Clinical trial NCT03594175
Conditions: Catheter Occlusion Thrombosis
Keywords: Central Venous Access Device (CVAD) CUSA-081 reteplase alteplase occluded catheters catheter occlusion thrombosis thrombotic occlusion CVAD
Sponsor: Chiesi Farmaceutici S.p.A.
Start date: 2020-02-12; Completion date: 2023-07-10
Enrollment: 462 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
To evaluate the efficacy and safety of CUSA-081 (diluted reteplase) in the restoration of central venous access device (CVAD) functionality in participants 18 years and older.

Detailed description:
This was a phase III, multinational, multicenter, randomized, double-blind, parallel-group, active and placebo-controlled study to examine CUSA-081 (diluted reteplase) versus placebo or alteplase in subjects with dysfunctional non-hemodialysis Central Venous Access Devices (CVADs).

During the study, the treatment period consisted of 1 visit that may have taken place on the same day as screening or on the following day. After complying with all inclusion criteria, subjects were randomized in a 9:1:6 ratio to CUSA-081 : placebo : alteplase treatment group. A follow-up assessment was performed on Day 30 (±2 days) after treatment with study drug. The end of the study was defined as the last follow-up contact of the last subject to receive study drug in the study.

Routine blood pressure measurement, heart rate and urine pregnancy test were performed before enrolment in the study. Throughout the study, safety assessment included evaluation of treatment emergent adverse events (TEAEs), adverse drug reactions (ADRs), and adverse events (AE) of Special Interest (AESI).

CUSA-081 (reteplase) is a recombinant tissue plasminogen activator (tPA), currently approved in the USA (trade name: RETAVASE®) for treatment of acute ST-elevation myocardial infarction (STEMI) to reduce the risk of death and heart failure.

Alteplase, a biosynthetic form of human tPA, Food and Drug Administration (FDA)-approved under the brand name ACTIVASE® for the treatment of acute ischemic stroke, acute myocardial infarction (AMI) to reduce mortality and incidence of heart failure, and acute massive pulmonary embolism for lysis.

Arms: arm 1: Participants received 1 or 2 doses of CUSA-081, 0.7 milligrams (mg) (0.4 units) per 2 milliliter (mL) directly into the catheter lumen. Participants received the first dose at minute (min) 0, and the second dose (if needed) at min 90. arm 2: Participants received 1 or 2 doses of placebo (normal saline) directly into the catheter lumen. Participants received the first dose at min 0, and the second dose (if needed) at min 90. arm 3: Participants received 1 or 2 doses of alteplase, 2 mg/mL, directly into the catheter lumen. Participants received the first dose at min 0, and the second dose (if needed) at min 90.
Interventions: intervention 1: CUSA-081 intervention 2: Placebo intervention 3: Alteplase
Intervention descriptions: intervention 1: Participants received 1 or 2 doses of CUSA-081 0.7 mg/2 mL directly into the catheter lumen intervention 2: Participants received 1 or 2 doses of placebo (normal saline) directly into the catheter lumen intervention 3: Participants received 1 or 2 doses of alteplase, 2 mg/2 mL, directly into the catheter lumen

Protocol documents:
Prot_000.pdf: 
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CLINICAL STUDY PROTOCOL 
STUDY CODE No.: CUSA-081-HEM-01
EUDRACT No. / IND No: 2019-002124-32 / 128551
READY 1:  A PHASE 3, RANDOMIZED, DOUBLE-BLIND, ACTIVE AND PLACEBO-
CONTROLLED STUDY ON THE USE OF CUSA-081 FOR DYSFUNCTIONAL CENTRAL 
VENOUS ACCESS DEVICES (CVADs)
Version No.: 8.0
Date: 21 February 2023
The information contained in this document is confidential and will not be disclosed to others without written 
authorization from Chiesi Farmaceutici S.p.A., except to the extent necessary to obtain informed consent from those 
persons to whom the drug may be administered or for discussions with local regulatory authorities, Ethics 
Committee/Investigational Review Boards, or people participating in the conduct of the study.
Chiesi Farmaceutici S.p.A.
Via Palermo 26/A
43122 Parma - Italy
NCT03594175


Clinical Study Protocol
Clinical Study Code No.: CUSA-081-HEM-01
Version No.: 8.0
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Date: 21 FEB 2023
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GENERAL INFORMATION
SPONSOR:
Chiesi Farmaceutici S.p.A.* 
Via Palermo 26/A
43122 Parma - Italy
+ 39 0521 2791
*also reported as Chiesi throughout
the text
SPONSOR MEDICAL EXPERT
(Clinical Research Physician)
 MD, PhD
Tel. 
Fax. 
E-mail: 
Mobile: 
*
*Readily available in case of medical
questions
MONITORING CRO
VERSION HISTORY
Version 
Date 
Change History
1.0
16 Jul 2018
First version of the protocol
2.0
17 Aug 2018
Clarification of inclusion and exclusion criteria, 
non-permitted concomitant medication, and use of 
other thrombolytics during treatment period
3.0
24 Sep 2018
Removed subjects less than 18 years of age from 
the study population
4.0
31 May 2019
Change of the Sponsor, protocol template updated, 
expansion of study to countries outside of the 
United States, updates to contact information, 
increased number of subjects enrolled to account 
for screen failures and early withdrawals, 
clarifications made to the eligibility criteria, and 
clarifications made regarding safety reporting
5.0
20 Nov 2020
Change in Sponsor Medical Expert. Revised 
inclusion and exclusion criteria. Clarifications on 
permitted and non-permitted concomitant 
medications related to catheter management. 
Addition of second manufacturer of placebo. 
Statement added for COVID-19. Revised study 
schedule for clarity including revision to language 
regarding urine pregnancy test. Revised language 
regarding determination of CVAD dysfunction and 
safety variables. Added definition for Adverse 


Clinical Study Protocol
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Version No.: 8.0
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Date:  21 FEB 2023
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Events of Special Interest. Additional revisions for 
clarification.
6.0
18 Dec 2020
Revised inclusion criteria to reinclude subject age 
of 18 years or older as it was inadvertently removed 
from protocol version 5.0. Added language to 
clarify pregnancy test requirement. Added 
additional language to define menopause and 
provide clarification. 
Clarifications on permitted and non-permitted 
concomitant medications related to catheter 
management. Revised language regarding 
determination of CVAD withdrawal and infusion 
functions. Minor administrative changes.
7.0
30 Sep 2022
Revised to include a change of the protocol to an 
adaptive study design.
A comparative interim analysis is planned to occur when 
approximately 416 patients have completed the study.
This interim analysis is composed of two separate 
evaluations.
1) To test whether CUSA-081 is superior to placebo in
the rate of treatment success following a single
administration of study drug with a dwell time up to 90
minutes.
2) To perform a futility analysis by assessing the
conditional power for the test of non-inferiority of
CUSA-081 compared to alteplase in the rate of treatment
success following a single administration of study drug
with a dwell time up to 90 minutes.
The study design may change based on the outcomes of
the interim analysis:

If CUSA-081 is demonstrated as superior to
placebo at the interim analysis, then the placebo
arm will be dropped, and the study will continue
enrolling patients to receive either CUSA-081 or
alteplase in a ratio of 9:6 CUSA-081: alteplase,
as described in Section 3.

If CUSA-081 is not demonstrated as superior to
placebo at the interim analysis, then the study
will continue without modifications.

If the futility analysis finds that it is unlikely to
demonstrate non-inferiority of CUSA-081
compared to alteplase, then the study will be
terminated early.
An independent and unblinded DMC will be established 
to review the data and provide a recommendation 
regarding the study design.
8.0
21FEB 2023
Revised to modify the interim analysis proposed in 
protocol v7.0 as a result of the interactions with the 
FDA. Modifications include the removal of the 
adaptive study design and the efficacy evaluation of 
the interim analysis that was introduced to test 


Clinical Study Protocol
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Version No.: 8.0
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Date:  21 FEB 2023
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whether CUSA-081 is superior to placebo in the 
rate of treatment success following a single 
administration of study drug with a dwell time up to 
90 minutes.  After these modifications, only the 
interim futility analysis is foreseen. 


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Version No.: 8.0
EUDRACT No./IND No.: 2019-002124-32/128551
Date:  21 FEB 2023
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PROTOCOL OUTLINE
Study title
READY 1: A Phase 3, Randomized, Double-Blind, Active and Placebo-
Controlled Study on the use of CUSA-081 for Dysfunctional Central Venous 
Access Devices (CVADs)
Sponsor
Chiesi Farmaceutici S.p.A. - Via Palermo 26/A 
43122 Parma - Italy 
Name of the Product
CUSA-081 (reteplase)
Centre(s)
Approximately 100 sites
Indication
Restoration of function to central venous access devices (CVADs)
Study design
Multinational, multicenter, prospective, randomized, double-blind, parallel-
group, active and placebo-controlled clinical trial
Study phase
Phase 3
Objectives
Primary Objective:
To demonstrate the superiority of CUSA-081 compared to placebo in the 
rate of treatment success following a single administration with a dwell 
time up to 90 minutes. Treatment success is defined as restoration of 
CVAD functionality measured as the ability to withdraw 3 mL of blood 
and infuse 5 mL of saline.
Secondary Objectives:
1.
To demonstrate the non-inferiority of CUSA-081 compared to
alteplase in the rate of treatment success following a single
administration with a dwell time up to 90 minutes.
2.
To demonstrate the superiority of CUSA-081 compared to placebo
in the rate of treatment success following a single administration
with a dwell time up to 60 minutes.
3.
To demonstrate the superiority of CUSA-081 compared to placebo
in the rate of treatment success following up to two administrations
with a dwell time up to 180 minutes.
4.
To demonstrate the superiority of CUSA-081 compared to alteplase
in the rate of treatment success following a single administration
with a dwell time up to 90 minutes.
5.
To evaluate the safety and tolerability of CUSA-081.
6.
To evaluate the rate of recurrent catheter dysfunction defined as first
re-occlusion within 30 days following administration of CUSA-081.
Treatment duration
Treatment configurations are summarized below:
Treatment 
Arm
Syringe 1 
(given at min 0)
Syringe 2 
(given, if needed, 
at min 90)
A
CUSA-081
CUSA-081
B
placebo
placebo
C
alteplase
alteplase


