Ea ratio, PAPs, 


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TAPSE, Right ventricle Sa, Inferior vena cava diameter and Mitral regurgitation 
(None/Mild/Moderate/Severe). 
 
No formal comparison between treatment groups on baseline characteristics will be 
done. 
 
 
7.7 
ANALYSIS OF EFFICACY 
 
7.7.1 Primary efficacy endpoint  
Analysis will be performed separately for each cohort on ITT and PP 
populations. 
 
E/Ea ratio will be summarized at each timepoint by treatment group by means 
of descriptive statistics. 
Comparisons between treatment groups will be done using an ANOVA with 
change from baseline in E/Ea at 24 hours as dependent variable, and treatment 
as covariate. 
The number of patients and the number of patients considered in the model 
will be provided by treatment. P-values of the fixed effects and covariates will 
also be presented. 
The adjusted means for treatments and 95% CIs will be presented. The 
difference between the adjusted means for Istaroxime groups versus Placebo 
will be calculated with the 95% CIs and p-values. 
The primary comparison will be Istaroxime 0.5 μg/kg/min versus Placebo at 
24 hours based on Cohort 1 subjects only. Highest dose of Istaroxime (1.0 
μg/kg/min) versus Placebo will be tested as a secondary comparison based on 
Cohort 2 subjects only.  
The same analysis will be repeated for change from baseline in E/Ea at 6 
hours. All treatment comparisons at 6 hours will be secondary analyses. 
 
E/Ea ratio will also be summarized separated by cohort at each timepoint by 
treatment group by means of descriptive statistics. 
 
7.7.2 Secondary efficacy endpoints 
For echocardiographic parameters, analysis will be performed separately for 
each cohort on ITT population. 
For all other endpoints, analysis will be performed using the combined 
cohorts on ITT population. 
 
Echocardiographic parameters 
The following secondary endpoints: 
o LVEF 


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o LV end systolic and end diastolic volumes 
o SVI, and CI 
o E, A, E/A ratio  
o Sa (tissue doppler parameters) 
o Ea and TAPSE 
will be summarized at each timepoint by treatment group by means of 
descriptive statistics.  
Comparison between treatment groups will be done using the ANOVA 
similar to the one used for the primary efficacy endpoint. Only change from 
baseline echocardiographic data at 6 hours and 24 hours values will be 
analyzed. 
 
The global test using multivariate analysis of variance (MANOVA) 
considering multiple echocardiographic parameters will also be performed 
separately at 6 and 24 hours and by cohort for echocardiographic parameters 
grouped into 3 types of cardiac mechanics:  
 Relaxation parameters such as: E/Ea, Ea 
 Contraction parameters such as: Sa, TAPSE 
 Overall cardiac pumping ability parameters such as: SVI, CI 
The MANOVA p-value from Pallai's Trace will be reported. ((Olson (1976) 
suggests that Roy's largest root is too likely produce Type I errors and should 
be avoided, that Wilk's lambda and Hotelling's trace are sensitive to violations 
of equal covariances in smaller samples, and he recommends Pillai's trace for 
general use.)  
 
Dyspnoea using VAS score 
Analysis will be performed only for patients with VAS score at screening 
different from 100 or missing. 
Dyspnoea using VAS score and changes from baseline will be summarized at 
each timepoint by treatment group by means of descriptive statistics. 
Changes in dyspnoea using VAS score will be analyzed using a MMRM with 
change from baseline in dyspnoea using VAS score at 3, 6, 12, 24, 48 hours as 
dependent variable, treatment, centre, timepoint, gender, baseline cTnT 
(normal <URL, abnormal ≥URL), atrial fibrillation (Yes/No) and 
treatment*timepoint 
interaction 
and 
baseline and baseline*timepoint 
interaction as covariates. An unstructured covariance matrix will be 
considered and the Kenward-Roger adjustment will be used for the degrees of 
freedom. Only pre-dose, 3, 6, 12, 24, 48 hours values after infusion start will 
be considered in the MMRM. 
 
AUC0-48h on change in dyspnoea by VAS score 
Analysis will be performed only for patients with VAS score at screening 
different from 100 or missing. 