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Test product 
dose/route/regimen
Treatment Arm A: One or two doses of CUSA-081 (reteplase), 0.70 mg 
(0.4 unit) / 2 mL, will be administered directly into the catheter lumen
Reference product 
dose/route/regimen
Treatment Arm B: One or two doses of placebo (normal saline) will be 
administered directly into the catheter lumen
Treatment Arm C: One or two doses of alteplase, 2 mg in 2 mL, will be 
administered directly into the catheter lumen
Number of subjects
A minimum of 841 randomized subjects:
Treatment Arm A (CUSA-081),
473 subjects
Treatment Arm B (placebo),
53 subjects
Treatment Arm C (alteplase),
315 subjects
Subjects will be randomized in a 9:1:6 ratio of CUSA-081: placebo: 
alteplase.
Study population
Subjects who are 18 years of age or older who have dysfunctional CVADs
Inclusion/exclusion criteria
Inclusion Criteria
Subjects must meet all of the following inclusion criteria to be eligible for 
enrollment into the study:
1.
Inability to have 3mL of blood withdrawn from the selected study
catheter;
2.
A single or multi-lumen CVAD, implanted ports or peripherally
inserted central catheters (PICCs) in place for > 24 hours and
documented as previously being patent and functional;
3.
Ability to designate one dysfunctional lumen of a multi-lumen
catheter to be used throughout the study for both study drug
instillation and assessment of CVAD function;
4.
Male and non-pregnant female subjects 18 years or older (see note
below);
5.
Able to have fluids infused at the volume necessary to instill study
drug into the CVAD (i.e., up to 2 mL);
6.
Informed consent form (ICF) signed and dated indicating that the
subject has been informed of and agreed with all pertinent aspects
of the study and is willing to comply with all study requirements
and procedures.
NOTE: A urine pregnancy test is required for all females of childbearing 
potential. Women in natural or surgical post-menopause do not need to be 
tested for pregnancy. Natural menopause is defined as the permanent 
cessation of menstrual periods, determined retrospectively after a woman 
has experienced 12 consecutive months of lack of menstruation 
(amenorrhea) without any other obvious pathological or physiological 
cause. Surgical menopause is defined as the removal of both ovaries 
(bilateral oophorectomy) before the natural menopause.
Exclusion Criteria
The presence of any of the following will exclude a subject from study 
enrollment:
1.
CVAD (any type) used for Hemodialysis;


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2.
CVAD known to be dysfunctional for more than 48 hours;
3.
Reasonable evidence of mechanical or non-thrombotic occlusion in
the selected study catheter (e.g., catheter malposition or migration,
sutures, kinks, or precipitates causing obstruction), radiographic
assessment is not required;
4.
Known or suspected catheter-related bloodstream infection
(CRBSI);
5.
Use of any intravenously administered fibrinolytic agent or
anticoagulant (e.g., alteplase, tenecteplase, reteplase, urokinase or
heparin) within 24 hours prior to the treatment period (first
instillation of study drug). Use of subcutaneous LMWH, UFH or
heparinoids for prophylaxis of thromboembolic events is allowed.
Furthermore, the use of oral anticoagulants is allowed;
6.
Known to be at high risk for bleeding events or embolic
complications in the opinion of the Investigator, or has a known
condition for which bleeding constitutes a significant hazard (e.g.
recent stroke, recent intracranial or intraspinal surgery or serious
head trauma, intracranial neoplasm, arteriovenous malformation or
aneurysm, known bleeding diathesis);
7.
Uncontrolled hypertension (systolic BP ≥160 or diastolic BP ≥110
mmHg) at screening;
8.
Clinically unstable in the opinion of the site investigator;
9.
Known to be pregnant or breastfeeding at screening;
10. Previously treated in this study (READY 1) or in study READY 2;
11. History of allergic reaction to reteplase, alteplase or vial ingredients
(excipients or diluents);
12. Use of any investigational drug or experimental medical device
within 28 days prior to treatment; non interventional observational
studies participation is allowed.
13. Not mentally, socially or otherwise able to complete the trial
assessments or not likely to survive beyond 30 days.
Study plan
A total of 2-3 visits may be performed during the study, as follows:

Screening (Baseline Visit) (Day 0 or Day 1)

Treatment Visit 1 (V1) (Day 1)

Follow-up Visit 2 (V2) (Day 30 ± 2 days) (Can be performed by
telephone)
An extra visit could be performed at Screening (Baseline Visit) for 
logistical reasons. Day 1 must be the following calendar day (for example, 
Monday and Tuesday; not Friday and Monday). Informed consent will be 
signed during this visit.
Most relevant allowed 
concomitant treatments
Catheter locking solutions (e.g. normal saline or low dose heparin, which 
requires a normal saline flush to clear the heparin from the IV line).
The use of subcutaneous LMWH, UFH or heparinoids for prophylaxis of 
thromboembolic events is allowed. Furthermore, the use of oral 
anticoagulants is allowed.
Most relevant forbidden 
concomitant treatments
The use of any intravenously administered fibrinolytic agent or 
anticoagulant within 24 hours prior to the beginning of the treatment period 
(first instillation of study drug).
Efficacy variables
(and/or pharmacokinetics 
variables)
Primary Efficacy Variable:
Percentage of subjects who have treatment success following a single 
instillation of study drug (CUSA-081, placebo, or alteplase) with a dwell 
time up to 90 minutes. Treatment success is defined as the restoration of 


Clinical Study Protocol
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Version No.: 8.0
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CVAD functionality measured as the ability to withdraw 3 mL of blood and 
infuse 5 mL of saline.
Secondary Efficacy Variables:
1.
Percentage of subjects who have treatment success following a
single instillation of study drug with a total dwell time up to 30 and
60 minutes;
2.
Percentage of subjects who have treatment success after up to two
instillations of study drug with a total dwell time up to 120, 150
and 180 minutes;
3.
The rate of recurrent catheter dysfunction defined as first re-
occlusion within 30 days following treatment with study drug.
Safety variables

Treatment Emergent Adverse Events (TEAEs), Adverse Drug
Reactions (ADRs), Serious Treatment Emergent Adverse Events
(SAEs), serious related TEAEs, TEAEs leading to study
withdrawal, and TEAEs leading to death

Treatment Emergent Adverse Events of Special Interest (AESIs),
as defined in Section 10.1
Sample size calculation
For the primary assessment of the superiority of CUSA-081 vs. placebo, a 
sample size of 450 in the active arm (CUSA-081), and 50 in the control arm 
(placebo), will provide >99% power to detect a difference of ≥35% using a 
two-sided test at significance level of 0.05. The expected placebo response 
rate is approximately 20%.
For the assessment of the non-inferiority of CUSA-081 vs. alteplase, a 
sample size of 450 in the active arm (CUSA-081), and 300 in the control 
arm (alteplase) will provide 87% power at the 2.5% significance level (that 
is by using one-sided 97.5% confidence intervals with a non-inferiority 
margin of -10%), assuming an equal response rate of 75% in the control arm 
(alteplase) and in the active (CUSA-081) arm. A sample size of 450 in the 
active (CUSA-081) arm and 300 in the control arm (alteplase) will also 
provide 92% power to detect a difference of 10 % between the success rates 
using a two-sided test at significance level of 0.05.
A minimum of 841 subjects will be randomized to achieve a minimum of 
800 subjects completing the study.
Statistical methods
Analysis Population:

Full analysis set (FAS): All randomized subjects who receive at
least one dose of study drug and with at least one available
evaluation of efficacy after the baseline. Analyses using the FAS
will be based on the treatment randomized. The FAS will be the
primary analysis set for all efficacy analyses.

Safety set (SS): All randomized subjects who receive at least one
dose of study drug. Subjects discontinued after dosing will be part
of the SS. Analyses using the SS will be based on the actual
treatment received. The SS will be the primary analysis set for all
safety analyses.

Intent-To-Treat analysis set (ITT): All randomized subjects
regardless if they received treatment with study drug.  The ITT will
be based on the randomized treatment allocation. The ITT will be
used for sensitivity analysis for the primary efficacy analyses.

Per-protocol analysis set (PPS): all subjects from the ITT
population without any major protocol deviation (i.e., wrong
inclusions, incomplete dosing, non-permitted concomitant


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medications). Exact definition of major protocol deviations will be 
discussed by the study team during the blind review of the data 
prior to the database lock. The PPS will be used for sensitivity 
analysis for the primary efficacy analyses and the non-inferiority 
analyses.
Efficacy:
The Primary efficacy comparison is to demonstrate the superiority 
in the rate of treatment success following a single administration of CUSA-
081 compared to placebo with a dwell time up to 90 minutes. The percentage 
of subjects with treatment success and two-sided 95% Wald confidence 
intervals (CIs) will also be provided by treatment group. The between 
treatment comparisons will be tested using a 2-sample z test. The between 
treatment group difference and associated 95% Wald confidence intervals 
will be provided. 
Similar analyses will be performed for the secondary efficacy 
objectives. 

The non-inferiority of CUSA-081 to alteplase in the rate of treatment
success following a single administration of study treatment with a
dwell time up to 90 minutes will be assessed based on the constructed
95% confidence interval for the difference in the rate of treatment
success between CUSA-081 vs alteplase, with non-inferiority
considered as demonstrated if the lower limit of the 95% confidence
interval for the difference in rate of success is greater than -10%.

The superiority in the rate of treatment success following a single
administration of CUSA-081 compared to placebo with a dwell time up
to 60 minutes will be tested using a 2-sample z test as described for the
primary efficacy comparison.

The superiority in the rate of treatment success following up to two
administrations of CUSA-081 compared to placebo with a dwell time
up to 180 minutes will be tested using a 2-sample z test as described for
the primary efficacy comparison.

The superiority in the rate of treatment success following a single
administration of CUSA-081 compared to alteplase with a dwell time
up to 90 minutes will be tested using a 2-sample z test as described for
the primary efficacy comparison.
A closed testing procedure will be used to control the overall Type 
I error rate at less than 5%. The hypothesis testing will start with the primary 
efficacy objective. Then the secondary efficacy objectives (1 – 4) will be 
tested, in the order listed in the study objectives, until a p-value ≥ 0.05 is 
observed (or lower limit of the two-sided 95% CI ≤ -10% for the non-
inferiority assessment).  
The rate of recurrent catheter dysfunction defined as first 
reocclusion within 30 days following treatment with CUSA-081 will be 
analyzed using the Kaplan-Meier method and summarized with median, 25th 
and 75th percentiles, and 95% confidence intervals. The rate of recurrent 
catheter dysfunction within 30 days will be estimated. This analysis will be 
based on all subjects with treatment success following up to two 
administrations of study drug with a total dwell time up to 180 minutes.


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Safety:

The number and percentage of subjects experiencing TEAEs, ADRs,
SAEs, serious related TEAEs, TEAEs leading to study withdrawal,
TEAE leading to death, and AESIs, will be presented by treatment
group and overall. Each of these groups of adverse events will also be
summarized by System Organ Class and Preferred Term.

Safety variables will be presented as the number and percent of
subjects with events and the number of events.
Interim Analysis 
A futility analysis is planned to occur when approximately 416 patients have 
completed the study.
The futility analysis will assess the conditional power for the test of non-
inferiority of CUSA-081 compared to alteplase in the rate of treatment 
success following a single administration of study drug with a dwell time up 
to 90 minutes.
If the futility analysis finds that it is unlikely to demonstrate non-inferiority 
of CUSA-081 compared to alteplase, then the study will be terminated 
early.
A separate independent and unblinded team will be established to perform 
the futility analysis and prepare the data for review (“Unblinded 
Statistical/Programming Team”).
An independent and unblinded Data Monitoring Committee (DMC) will be 
established to review the data of the futility analysis and provide a 
recommendation to stop or continue the study. Details of the DMC, including 
roles, responsibilities, communication processes and frequency of meetings 
will be provided in a separate DMC Charter.
The DMC will provide a recommendation regarding the continuation of the 
study as follows:

the study should continue without modification;

the study should be terminated prematurely.
The DMC will also review the safety data at the time of the futility analysis.
The study team and the other blinded team members will remain blinded 
throughout the futility analysis.


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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS
The following abbreviations are used in this study protocol.
Table 1: Abbreviations
ADR
Adverse Drug Reaction
AE
Adverse Event
AESI
Adverse Events of Special Interest
AMI
Acute Myocardial Infarction
ATC
Anatomically Therapeutic Chemical 
BP
Blood Pressure
cGCP
Current Good Clinical Practices
cGMP
Current Good Manufacturing Practices
CI
Confidence Interval
CIOMS
Council for International Organizations of Medical Sciences
CRA
Clinical Research Associate
CRBSI
Catheter Related Bloodstream Infection
CRO
Contract Research Organization
CTA
Clinical Trial Agreement
CVAC
Central Venous Access Catheter
CVAD
Central Venous Access Device
CVC
Central Venous Catheter
DMC
Data Monitoring Committee
EC
Ethics Committee
eCRF
Electronic Case Report Form
EMA
European Medicines Agency
FAS
Full Analysis Set
FDA
Food and Drug Administration
IB
Investigator’s Brochure
ICF
Informed Consent Form
ICH
International Conference on Harmonization
IEC
Independent Ethics Committee
IMPS
Investigational Medicinal Product Supplies
INR
International Normalized Ratio
IRB
Institutional Review Board
ITT
Intent to Treat analysis set
IUD
Intrauterine device
IUS
Intrauterine system
I.V.
Intravenous
IRT
Interactive Response Technology
LMWH
Low Molecular Weight Heparin
MedDRA
Medical Dictionary for Regulatory Activities
NI
Non-inferiority
PE
Pulmonary Embolism
PICC
Peripherally Inserted Central Catheter
PPS
Per-protocol analysis set
r-PA
Recombinant Plasminogen Activator
rt-PA
Recombinant Tissue Plasminogen Activator
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SMP
Safety Management Plan


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SS
Safety Set
SUSAR
Suspected Unexpected Serious Adverse Reaction
STEMI
ST-Elevation Myocardial Infarction
SWFI
Sterile Water For Injection
t-PA
Tissue Plasminogen Activator
TEAE
Treatment Emergent Adverse Event
TFL
Tables, Figures, and Listings
UFH
Unfractionated Heparin 


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CONTENT
GENERAL INFORMATION..........................................................................................................................2
VERSION HISTORY.......................................................................................................................................2
PROTOCOL OUTLINE ..................................................................................................................................5
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS................................................................11
CONTENT.......................................................................................................................................................13
1.
INTRODUCTION..................................................................................................................................15
1.1 BACKGROUND INFORMATION.................................................................................................................15
1.2 STUDY RATIONALE.................................................................................................................................16
1.3 RISK/BENEFIT ASSESSMENT ...................................................................................................................17
2.
STUDY OBJECTIVES..........................................................................................................................18
2.1 PRIMARY OBJECTIVE(S)..........................................................................................................................18
2.2 SECONDARY OBJECTIVE(S).....................................................................................................................19
3.
STUDY DESIGN....................................................................................................................................19
4.
SUBJECT SELECTION CRITERIA ..................................................................................................22
4.1 SUBJECT RECRUITMENT..........................................................................................................................22
4.2 INCLUSION CRITERIA ..............................................................................................................................22
4.3 EXCLUSION CRITERIA.............................................................................................................................22
4.4 SUBJECT WITHDRAWALS........................................................................................................................23
5.
CONCOMITANT MEDICATIONS ....................................................................................................23
5.1 PERMITTED CONCOMITANT MEDICATIONS RELATED TO CATHETER MANAGEMENT ...........................23
5.2 NON-PERMITTED CONCOMITANT MEDICATIONS TO CATHETER MANAGEMENT...................................24
6.
TREATMENT(S) ...................................................................................................................................24
6.1 APPEARANCE AND CONTENT..................................................................................................................24
6.2 DOSAGE AND ADMINISTRATION.............................................................................................................25
6.3 PACKAGING.............................................................................................................................................27
6.4 LABELING................................................................................................................................................28
6.5 TREATMENT ALLOCATION......................................................................................................................28
6.6 TREATMENT CODE..................................................................................................................................28
6.7 TREATMENT COMPLIANCE......................................................................................................................29
6.8 DRUG STORAGE ......................................................................................................................................29
6.9 DRUG ACCOUNTABILITY ........................................................................................................................29
6.10 PROVISION OF ADDITIONAL CARE........................................................................................................30
7.
STUDY PLAN ........................................................................................................................................30
7.1 STUDY SCHEDULE...................................................................................................................................31
7.2 INVESTIGATIONS.....................................................................................................................................35
8.
EFFICACY ASSESSMENTS ...............................................................................................................35
8.1 PRIMARY EFFICACY VARIABLE..............................................................................................................35
8.2 SECONDARY EFFICACY VARIABLES .......................................................................................................36
9.
SAFETY VARIABLES..........................................................................................................................36
10.
ADVERSE EVENT REPORTING.......................................................................................................36
10.1 DEFINITIONS..........................................................................................................................................36


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10.2 EXPECTEDNESS .....................................................................................................................................38
10.3 INTENSITY OF ADVERSE EVENT............................................................................................................38
10.4 CAUSALITY ASSESSMENT .....................................................................................................................38
10.5 ACTION TAKEN WITH THE STUDY DRUG DUE TO AN AE.....................................................................39
10.6 OTHER ACTIONS TAKEN.......................................................................................................................39
10.7 OUTCOME..............................................................................................................................................39
10.8 RECORDING ADVERSE EVENTS.............................................................................................................39
10.9 REPORTING SERIOUS ADVERSE EVENTS AND ADVERSE EVENTS OF SPECIAL INTEREST ....................40
10.10 REPORTING SERIOUS ADVERSE EVENTS TO REGULATORY AUTHORITIES/ETHICS COMMITTEES/IRB
......................................................................................................................................................................40
10.11 GENERAL NOTES.................................................................................................................................41
11.
DATA MANAGEMENT .......................................................................................................................42
12.
STATISTICAL METHODS .................................................................................................................43
12.1 SAMPLE SIZE.........................................................................................................................................43
12.2 POPULATIONS FOR ANALYSIS...............................................................................................................46
12.3 STATISTICAL ANALYSIS........................................................................................................................47
13.
ETHICS COMMITTEE/INSTITUTIONAL REVIEW BOARD APPROVAL ..............................50
14.
REGULATORY REQUIREMENTS ...................................................................................................51
15.
INFORMED CONSENT .......................................................................................................................51
16.
SOURCE DOCUMENTS/DATA..........................................................................................................51
16.1 RECORDING OF SOURCE DATA .............................................................................................................51
16.2 DIRECT ACCESS TO SOURCE DOCUMENT/DATA ..................................................................................52
17.
STUDY MONITORING........................................................................................................................52
18.
QUALITY ASSURANCE......................................................................................................................52
18.1 PROTOCOL DEVIATIONS........................................................................................................................52
19.
INSURANCE AND INDEMNITY........................................................................................................53
20.
CONFIDENTIALITY............................................................................................................................53
21.
PREMATURE TERMINATION OF THE STUDY...........................................................................53
22.
CLINICAL STUDY REPORT..............................................................................................................53
23.
RECORD RETENTION........................................................................................................................53
24.
PUBLICATION OF RESULTS............................................................................................................54
25.
REFERENCES.......................................................................................................................................55
APPENDIX 1 - APPROVAL OF THE PROTOCOL BY CLINICAL INVESTIGATOR(S) ..................................58