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AUC0-48h on change in dyspnoea by VAS score will be summarized by 
treatment group by means of descriptive statistics. 
Comparison between treatment groups will be performed by means of an 
ANCOVA model with AUC0-48h on change in dyspnoea by VAS score as 
dependent variable, treatment, centre, gender, baseline cTnT (normal <URL, 
abnormal ≥URL) and atrial fibrillation (Yes/No) as fixed effects and baseline 
dyspnoea as covariate. 
The number of patients and the number of patients considered in the model 
will be provided by treatment. P-values of the fixed effects and covariate will 
also be presented. 
The adjusted means for treatments and the 95% CIs will be presented. The 
differences between the adjusted means for Istaroxime groups versus Placebo 
will be calculated with the 95% CIs and p-values. 
 
BNP 
BNP and change from baseline will be summarized at each timepoint by 
treatment group by means of descriptive statistics. 
Comparison between treatment groups will be performed by means of an 
ANCOVA model with change from baseline in BNP at 24 hours as dependent 
variable, treatment, centre, gender, baseline cTnT (normal <URL, abnormal 
≥URL) and atrial fibrillation (Yes/No) as fixed effects and baseline BNP as 
covariate 
The number of patients and the number of patients considered in the model 
will be provided by treatment. P-values of the fixed effects and covariate will 
also be presented. 
The adjusted means for treatments and the 95% CIs will be presented. The 
differences between the adjusted means for Istaroxime groups versus Placebo 
will be calculated with the 95% CIs and p-values. 
 
Patients with hospital readmissions or emergency visits for cardiovascular 
reasons within Day 30 
The number and percentage of patients with hospital readmissions or 
emergency visits for cardiovascular reasons within Day 30 will be 
summarized by treatment groups using descriptive statistics.  
 
Patients with episodes of worsening heart failure during the hospitalization 
The number and percentage of patients with episodes of worsening heart 
failure during the hospitalization will be summarized at Days 2, 3 and 4 by 
treatment groups using descriptive statistics. 
 
Length of hospitalization 
Length of hospitalization and the number days of hospitalization after day 4 
will be summarized by treatment group using descriptive statistics.  


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7.8 
ANALYSIS OF SAFETY  
Analysis will be performed on Safety population. 
 
Adverse events 
Pre-treatment AEs and TEAEs will be presented separately. Pre-treatment AEs will 
be presented in the listings only. 
The number of treatment-emergent AEs, SAEs, ADRs, AEs leading to 
discontinuation and AEs leading to death, and the number and the percentage of 
patients experiencing treatment-emergent AEs, SAEs, ADRs, AEs leading to 
discontinuation and AEs leading to death will be summarised by treatment group.  
AEs will be coded using the MedDRA dictionary (version 16.0). The SOCs and PTs 
will be used for tabulation. The number of AEs and the number and the percentage of 
patients with at least one AE will be presented by SOC and PT for treatment-
emergent AEs, SAEs, ADRs, AEs leading to discontinuation and AEs leading to 
death by treatment group. 
 
Vital signs 
Vital signs and change from pre-dose will be summarised by treatment group at each 
timepoint by means of descriptive statistics.  
 
Biomarkers 
NT-proBNP and cTNT and increase of cTnT will be summarised by treatment group 
at screening visit and at all timepoints during the study by means of descriptive 
statistics. 
The number and percentage of patients with a percent change from pre-dose in cTnT 
>50% and >20% will be summarized at each post-dose timepoints by treatment 
group. 
The number and percentage of patients with a normal cTnT value (<14 ng/L) at 
screening and with a percent change from pre-dose in cTnT >50% and the number 
and percentage of patients with an abnormal value of cTnT (≥14 ng/L) at screening 
and with a percent change from pre-dose in cTnT >20% will also be summarized at 
each post-dose timepoints by treatment group. 
 
24 hours digital ECG Holter  
Premature ventricular complexes, ventricular tachycardia, new onset atrial fibrillation 
and incidence of 
clinically or hemodynamically significant episodes of 
supraventricular or ventricular arrhythmias will be summarised by treatment group at 
screening and Days 1 and 2 by means of descriptive statistics. 
 
ECG 


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ECG parameters derived from 12-lead ECG will be summarized by treatment group 
at screening and Days 1, 2, 3 and 4 by means of descriptive statistics.  
 
Laboratory parameters 
Shift tables presenting the number and the percentage of patients in each bivariate 
category (screening and Day 3) with regards to investigator’s interpretation (normal, 
Not Clinically Significant (NCS), Clinically Significant (CS), No result (NR)) will be 
provided for all laboratory parameters (excluding potassium, eGFR, creatinine and 
creatinine clearance).   
 