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1.
INTRODUCTION
1.1  Background Information
Central venous access devices (CVADs) are essential for managing chronic and acute conditions for 
which long-term medication is required, for example, the treatment of pain, cancer, infection, or to 
supply nutrition. In addition to the infusion of therapeutic agents, CVADs also allow for the 
withdrawal of blood and are therefore useful in reducing the need for repeated venipuncture.
Each year, as many as 25% of the approximately 7 million catheters that are placed in patients to 
deliver chemotherapy, nutritional support, antibiotics, or blood products become occluded (Brown, 
2004; Richardson, 2007). Catheter occlusion can result in patient discomfort, deep vein thrombosis, 
hospitalization and possibly the need for invasive procedures, along with increased costs. Moreover, 
occluded catheters can result in delayed or missed therapies and often requires surgical replacement. 
The majority of occlusions are caused by formation of a thrombus within or around the catheter. A 
fibrin sheath is one of the most common causes of thrombotic obstruction. It can occur within 24 
hours after CVAD placement and usually develops within 2 weeks. The fibrin sheath does not usually 
affect catheter function but may cause a partial obstruction by creating a one-way valve over the 
catheter tip. Intraluminal clots account for 5–25% of all catheter occlusions and may cause complete 
catheter obstruction. Catheter-related venous thrombosis refers to a thrombus that develops in 
proximity to a CVAD. A mural thrombus is a blood clot that adheres to the vessel wall and can 
occlude the tip of the catheter but does not completely occlude the vein in which the catheter is 
positioned.
After ruling out mechanical dysfunction and medication or parenteral nutrition-related causes, the 
next step is to exclude thrombotic obstruction. Although diagnostic imaging techniques or linograms 
are available, a common practice is to treat suspected thrombus-related occlusions empirically with 
thrombolytics (fibrinolytics with thrombolytic agents) which allows catheter function to be restored 
efficiently and is a cost-effective alternative to replacing the catheter. In the United States, the current 
standard treatment for CVAD occlusions is intra-catheter instillation of alteplase 2 mg/2 mL (Baskin 
et al, 2009).
Fibrinolytic agents, sometimes referred to as plasminogen activators, are classified into two types: 
fibrin-specific and non–fibrin-specific agents. Fibrin-specific agents, which include alteplase (tPA), 
reteplase (recombinant plasminogen activator [r-PA]), and tenecteplase, produce limited plasminogen 
conversion in the absence of fibrin. Non–fibrin-specific agents (e.g., streptokinase) catalyze systemic 
fibrinolysis (Rivera-Bou, 2017).
Tissue plasminogen activator (tPA) is a naturally occurring fibrinolytic agent found in vascular 
endothelial cells and is involved in the balance between thrombolysis and thrombogenesis. It exhibits 
significant fibrin specificity and affinity. At the site of the thrombus, the binding of t-PA and 
plasminogen to the fibrin surface induces a conformational change that facilitates conversion of 
plasminogen to plasmin and dissolves the clot (Ouriel, 2004). 
Alteplase has a plasma half-life of 4-6 minutes. It is FDA-approved under the brand name 
ACTIVASE® for the treatment of acute ischemic stroke (AIS), acute myocardial infarction (AMI) to 
reduce mortality and incidence of heart failure, and acute massive pulmonary embolism (PE) for lysis. 
At present, alteplase is the only thrombolytic agent approved in the US under the name CATHFLO® 
ACTIVASE® for the management of occluded CVADs (CATHFLO ACTIVASE USPI, 2017).
Reteplase is a recombinant tissue plasminogen activator (rt-PA) currently approved in the US (trade 
name: RETAVASE®), for treatment of acute ST-elevation myocardial infarction (STEMI) to reduce 
the risk of death and heart failure. Reteplase consists of the protease and Kringle 2 domains of human 


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tissue plasminogen activator (tPA), which are associated with fibrin specificity and low fibrin-binding 
affinity, respectively. Reteplase lacks the fibronectin-like finger domain of alteplase that promotes 
high fibrin binding affinity (Van Zonneveld et al, 1986). At high concentrations, reteplase does not 
compete with plasminogen for fibrin-binding sites, allowing plasminogen at the site of the clot to be 
transformed into clot-dissolving plasmin. These characteristics help explain why clots resolve faster 
in patients receiving reteplase than in those receiving alteplase (Rivera-Bou, 2017). Compared to 
alteplase, reteplase has a lower fibrin binding affinity, similar fibrin specificity, and longer half-life 
than alteplase (18 min, almost 4-fold greater than alteplase). These differences in interactions with 
fibrin may facilitate the in vivo clot penetration and faster clot lysis time observed with reteplase but 
it does not necessarily predict reperfusion rates (Fischer and Kohnert, 1997).
This lower fibrin affinity with reteplase has also been linked to a more pronounced attenuation of 
platelet aggregation in the early phase (2 hr) (Moser et al, 1999) and to a markedly greater degree of 
platelet activation in the late phase (24 hr) after thrombolytic therapy compared to alteplase (Gurbel 
et al, 1998).
1.2 Study Rationale 
Chiesi is conducting a clinical development program to support the approval of CUSA-081 (reteplase) 
for the restoration of function to occluded central venous access devices not used in the hemodialysis 
setting. The Investigator’s Brochure (IB) summarizes the data available from the medical literature 
regarding the use of reteplase for the restoration of function to CVADs as well as the known efficacy, 
safety, and immunogenicity results from the clinical program for the currently approved indication 
(STEMI) for reference.
The results of nine studies (7 in adults, 2 in pediatrics) from the medical literature provide supportive 
evidence that reteplase may be a safe and effective treatment option when used to restore functionality 
in occluded CVADs in the hemodialysis and no hemodialysis setting. Based on the data presented, it 
appears that a reteplase dose of 0.4 U, with a concentration of 0.2 U (0.35 mg)/ mL, instilled in a 
volume of ≤ 2 mL to fill the lumen of the CVAD is effective in adults, with a dwell time of 90 minutes 
and an assessment of patency at 30 minute intervals. The dose (0.4 U) of reteplase can be repeated 
one time if catheter function is not restored at 90 minutes after the initial instillation.
Overall success rates in the adult studies ranged from 67% to 100% and longer dwell times were 
generally associated with higher patency rates.
All these studies demonstrated that the safety profile of reteplase is predictable and similar in adult 
patients. The incidence and severity of AEs during these studies varied from none reported to bleeding 
from prior puncture site and minor complications. No instances of allergic reactions were noted. In 
Table 2, main efficacy and safety findings of reteplase in restoring patency of dysfunctional CVADs 
are reported. 


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Table 2: Summary of Available Literature of Reteplase Catheter Studies in Adults 
Study
Catheter Use
Dose per 
Lumen 
(units/mg))
Dwell Time
Efficacy
Safety
Liu et al, 
2004
Fluids/drugs
0.4/0.70
0.4/0.70
0.8/1.40
0.8/1.40
30 min
60 min
90 min
120 min
93/139 (66.9%)
123/139 (88.5%)
126/133 (94.7%)
126/133 (94.7%)
No treatment-
associated AEs
Owens, 
2002
Fluids/drugs and 
Dialysis
0.4/0.70
39.9±34.4 min
81/84 (96%)
No AEs noted
Castner, 
2001
Dialysis
0.4/0.70
20-30 min
171/199 (85.9%)
No AEs were 
noted
Hilleman et 
al, 2003
Dialysis
0.5/0.87
2/3.48
3/5.22
33±10 hrs
32±7 hrs
32±7 hrs
38/45 (84%)
18/20 (90%)
18/20 (90%)
No AEs 
reported
Falk et al, 
2004
Dialysis
0.4-0.8/0.70-
1.40
30-60 min
44/50 (88%)
No AEs 
reported
Hyman et 
al, 2004
Dialysis
0.4-0.8/0.70-
1.40
30-60 min
50/59 (85%)
No bleeding or 
allergic 
reactions noted
Wolf et al, 
2000
Dialysis
5/8.70
60 min
8/8 (100%)
No evidence of 
hemorrhage, 
no blood 
transfusions 
required
1.3 Risk/Benefit Assessment
Two clinical studies evaluating efficacy and safety of reteplase in restoring patency of dysfunctional 
non-hemodialysis CVADs were conducted in a total of 166 subjects reporting high patency success 
rate after a single instillation.
In a Phase 2, open-label, single arm, prospective study conducted at the 
 
 from January 2001 through August 2002 
reteplase was administered to 139 oncology patients (Liu et al, 2004). The primary efficacy 
outcome was the restoration of CVC function after a single administration of reteplase with a 30-
minute dwell time. Catheter function was considered normal if blood could be withdrawn and 
solution could be infused without resistance or discomfort.
The 10 U vial of reteplase was reconstituted with 10 mL of SWFI to a concentration of 1 U/mL. 
The reconstituted reteplase solution was frozen in a 3-mL syringe containing 0.4 U/0.4 mL. After 


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thawing, the reteplase solution was diluted with 0.9% sodium chloride solution without 
bacteriostatic or fungistatic agents to the final concentration of 0.4 U/2 mL.
The intravenous drug therapy nurse instilled the ≤ 2 mL volume of 0.4 U of reteplase in each lumen 
of the catheter for 30 minutes. The reteplase dose was permitted to dwell for an additional 30 
minutes if the catheter remained occluded. This procedure was repeated once if patency was not 
restored after 60 minutes. The success rate after the 30-minute dwell time was 66.9% (93/139 
CVCs; 95% confidence interval [CI]: 0.59-0.74). After an additional 30-minute dwell time, 88.5% 
of the catheters were patent (123/139 CVCs; 95% CI: 0.82-0.93). Of the 16 CVCs that remained 
occluded, 10 were treated with a second dose of up to 0.4 U of reteplase, which was allowed to 
dwell for a total of 60 minutes (120 minutes cumulative time). Success rate after 120 minutes of 
cumulative time was 94.7% (126/133 CVCs; 95% CI: 0.90-0.98).
Both active IMPs, reteplase and alteplase, are tissue plasminogen activators, fibrinolytic agents 
already approved for the treatment of acute myocardial infarction and pulmonary embolism. 
Alteplase is approved for the treatment of occluded CVADs. In this phase III study, alteplase will 
be administered according to the prescribing information instructions. Reteplase will be 
administered intravenously (as per current in label indication) at a dose 1/25th the dose currently 
used for the approved indication (i.e. treatment of acute STEMI). Moreover, study visits, procedures 
and assessments are in accordance with well-established guidelines and clinical practice. Finally, 
both agents have been investigated in humans and have shown good safety and tolerability. In light 
of the above considerations no additional risk for enrolled subjects is thought to be associated to the 
participation into the study.
The study will focus on subjects with occluded CVADs. Previous data showed an optimal efficacy 
and safety profile of reteplase when administered in subjects with dysfunctional CVADs with the 
aim of restoring catheter patency. In vitro and preliminary clinical data, although with some 
limitation, seem to be suggestive of a possible faster onset of action when compared with alteplase, 
current standard of care in the treatment of dysfunctional CVAD for thrombosis.
The efficacy and safety endpoints are standard for evaluation of this drug class in the setting of 
dysfunctional CVADs management and will allow to determine the effect of IMP on clearance of 
CVADs.
Moreover, a futility analysis is planned to assess the conditional power for the test of non-inferiority 
of CUSA-081 compared to alteplase in the rate of treatment success following a single 
administration of study drug with a dwell time up to 90 minutes. The aim of the futility analysis is 
to reduce the exposure to the investigational study drug, CUSA-081, in case the study is unlikely to 
demonstrate non-inferiority compared to alteplase. 
This trial will be conducted in compliance with the Declaration of Helsinki (1964 and amendments) 
current ICH E6 Good Clinical Practices and all other applicable laws and regulations.
2.
STUDY OBJECTIVES
2.1 Primary Objective(s)
To demonstrate the superiority of CUSA-081 compared to placebo in the rate of treatment success 
following a single administration with a dwell time up to 90 minutes. Treatment success is defined as 
restoration of CVAD functionality measured as the ability to withdraw 3 mL of blood and infuse 5 
mL of saline. 