Renal function 
Renal function will be evaluated by means of potassium, eGFR, creatinine and 
creatinine clearance parameters; for these parameters, the number and percentage of 
patients by investigator’s interpretation (Normal, NCS, CS, NR) will be summarised 
by treatment group at screening and Days 2, 3, 4 and 30. In addition, the continuous 
values of eGFR will be summarized by means of descriptive statistics.   
 
7.8.1 Extent of exposure  
Extent of exposure will be summarized by treatment group by means of 
descriptive statistics.  
 
 
7.9 
OTHER PLANNED ANALYSIS 
None 
 
8.0 
GENERAL CONSIDERATIONS 
 
8.1 
SOFTWARE TO BE USED 
All statistical analyses and data processing will be performed using Statistical 
Analysis Systems (SAS®) Software (release 9.4) on a Windows 7 operating system. 
 
8.2 
PROGRAMS AND TABLES QUALITY CONTROL 
The statistician-programmer of the tables, listings and figures will carefully review 
the programs and will verify that no error message is highlighted in the ‘LOG’ file.  
Moreover, a second statistician-programmer will verify the internal consistency of 
each table and figure by checking the results using different SAS programs.  
 
The following level of validation will be implemented: 
 Tables:    
- Check the absence of errors and warning messages in the SAS LOG; 
- Check the 100% of data contained in the table through double 
programming; 


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- Check the layout of the table (including titles, footnotes) versus the one 
detailed in the mock-up/SAP. 
 Listings: 
- Check the absence of errors and warning messages in the SAS LOG; 
- Check the correctness of the SAS code used for the production of the 
listings (e.g. the correct variables were used, the correct selections and 
formats were applied, etc); 
- Check the layout of the listing (including titles, footnotes) versus the one 
detailed in the mock-up/SAP. 
 
 
9.0 
PROGRAMMING SPECIFICATIONS 
The following SAS code will be used to calculate the age of patient: 
 
age = floor((intck('month', date of birth, date of screening)-(day(date of screening)<day(date 
of birth)))/12) 
 
 
The following SAS code will be used to estimate the MMRM: 
 
proc mixed data = dataset; 
class tmt timepoint centre gender cTnT atr_fib patient; 
model change = tmt timepoint tmt*timepoint centre cTnT atr_fib gender baseline 
baseline*timepoint / ddfm=kr; 
repeated timepoint / subject=patient type=un; 
lsmeans tmt tmt*timepoint / cl; 
run; 
 
where change represents the change from baseline to post-dose values of the variable, tmt the 
treatment group, timepoint the timepoint, centre the centre, gender the gender (male/female), 
cTnT the baseline cTnT (normal <URL, abnormal ≥URL), atr_fib the atrial fibrillation 
(Yes/No), baseline the baseline value of the variable and patient the Patient Number. 
The variable tmt will be ordered in SAS in the following order: 
 Istaroxime 0.5 μg/kg/min  
 Istaroxime 1.0 μg/kg/min  
 Placebo 
 
 
The following SAS code will be used to estimate the ANCOVA model: 
 
 proc mixed data = dataset; 
 class tmt centre gender cTnT atr_fib; 
 model variable = tmt centre gender cTnT atr_fib predose; 


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 lsmeans tmt / cl; 
run; 
 
where variable represents the efficacy variable (AUC on changes in dyspnoea by VAS 
score or changes in BNP), tmt the treatment group, centre the centre, gender the gender 
(male/female), cTnT the baseline cTnT (normal <URL, abnormal ≥URL), atr_fib the atrial 
fibrillation (Yes/No), predose the pre-dose of efficacy variable. 
The variable tmt will be ordered in SAS in the following order: 
 Istaroxime 0.5 μg/kg/min  
 Istaroxime 1.0 μg/kg/min  
 Placebo 
 
 
 
The following SAS code will be used for the Wilcoxon rank sum test for Tmax: 
 
proc npar1way wilcoxon hl alpha=0.1 data = dataset; 
class tmt; 
var Tmax; 
run; 
 
where tmt represents the treatment groups and Tmax the Tmax parameter value for each 
patient. 
 