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2.2 Secondary Objective(s)
The secondary objectives of the study are as follows:
1. To demonstrate the non-inferiority of CUSA-081 compared to alteplase in the rate of
treatment success following a single administration with a dwell time up to 90 minutes;
2. To demonstrate the superiority of CUSA-081 compared to placebo in the rate of treatment
success following a single administration with a dwell time up to 60 minutes;
3. To demonstrate the superiority of CUSA-081 compared to placebo in the rate of treatment
success following up to two administrations with a dwell time up to 180 minutes;
4. To demonstrate the superiority of CUSA-081 compared to alteplase in the rate of treatment
success following a single administration with a dwell time up to 90 minutes;
5. To evaluate the safety and tolerability of CUSA-081;
6. To evaluate the rate of recurrent catheter dysfunction defined as first re-occlusion within 30
days following treatment with CUSA-081.
3.
STUDY DESIGN
This is a Phase 3 multinational, multicenter, prospective, randomized, double-blind, parallel-group, 
active and placebo-controlled study examining the effect of instillations of CUSA-081 versus 
placebo or alteplase in subjects with dysfunctional non-hemodialysis CVADs. Restoration of 
CVAD function is defined as follows: the ability to both withdraw at least 3 mL of blood and infuse 
5 mL of saline. 
Since the goal is to acutely identify and treat a dysfunctional catheter, the treatment period consists 
of one visit and may take place on the same day as screening or on the following day. There will be 
a follow-up assessment performed on Day 30 (±2 days) after treatment with study drug. The 
assessment may be conducted by telephone interview. 
Catheters with multiple lumens, ports and peripherally inserted central catheters (PICCs) will be 
allowed. If multiple lumens are dysfunctional, only one lumen will be designated by the 
Investigator for treatment in this study. The lumen chosen will be documented and indicated using a 
study lumen tag to ensure that the same lumen will be used for all study drug instillations and study 
assessments. No other thrombolytics may be used to treat other dysfunctional lumens during the 
treatment period (0 – 180 minutes).
Subjects will be screened according to the Study Inclusion and Exclusion criteria (sections 4.2 and 
4.3) and will be candidates for randomization if all eligibility criteria are met. Subjects will be 
randomized in a 9:1:6 ratio of CUSA-081: placebo: alteplase (
Table 3).
Table 3: Study Treatment Arms
Treatment 
Arm
Syringe 1 
(given at min 0, 
assessed at minutes 30, 60, 90)
Syringe 2 
(given, if needed, at minutes 90, 
assessed at min 120, 150, 180)
A
CUSA-081
CUSA-081
B
placebo
placebo
C
alteplase
alteplase
NOTE: Refer to the Study Treatment Schema in 


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Figure 1 for more detail.
A futility analysis is planned to occur when approximately 416 patients have completed the study 
(see Section 12.3.7 for additional details). The futility analysis will assess the conditional power for 
the test of non-inferiority of CUSA-081 compared to alteplase in the rate of treatment success 
following a single administration of study drug with a dwell time up to 90 minutes.
If the futility analysis finds that it is unlikely to demonstrate non-inferiority of CUSA-081 compared 
to alteplase, then the study will be terminated early.
A separate independent and unblinded team will be established to perform the futility analysis and 
prepare the data for review (“Unblinded Statistical/Programming Team”).
An independent and unblinded DMC will be established to review the data and provide a 
recommendation regarding the continuation of the study. Details of the DMC, including roles, 
responsibilities, communication processes, and frequency of meetings will be provided in a separate 
DMC Charter.
The DMC will provide a recommendation regarding the study continuation as follows:

the study should continue without modification;

the study should be terminated prematurely.
The study team and the other blinded team members will remain blinded throughout the futility 
analysis.
If the study is terminated prematurely, investigators, Competent Authorities and Ethics Committees 
(EC)/ Institutional Review Boards (IRBs) will be informed.
Safety will be monitored throughout the study. The DMC will also review safety data at the same 
time as the futility analysis.
The end of the trial is defined as the last follow-up contact of the last subject to receive study drug in 
the trial.


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Figure 1: Treatment Schema
Study Druga Instillationb1 at 0 min
Assess CVAD function at 30 min
Treatment Success
Dysfunctional
Assess CVAD function at 60 min
Treatment Success
Assess CVAD function at 90 min
Treatment Success
Dysfunctional
Dysfunctional
Study Druga Instillationb2 at 90 min
Assess CVAD function at 120 min
Treatment Success
Dysfunctional
Assess CVAD function at 150 min
Treatment Success
Assess CVAD function at 180 min
Treatment Success
Dysfunctional
Dysfunctional
Exit the 
Treatment 
Algorithim
a 9:1:6 randomization of reteplase:placebo:alteplase
reteplase: 0.70 mg / 2 mL, alteplase: 2 mg / 2 mL, placebo: 2 mL normal saline
b Only one lumen of the catheter is to be used throughout the study for both study drug 
instillation and assessment of CVAD function


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4.
SUBJECT SELECTION CRITERIA
4.1 Subject Recruitment 
A minimum of 841 subjects will be randomized in a 9:1:6 ratio of CUSA-081: placebo: alteplase in 
order to achieve a minimum of 800 subjects completing the study. A screen failure rate of 
approximately 5% is assumed, which implies that approximately 885 subjects should be enrolled in 
order to reach the required number of completed subjects.
4.2 Inclusion Criteria 
Subjects must meet all of the following inclusion criteria to be eligible for enrollment into the study:
1. Inability to have 3mL of blood withdrawn from the selected study catheter;
2. A single or multi-lumen CVAD, implanted ports and peripherally inserted central catheters
(PICCs) in place > 24 hours and documented as previously being patent and functional;
3. Ability to designate one dysfunctional lumen of a multi-lumen catheter to be used
throughout the study for both study drug instillation and assessment of CVAD function;
4. Male and non-pregnant female subjects 18 years or older (see note below);
5. Able to have fluids infused at the volume necessary to instill study drug into the CVAD
(i.e., up to 2 mL);
6. Informed consent form (ICF) signed and dated indicating that the subject has been
informed of and agreed with all pertinent aspects of the study and is willing to comply with
all study requirements and procedures.
NOTE: A urine pregnancy test is required for all females of childbearing potential. Women in natural 
or surgical menopause do not need to be tested for pregnancy. Natural menopause is defined as the 
permanent cessation of menstrual periods, determined retrospectively after a woman has 
experienced 12 consecutive months of lack of menstruation (amenorrhea) without any other obvious 
pathological or physiological cause. Surgical menopause is defined as the removal of both ovaries 
(bilateral oophorectomy) before the natural menopause.  
4.3 Exclusion Criteria
The presence of any of the following will exclude a subject from study enrolment:
1.
CVAD (any type) used for hemodialysis;
2. CVAD known to be dysfunctional for more than 48 hours;
3. Reasonable evidence of mechanical or non-thrombotic occlusion in the selected study
catheter (e.g., catheter malposition or migration, sutures, kinks, or precipitates causing
obstruction), radiographic assessment is not required;
4. Known or suspected catheter related bloodstream infection;
5. Use of any intravenously administered fibrinolytic agent or anticoagulant (For example,
but not limited to, alteplase, tenecteplase, reteplase, urokinase or heparin) within 24 hours
prior to the treatment period (first instillation of study drug). Use of subcutaneous LMWH,
UFH or heparinoids for prophylaxis of thromboembolic events is allowed. Furthermore, the
use of oral anticoagulants is allowed;
6. Known to be at high risk for bleeding events or embolic complications in the opinion of the
Investigator, or has a known condition for which bleeding constitutes a significant hazard
(e.g. recent stroke, recent intracranial or intraspinal surgery or serious head trauma,
intracranial neoplasm, arteriovenous malformation or aneurysm, known bleeding
diathesis);


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7. Uncontrolled hypertension (systolic BP ≥ 160 or diastolic BP ≥ 110 mmHg) at screening;
8. Clinically unstable in the opinion of the site Investigator;
9. Known to be pregnant or breastfeeding at screening;
10. Previously treated in this study (READY 1) or in study READY 2;
11. History of allergic reaction to reteplase, alteplase or vial ingredients (excipients or
diluents);
12. Use of any investigational drug or experimental medical device within 28 days prior to
treatment; non interventional observational studies participation is allowed.
13. Not mentally, socially or otherwise able to complete the trial assessment or not likely to
survive beyond 30 days
4.4 Subject Withdrawals
Subjects may be discontinued from the study for any of the following reasons:

An adverse event occurs that, in the opinion of the investigator, makes it unsafe for the subject
to continue in the study. Should serious bleeding in a critical location (e.g., intracranial,
gastrointestinal, retroperitoneal, pericardial) occur, treatment with study drug should be
stopped and the study drug should be withdrawn from the catheter.

The subject withdraws consent.

The subject is lost to follow-up.

The subject's safety is affected by violation of inclusion or exclusion criteria or use of not-
permitted concomitant medication.

The subject is unwilling or unable to adhere to the study requirements, i.e, non-compliance.

The sponsor or the regulatory authorities or the Ethics Committee(s), for any reason,
terminates the entire study, or terminates the study for this trial site or this particular subject.
When a subject discontinues the study, all efforts will be made to complete and report the observations 
during study participation.
If a subject is withdrawn/drops-out of the study after receiving the study drug, the subject study 
number and corresponding study drug should not be reassigned to another subject.
It is understood by all concerned that an excessive rate of withdrawals can render the study 
uninterpretable; therefore, unnecessary withdrawals of subjects should be avoided.
However, should a subject discontinue the study, all efforts will be made to complete and report the 
observations as thoroughly as possible.
In case of withdrawal, the Investigator must fill in the “Study Termination” page in the CRF, reporting 
the main reason for withdrawal.   
5.
CONCOMITANT MEDICATIONS
5.1 Permitted Concomitant Medications Related to Catheter Management
The use of catheter locking solutions (e.g. normal saline, low dose heparin which requires a normal 
saline flush to clear the heparin from the IV line) is permitted.


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The use of subcutaneous LMWH, UFH or heparinoids for prophylaxis of thromboembolic events is 
allowed. Furthermore, the use of oral anticoagulants is allowed.
5.2 Non-permitted Concomitant Medications to Catheter Management
The use of any intravenously administered fibrinolytic agent or anticoagulant within 24 hours prior 
to the beginning of the treatment period (first instillation of study drug) is not permitted. 
No other fibrinolytic agent may be used to treat other dysfunctional lumens during the treatment 
period (0 – 180 minutes).
6.
TREATMENT(S)
Chiesi will be responsible for supplying Study Drugs to the study sites. The study sites will be 
responsible for the safe storage of all Study Drugs assigned to this study, in a secure place with 
restricted access, and maintained within the appropriate ranges of temperature.  
6.1 Appearance and Content 
6.1.1 CUSA-081
Study drug will be prepared in 2 separate 10 mL syringes with a blinded label by an unblinded 
pharmacist in order to maintain the blind to study personnel for administration to the subject. All 
other study personnel at the study sites will be blinded. CUSA-081 information is described in 
Table 4.
6.1.2 Placebo
The placebo will be identical in appearance to the active drugs in order to keep the treatments 
blinded. The placebo will be prepared in 2 separate 10 mL syringes with a blinded label by an 
unblinded pharmacist in order to maintain the blind to study personnel for administration to the 
subject. The placebo information is described in Table 4.
6.1.3 Alteplase
Alteplase, the active comparator study drug, is being supplied as commercially available CATHFLO 
ACTIVASE. Study drug will be prepared in 2 separate 10 mL syringes with a blinded label by an 
unblinded pharmacist in order to maintain the blind to study personnel for administration to the 
subject.  All other study personnel at the study sites will be blinded.  Alteplase information is 
described in Table 4.


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Table 4: Appearance and Content
Product Name
CUSA-081
Placebo
Alteplase
Active ingredient
Reteplase 10 U / vial
None
Alteplase 2 mg / vial
Excipients
Tranexamic acid, 
Phosphoric acid, 
Polysorbate 80, Sucrose, 
Dipotassium Hydrogen 
Phosphate
Normal Saline
L-arginine,
Polysorbate 80, 
Phosphoric Acid
Presentation
Reconstituted and diluted 
RETAVASE provided in 
2 syringes, each 
containing 0.4 U (0.70 
mg) / 2 mL reteplase 
2 syringes each 
containing 2 mL of 
normal saline
Reconstituted 
CATHFLO 
ACTIVASE provided 
in 2 syringes, each 
containing 2 mg / 
2 mL alteplase
6.2 Dosage and Administration 
6.2.1 Selection of Doses in the Study
The dose of 0.70 mg / 2 mL of reteplase was selected based on previous clinical experience and 
published literature in dysfunctional CVADs. 
Most of the studies in the literature where reteplase was used to restore functionality in CVADs 
tested a dose of up to 0.4 U/2 mL per lumen (equivalent to 0.70 mg / 2 mL), with an additional dose 
after 30 to 60 minutes of dwell time if needed (Castner, 2001; Falk et al, 2004; Freyer et al, 2002; 
Freyer et al, 2003; Hilleman et al, 2003; Hyman et al, 2004; Liu et al, 2004; Owens , 2002; Terrill et 
al, 2003; Wolf et al, 2000). 
This dosing was empirically based on a ratio of 1:5 between reteplase and alteplase dose (20 units : 
100 mg) used for the treatment of AMI in the GUSTO III clinical program (GUSTO III, 1997), as 
well as the doses used in a study comparing the hemodynamic effects of reteplase and alteplase in 
massive pulmonary embolism (PE) (Tebbe et al, 1999). In those trials, little difference was noted 
between the two drugs as far as safety, mortality, stroke, intracranial hemorrhage, or clinical success 
rates. 
Based on the dosing ratio in GUSTO III and the Pulmonary Embolism study, and the clinical 
success seen with 2 mg of alteplase in restoring functionality to CVADs (Haire et al, 1994; Ponec, 
2001; Deitcher et al, 2002; Semba et al, 2002), the reteplase 0.4 units dose was the dose used most 
often in the studies found in the literature where reteplase was used to restore functionality in 
CVADs (Note: 0.4 units of reteplase to 2 mg of alteplase retains the ratio of 1:5). Higher doses were 
also utilized in various studies, but 0.4 units would seem to be a lower yet effective dose in adults. 
A single dose of the reteplase solution is instilled in a volume of ≤ 2 mL to fill the lumen of the 
CVAD and allowed to dwell for a minimum of 30 minutes (repeated once if catheter function is not 
restored) and subsequently removed by aspiration. 
Therefore, in this study, a reteplase dose of 0.4U (0.7 mg) / 2mL will be used. 
If catheter function is not restored at 90 minutes after one dose of reteplase, a second 0.4U/2 mL 
dose may be instilled.


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6.2.2 Dosage
6.2.2.1 Treatment A: CUSA-081
RETAVASE (reteplase) for Injection 10 U / vial, reconstituted in Sterile Water for Injection 
(SWFI) and diluted to 0.4 U (0.70 mg) / 2 mL total dose (CUSA-081). One RETAVASE vial will 
be used to reconstitute up to two doses of CUSA-081.

Subjects will receive the first instillation of study drug (CUSA-081) into the catheter via the
single lumen designated for this study.

If patency is not restored after 90 minutes following the first instillation, a second dose of
study drug (CUSA-081) will be administered.
6.2.2.2 Treatment B: Placebo
Placebo will be 2 mL of Normal Saline (0.9% sodium chloride, USP). Normal saline was chosen 
instead of SWFI due to its isotonic property and to avoid risk of hemolysis with SWFI which may 
risk unblinding the study.

Subjects randomized to Treatment Arm B will receive the first instillation of blinded study
drug (placebo) into the catheter via the single lumen designated for this study.

If patency is not restored after 90 minutes following the first instillation, a second dose of
blinded study drug (placebo) will be administered.
6.2.2.3 Treatment C: Alteplase
Alteplase, 2 mg/ 2 mL single dose vials reconstituted in SWFI according to manufacturer’s label.

Subjects randomized to Treatment Arm C will receive the first instillation of blinded study
drug (alteplase) into the catheter via the single lumen designated for this study.

If patency is not restored after 90 minutes following the first instillation, a second dose of
blinded study drug (alteplase) will be administered.
6.2.2.4 Randomized Treatment Period
To assure blinding of the study staff and subjects, the study pharmacist (unblinded) will prepare two 
10 mL graduated syringes, each labelled appropriately, to be administered per protocol in sequential 
order for each subject after randomization. Preparation of syringes will be performed as follows:
Table 5:
Study Treatment Arms
Treatment Arm
Syringe 1 
(given at min 0)
Syringe 2 
(given if needed, at min 90)
A
CUSA-081
CUSA-081
B
Placebo
Placebo
C
Alteplase
Alteplase


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6.2.3 Preparation of Study Drug for Administration
6.2.3.1 CUSA-081 
Aseptic technique will be used throughout the study drug preparation.  Each RETAVASE vial of 10 
units sterile lyophilized reteplase powder will be reconstituted and diluted with Sterile Water for 
Injection (SWFI) to reach the correct concentration of CUSA-081 for instillation for catheter 
clearance.
The final CUSA-081 concentration in each 10 mL graduated syringe will be 0.2 units/mL. Each 
syringe of CUSA-081 will contain 2 mL or 0.4 units of reteplase.
When reconstituted as directed, the CUSA-081 solution is required to be used (instilled) within 4 
hours when stored at 2° to 30° C (36°F to 86°F).
Detailed instructions for the reconstitution and dilution of RETAVASE resulting in the CUSA-081 
dosing syringes for administration are provided in the study Pharmacy Manual.
6.2.3.2 Placebo
Placebo syringes will be prepared by withdrawing 2 mL of Normal Saline (0.9% sodium chloride, 
USP) into two unused sterile 10 mL graduated syringes. Each syringe should be filled to deliver 2 
mL of Normal Saline. 
Detailed instructions for preparation of the Placebo dosing syringes for administration are included 
in the study Pharmacy Manual.
6.2.3.3 Alteplase
Alteplase, 2 mg/2 mL single dose vials will be reconstituted with SWFI according to the FDA 
approved package insert for CATHFLO ACTIVASE.  Two syringes will be prepared, one for each 
dose.
The alteplase solution may be used for intra-catheter instillation within 4 hours following 
reconstitution when stored at 2-30°C (36-86°F).
Detailed instructions for the preparation of the alteplase dosing syringes for administration are 
provided in the study Pharmacy Manual.
6.3 Packaging
All investigational products will be prepared in accordance with current Good Manufacturing 
Practices (cGMP) as required by the current Good Clinical Practices (cGCP).  Chiesi will supply all 
study drug Sterile Water for Injection (SWFI) and syringes.
6.3.1 Primary Packaging
Study drugs (CUSA-081, placebo and alteplase) will be supplied with the appropriate clinical labels 
in a unique kit for each treatment arm. A corresponding ancillary kit for each treatment arm that 
includes the ancillary supplies required for the reconstitution, dilution, and preparation of the study 
drug will also be provided. The contents of the study drug and ancillary kits are unblinded to the 
pharmacy study personnel. 