The following SAS code will be used for the ANOVA: 
 
proc glm data = dataset; 
 class tmt; 
 model variable = tmt; 
 lsmeans tmt / cl; 
run; 
 
The following SAS code will be used for the MANOVA: 
 
proc glm data = dataset; 
 class tmt; 
 model variable = tmt; 
contrast 'active vs placebo' group 1 -1; 
manova h=tmt; 
 lsmeans tmt / cl; 
run; 
 
The global test p-value from Pallai's Trace will be reported.  


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PROGRAMMING CONVENTIONS 
All tables/figures/listings will be presented in landscape format.  
The standard font size is 9 points Courier New for all tables. Listings will be presented with 
an 8 or 7 points Courier New.  
 
Titles and footnotes will be left-aligned.  
Each table/figure/listing will have 2 titles: 
 The 1st title will be the table/figure/listing number with the description of the 
table/figure/listing;  
 The 2nd title will be a description of the study population presented in the 
table/figure/listing. 
Some tables will have a third title (before 2nd title) with a description of the statistical 
method used in those tables. 
 
Tables identified by a “#” at the end of 1st title will also be presented in the Key First 
Results. 
 
Any footnote added to explain the table/listing/figure contents will be presented in the 
following format: 
Note 1: Percentages are calculated on the number of patients (N). 
Note 2: A serious adverse event is an .... 
Note 3: ………. 
 
The last two footnotes of each table/figure will be footers indicating: 
 the reference listing of the data; 
 the program name, the date and time of generation and the SAS® version used. 
The last footnote of each listing will be a footer indicating the program name, the date and 
time of generation and the SAS® version used. 
 
In the tables, listing and figures the treatment under comparison will be labelled as 
“Istaroxime 0.5 μg/kg/min”, “Istaroxime 1.0 μg/kg/min” and “Placebo”. 
 
Except for the listing 16.2.1-2.1 (based on screening failure patients) all the listings will be 
based on the randomized population. 
Unless otherwise stated, listings will be presented by randomized treatment group, and sorted 
by the patient number (5-digit).  
In all the listings on safety variables, a column with a flag (§) for treatment misallocation will 
identify the treatment misallocations. 
The derived variables will be identified in the listings with a flag (*). 
 
In general, dates will be presented on listings in the format ddmmmyyyy (date9.) and time in 
the format hh:mm (time5.). In case of partial dates or times, missing information will be 


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replaced by dashes. Numeric variables will be listed generally with the same number of 
decimal places as in the actual data. 
 
The following rules on decimal places will be considered in the listings for the derived 
variables (in the analyses approximations will not be performed): 
 Age (years), length of hospitalization (days): 0 decimal place; 
 Extent of exposure (hours), AUC0-48h on changes in dyspnoea: 1 decimal place; 
 Change from pre-dose/baseline: same as the variable considered. 
 
The following rules on decimal places will be considered for the results of the analyses (if the 
analyses are performed on derived variables, the level of precision of the actual data is 
derived from the previous list): 
 Min, max: same as actual data; 
 Mean and its confidence limits (unadjusted and adjusted), adjusted difference between 
means and its confidence limits, SD, median: actual data + 1 decimal; 
 Percentage: 1 decimal place. 
 
 
10.0 
ARCHIVING 
These deliverables will be archived in CROS NT study file. The paper TMT will be sent 
back to the Sponsor after completion of the study.    
 
Deliverable 
Electronic 
Paper 
Statistical Analysis Plan 
X 
X(*)  
Randomization List  
X 
X(*)  
Data Review Plan  
X 
X (*) 
Data Review Listings  
X 
 
Data Review Report  
X 
X(*) 
Derived datasets 
X 
 
Key First Results 
X 
 
Tables, Listings and Figures 
X 
X(*) only for 
approval page 
Tables, Listings, Figures validation
 
X(*)(^) 
Statistical Report 
X 
X(*) 
(*) Flag for original documents   
(^) Only tables and figures 
 
 
11.0 
REFERENCES 
 European Medicines Agency (EMEA), International Conference on Harmonisation (ICH) E3 
Harmonised Guideline (1996) “Structure and Content of Clinical Study Reports”; 
 EMEA, ICH E9 Harmonised Guideline (1998) “Statistical Principles for Clinical Trials”. 


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APPENDIX 1: MOCK-UPS OF TABLES, LISTINGS AND FIGURES 
 
See document “AppendixI_CVT-CV-002_1.0_181030.docx”. 
Olson, C. L. (1976). On choosing a test statistic in multivariate analysis of variance. Psychological 
Bulletin, 83, 579-586.