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Table 6:
Study Drug Kit Components
Treatment Arm
Study Drug Kit Contents
A
One 10 U vial of reteplase
B
One 10 mL vial of normal saline
C
Two single dose vials of alteplase 
6.3.2 Secondary Packaging
Study drug and ancillary kits will be packaged to be visually identical with identical labelling and 
storage requirements, in order to maintain the blind for any staff that are not unblinded. 
The study drugs and ancillary kits will be packaged in separate boxes. Each kit will have a unique kit 
identification number on the label, traceable to the contents of the kits. Upon randomization of a 
subject in the IRT system, the unblinded pharmacist will receive the study drug and ancillary kit ID 
numbers corresponding to the treatment arm assigned to the subject. 
6.4 Labeling
All the supplies will be labelled according to local law and regulatory requirements and will be 
compliant with Annex 13 to the Volume 4 of the GMP and 21 CRF part 312.6. All labels will be 
available in the local language.
For all applicable labels, the subject identification is expressed by the kit number. This number is 
assigned by the IRT System which allows full traceability of the essential details such as: site 
identification number, investigator’s name, subject number, visit number and, randomization number. 
6.5 Treatment Allocation
A balanced block randomization scheme will be prepared via a computerized system. Once the 
subject meets all inclusion and no exclusion criteria and has provided informed consent, the 
investigational pharmacy or designee at the study site will enroll the subject and request the study 
drug assignment using the Interactive Response Technology (IRT) at the time of randomization. 
Randomization of study drug must only occur on the day of study drug administration.
Assignment to double-blind treatment arms will be determined by the randomization schedule 
programmed into the IRT. Once a subject is randomized, the study site unblinded Pharmacist will 
be provided a study drug kit allocation and the appropriate ancillary kit allocation via the IRT. The 
unblinded Pharmacist will prepare the study drug and affix a blinded label to the syringes for 
dispensation to the blinded site personnel for administration to the subject. 
6.6 Treatment Code
The Sponsor’s clinical team will be blinded during the study as they will not have direct access to the 
randomization list.
In case of emergency, unblinding of the treatment code will be done through IRT. The treatment 
group will be disclosed, and confirmation will follow (by fax and/or notification email). The IRT will 
be designed to send a confirmation (by fax and/or notification email) to the site for every transaction 
performed by the Investigators. 
When possible, the CRO’s Medical Monitor should be consulted prior to unblinding the treatment 
code. The IRT will promptly notify the Sponsor, CRO’s Medical Monitor and the Clinical Site 


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Monitor whenever a treatment code is unblinded; however, the blinded treatment code will remain 
intact in these notifications.
Users from Chiesi Global Pharmacovigilance will have their own credentials to unblind subjects in 
case of SUSARs to be reported to the competent Regulatory Authorities and Ethics Committees/IRB. 
Subjects will be provided a card with the phone numbers of the Investigator or designee to be called 
in case of emergency. 
For interim futility analysis purposes, (see Section 12.3.7 for details), treatment allocation codes will 
be unblinded only for patients included in the analysis, i.e. those who have completed the study or 
those who have prematurely discontinued from the study within the pre-determined cut-off date. 
Unblinded information will be available only to the Unblinded Statistical/Programming Team and to 
the independent DMC members and only following the interim database lock.
6.7 Treatment Compliance
Not applicable
6.8 Drug Storage 
The unblinded pharmacist at the Investigator’s site will be responsible for the safe storage of all 
medications assigned to this study, in a secure place with restricted access limited to those individuals 
authorized to dispense the study drug and maintained within the appropriate ranges of temperature 
and humidity.
All study drug kits will be stored at refrigerated temperatures at 2°C to 8°C (36°F to 46°F). The 
Pharmacy Manual contains additional information for storage and drug accountability. Ancillary kits 
will be stored at controlled room temperature 20°C to 25°C; excursions 15°C to 30°C (59°F to 86°F) 
permitted. The Pharmacy Manual contains additional information for storage and drug accountability. 
For all Investigational Medicinal Product Supplies (IMPS) after reconstitution/preparation, they will 
have to be used (instilled) within 4 hours when stored at 2°C to 30°C (36 °F to 86 °F).
The Investigator or his/her designee is responsible for explaining the correct use of the study drugs 
to the site personnel, verifying that instructions are followed properly, maintaining accurate records 
of study drug dispensing, administration and collection, and returning all unopened and unused study 
drugs to the Sponsor or Sponsor’s designee at the end of the study.
The unblinded pharmacist or unblinded designee must check the Min/Max temperatures once daily 
for adequate storage of refrigerated drug kits and ambient ancillary kits. The Min/Max temperatures 
must be recorded on a dedicated temperature tracking form. The temperature can also be checked/ 
controlled by the constant use of an automated temperature controller system. Any deviation from 
the temperature requirement for storage will be promptly reported and Sponsor shall assess if the 
affected study drugs can be used.
Note: The refrigerated study drug vials must never be warmed by artificial means.
6.9 Drug Accountability
The unblinded pharmacist at the Investigator’s site is responsible for the management of all the study 
medication and ancillary kits to be used in the study. Study medication should be stored in a locked, 
secure storage facility with access limited to those individuals authorized to dispense study 
medication.


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An inventory will be maintained by the unblinded study pharmacist (or designee), to include a signed 
account of all the study medication(s) received, dispensed, administered and returned for each subject 
during the trial. 
At the conclusion or termination of the study, the unblinded study pharmacist (or designee) shall 
conduct and document a final drug supply (used and unused) inventory. An explanation will be given 
for any discrepancies.
Prior sponsor authorization for destruction of unused clinical trial materials by the institution is 
expected. A certificate of destruction is required to be provided to the Sponsor. 
6.10 Provision of Additional Care
At completion of subject’s study participation, it is the Investigator’s responsibility to prescribe 
standard of care for catheter clearance if patency has not been achieved.
Due to the double-blind design of the study, it is not possible for the investigator to ascertain if a 
randomized subject has received reteplase, alteplase, or placebo without breaking the blind.  If 
CATHFLO® ACTIVASE®(alteplase) is prescribed as standard of care following documentation of 
treatment failure at 90 minutes after the second instillation of study drug, or upon Early 
Withdrawal, it is worth noting that according to the FDA-approved product label, studies have not 
been performed with administration of total doses greater than 4 mg (two 2 mg doses).
7.
STUDY PLAN
Due to the short nature of the treatment period and the ability for the follow up visit to be conducted 
via telephone, no missing data or modifications are anticipated due to COVID-19. Additionally, no 
modifications to study procedures or study drug administration are anticipated. In the event that a 
subject withdraws from the study due to COVID-19, this reason will be documented. 


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7.1 Study Schedule
The study schedule (Table 7) summarizes the study assessments by visit and time point.
Table 7: Study Schedule
Screening and/or Treatment Visit1 (Day 1)2
Visit 2 -
Follow-Up 
Assessment3
Syringe 1 First Dose (at 0 min)
Syringe 2 Second Dose (at 90 min, if given)
Assessment or Procedure
Screen
Day 0
Or 
Day 1
0 Min
30 Min
60 Min
90 Min
90 Min
120 Min
150 Min
180 Min
Day 30 (±2 
days) after 
Treatment4
Informed consent
X
Inclusion/exclusion criteria
X
Demographic data
X
Medical history
X
Body weight and height
X
Blood pressure measurement 
and heart rate
X2
Urine pregnancy test5
X
Concomitant 
medications/procedures
X1
X6
CVAD history
X
CVAD function assessment
X1,2
X7, 8
X7, 8
X7, 8
X7, 8
X7, 8
X7, 8
X4,9
Enrollment into IRT
X
Study drug administration 
X8
X8
Adverse events
X1, 10
X10
X10
X10
X10
X10
X10
X10
X10
X10
CVAD = central venous access device; d = day; IRT = interactive response technology, min = minutes.


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1 If at any time after the first instillation of study drug, a subject is withdrawn from the study (due to subject’s wishes or at the Investigator’s discretion), safety monitoring for that 
subject should still occur for up to 30 days (± 2 days) after the last withdrawal of study drug from the catheter (see adverse event section [AE] collection period in Section 10).
2 Subjects will be screened for eligibility and treated over the course of one or two study visits.  Randomization only occurs on day of treatment and cannot occur on screening 
visit if not the same as the treatment day. Therefore, the Screening Visit may occur on Day 0 or Day 1 (for example Monday and Tuesday; not Friday and Monday) and the 
Treatment Visit will occur on Day 1. Screening assessments will occur prior to the first dose of study medication. Blood pressure, heart rate, CVAD assessment and all other 
eligibility for inclusion criteria must be reassessed if screening and treatment are conducted on separate days.
3 Subjects who are unable to be reached for follow-up assessment after discontinuing study drug will be categorized as lost to follow up from the study
4 The Day 30 (± 2 days) follow-up assessment will occur via phone or may occur in person if the subject has a scheduled clinic visit. The follow-up period is calculated from the 
date of the randomization/treatment Day.
5 A urine pregnancy test is required for all females of childbearing potential. Women in natural or surgical post-menopause do not need to be tested for pregnancy.
6 Only concomitant medications and/or procedures ongoing at the time of consent, or stopped within 30 days prior to informed consent, and only new concomitant medications 
and/or procedures related to spontaneously reported SAEs/AESIs will be collected after randomization. 
7 Note that all catheter assessments are based on the dwell time from the time of study drug instillation (i.e., from “0 min”; NOT from the time of the prior assessment). All 
efforts should be made to conduct the assessment at the required time point. The window of ± 5 minutes is also from the time of study drug instillation and not time of the prior 
assessment. Assessments of catheter dysfunction are described in Sections 7.1.1 and 7.2.3. In the event a 2nd dose of study drug is needed, it should be administered within 15 
min of withdrawal of the 1st dose from the CVAD.
8 Once CVAD function has been restored (i.e. the ability to both withdraw at least 3 mL of blood and infuse 5 mL of saline solution), the subject will be deemed a Treatment 
Success and will exit the treatment algorithm (


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Figure 1). Therefore, subsequent dosing and catheter function checks will not be performed during V1. At 180 minutes, or in the case of Early Withdrawal, if treatment success is 
not achieved, any subsequently administered standard of care and related outcome will be documented.
9 Assessment of recurrent catheter dysfunction as described in Section 7.1.2. Information including, but not limited to, the need for other thrombolytic administration and/or 
catheter removal or replacement will be collected beyond study drug administration and up through Day 30 (± 2 days). Subjects will be requested to document the date of re-
occlusion and any intervention or the date the catheter was removed in order to provide the data when contacted by site personnel.
10 Collection of Aes and SAEs will begin from the time of Informed Consent signature. All AEs, TEAEs and SAEs on-going at the time the subject’s study treatment ends should 
be evaluated up to 30 days after last administration of Study Drug.  After this period, all unresolved TEAEs will be reported as “ongoing” in the CRF.  


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7.1.1 Visit 1
A screening visit will be conducted to identify eligible subjects to consent for the study. 
Screening will generally occur on the day of treatment (Day 1) but can also be performed on 
the day prior to treatment (Day 0) if needed for logistical reasons. Day 1 must be the 
following calendar day in this situation (for example Monday and Tuesday; not Friday and 
Monday).
Prior to any study assessments, examinations, or procedures being performed, the subject will 
sign the ICF.
It is imperative to confirm both eligibility and the subject’s ability to undergo study drug 
instillations and assessments potentially lasting 3 to 4 hours. In the occasional circumstance 
where randomization needs to be rescheduled for the following calendar day, eligibility 
should be reconfirmed. The pharmacist or designee will access the IRT to obtain 
randomization allocation.
During the Screening Visit, the following procedures and assessments will occur:

Written informed consent for each subject

Review of the inclusion and exclusion criteria

Collection of demographic data (birth date, sex, race/ethnicity)

Medical history

Weight and height

Blood pressure measurement and heart rate

A urine pregnancy test is requested for all females of childbearing potential. Women
in physiological or surgical post-menopause do not need to be tested for pregnancy.

AEs and concomitant medications/procedures to be assessed and recorded.

CVAD history, including type, indication for insertion, and the number of hours the
catheter has been known to be dysfunctional

Determination of CVAD dysfunction, defined as the inability to withdraw 3 mL of
blood (section 7.2.3), should be performed by a trained health care professional after
the usual assessments, with repositioning and flushes, to confirm that an easily
reversible or mechanical reason is not the suspected reason for poor catheter function.
Note: For multi-lumen catheters, the Investigator or designee will designate one lumen only 
to be used throughout the study for both study drug instillation and assessment of CVAD 
function. The lumen chosen will be documented and indicated using a study lumen tag to 
ensure that the same lumen will be used for all study drug instillations and study assessments.

Subjects who qualify for the study, following completion of all screening assessments,
will be administered study medication. Note that all assessment time points / dwell
times have a window of ± 5 minutes from the time of study drug instillation (0
min).

After the first instillation of study drug, the CVAD will remain undisturbed for
30 minutes, at which time an assessment of function will be performed. Assessment of


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function will be performed as described in section 7.2.3 by a trained (as deemed by the 
participating institution) health care professional. Restoration of CVAD function (i.e., 
treatment success) is defined as follows: the ability to both withdraw at least 3 mL of 
blood and infuse 5 mL of saline solution.

If CVAD function has not been restored at 30 minutes after the first instillation of
study drug, any aspirated or withdrawn study drug will be re-instilled, and the CVAD
will remain undisturbed for an additional 30 minutes and an assessment of function
will again be performed at 60 minutes after this first instillation of study drug.

If CVAD function has not been restored at 60 minutes after the first instillation of
study drug, any aspirated or withdrawn study drug will be re-instilled, and the CVAD
will remain undisturbed for an additional 30 minutes and an assessment of function
will again be performed at 90 minutes after this first instillation of study drug.

If CVAD function has not been restored by 90 minutes after the first instillation of
study drug, the first drug instillation will be withdrawn and discarded, and a second
instillation of study drug will be administered within 15 minutes from the withdrawal
of the 1st dose. Similarly, subsequent CVAD function will be assessed as described in
section 7.2.3 (i.e., after 30 minutes, 60 minutes and then after 90 minutes from
instillation of the 2nd dose, if needed).
Subjects will exit the treatment algorithm when treatment success is achieved at the specified 
time points, or after assessment of CVAD function 90 minutes following the second 
instillation of study drug (time point 180 min), whichever occurs first. Once a subject exits 
the treatment algorithm, assessments of CVAD function are completed for Day 1. In 
unsuccessful cases, further treatments for restoration of catheter functionality may be 
provided per the site’s standard of care for CVAD. At 180 minutes, or in the case of Early 
Withdrawal, if treatment success is not achieved, any subsequently administered standard of 
care and related outcome will be documented.
Subjects will be monitored for AEs (including SAEs and AESIs) during the treatment period 
(Day 1) from the time the subject is administered the first dose of study drug until the 
withdrawal of study drug at V1, and for spontaneously reported SAEs and AESIs afterwards 
up until the Day 30 (±2 days) follow up assessment. 
Concomitant medications and procedures will also be collected as part of the SAE reporting 
requirements.
At the end of Visit 1, subjects will be discharged with instructions on how to record and report 
the status of their CVAD during the 30-day follow-up period, including:

Date of first recurrent catheter re-occlusion (for those with treatment success at V1),

Date and type of any additional treatment they received for catheter dysfunction, and

Date of catheter removal or replacement.
7.1.2 Visit 2 
The Day 30 (±2 days) follow-up assessment will occur by phone or in person if the subject has 
a scheduled clinic visit. Spontaneously reported SAEs, AESIs, and related concomitant 
medications will be recorded and the status of the CVAD function will be determined by asking 
the subject to respond to the questions provided at the V1 discharge.


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7.2 Investigations
7.2.1 Pregnancy Test
A urine pregnancy test is required for all females of childbearing potential at Visit 1. 
Women in natural or surgical post-menopause do not need to be tested for pregnancy at Visit 
1.
7.2.2 Blood Pressure and Heart Rate
Blood pressure and heart rate will be measured at screening in supine position after 5-min rest. 
If blood pressure and heart rate are collected as part of Standard of Care on the same day of 
informed consent signing, then assessment taken prior to consent can be used. Blood pressure 
must be reassessed if screening and treatment are conducted on separate days.
7.2.3 Determination of CVAD Withdrawal and Infusion Functions
Withdrawal function will be determined by the following method:

An empty 10mL graduated syringe will be attached to the catheter, forming an airtight
seal. The syringe plunger will be pulled back to the 5 mL mark to attempt to withdraw
blood. The CVAD is considered to have withdrawal function if at least 3 mL of blood
(blood and any previously infused fluids) fills the syringe.
Infusion function will be determined by the following method:

A 10-mL graduated syringe with 5 mL normal saline solution will be attached to the
catheter to gently infuse the saline solution. The investigative site will be permitted to
use saline from their own supply for the infusion function assessment. The CVAD is
considered to have infusion function if the entire content of the syringe could be infused
without significant resistance.
CVAD dysfunction, assessed for determining patient’s eligibility, is defined as the inability to 
withdraw 3 mL of blood.
Restoration of CVAD function (patency), assessed for determining treatment efficacy, is 
defined by the two following actions:

Ability to have 3 ml of blood (blood and any previously infused fluids) withdrawn
AND 

Ability to gently infuse 5 ml of saline
Therefore, restoration of CVAD function (patency) is assessed by first attempting to withdraw 
3 ml of blood and if successful, then attempting to gently infuse 5 ml of normal saline solution. 
To further clarify, if the 3 ml of blood (blood and any previously infused fluids) cannot be 
withdrawn, the CVAD is considered as dysfunctional, and further assessments are required as 
per protocol section 7.1.1 at the specified time points.
8.
EFFICACY ASSESSMENTS
8.1 Primary Efficacy Variable
The primary efficacy variable is the percentage of subjects who have treatment success 
following a single instillation of study drug (CUSA-081, alteplase, or placebo) with a dwell 


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time up to 90 minutes. Treatment success is defined as the restoration of CVAD functionality 
measured as the ability to withdraw 3 mL of blood and infuse 5 mL of saline.
8.2 Secondary Efficacy Variables
The secondary variables of the study are as follows:
1.
Percentage of subjects who have treatment success following a single instillation of
study drug with a total dwell time up to 30 and 60 minutes;
2.
Percentage of subjects who have treatment success after up to two instillations of
study drug with a total dwell time up to 120, 150 and 180 minutes;
3.
Rate of recurrent catheter dysfunction (defined as first re-occlusion) within 30 days
following treatment with study drug.
9.
SAFETY VARIABLES
The safety variables of the study are as follows:

Treatment Emergent Adverse Events (TEAEs), Adverse Drug Reactions (ADRs),
Serious Treatment Emergent Adverse Events (SAEs), serious related TEAEs, TEAEs
leading to study withdrawal, and TEAEs leading to death

Treatment Emergent Adverse Events of Special Interest (AESI), as defined in Section
10.1
10.
ADVERSE EVENT REPORTING
10.1 Definitions
An Adverse Event is “any untoward medical occurrence in a patient or clinical trial subject 
administered a medicinal product and which does not necessarily have a causal relationship 
with this treatment”.
An adverse event can therefore be any unfavourable and unintended sign (including abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product.
An Adverse Drug Reaction is an “untoward and unintended responses to an investigational 
medicinal product related to any dose administered”.
All adverse events judged by either the reporting Investigator or the Sponsor as having a 
reasonable causal relationship to a medicinal product qualify as adverse reactions. The 
expression “reasonable causal relationship” means to convey in general that there are facts 
(evidence) or arguments meant to suggest a causal relationship.
The definition covers also medication errors and uses outside what is foreseen in the protocol, 
including misuse and abuse of the product.
A. Serious Adverse Event (SAE)/Serious Adverse Drug Reaction is any untoward medical
occurrence or effect that at any dose falls in one or more of the following categories:

Results in death
Death is not an adverse event but an outcome. It is the cause of death that should be regarded 
as the adverse event. The only exception to this rule is “sudden death” where no cause has been 


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established; in this latter instance, “sudden death” should be regarded as the adverse event and 
“fatal” as its reason for being serious.

Is life-threatening
Life-threatening refers to an event in which the subject was at risk of death at the time of the 
event (e.g., aplastic anemia, acute renal failure, and anaphylaxis). The term does not refer to an 
event which hypothetically might have caused death if it were more severe.

Requires hospitalization or prolongation of existing hospitalization
Hospitalization refers to a situation whereby an AE is associated with unplanned formal 
overnight admission into hospital, usually for purpose of investigating and/or treating the AE. 
Hospitalization for the treatment of a medical condition that occurs on an “elective” or 
“scheduled” basis or for a pre-existing condition that did not worsen during the study should 
not necessarily be regarded as an AE. Complications that occur during the hospitalization are 
AEs. If a complication prolongs hospitalization, the event is an SAE. Emergency room visits 
that do not result in a formal admission into hospital should be evaluated for one of the other 
seriousness criteria (e.g., life-threatening; persistent or significant disability or incapacity; 
medically significant).

Results in persistent or significant disability or incapacity.
The term significant disability should be viewed as any situation whereby an AE has a clinically 
important effect on the subject’s physical or psychological well-being to the extent that the 
subject is unable to function normally.

Is a congenital anomaly or birth defect

Is a medically significant adverse event
This criterion allows for any situations in which important adverse events/reactions that are not 
immediately life-threatening or do not result in death or hospitalization may jeopardize the 
subject’s health or may require intervention to prevent one of the above outcomes.
Examples of such events are intensive treatment in an emergency room or at home for allergic 
bronchospasm; blood dyscrasias or convulsions that do not result in hospitalisation; or 
development of drug dependency or drug abuse.
Medical and scientific judgment should be exercised in deciding whether an event is serious 
because medically significant.
Any suspected transmission via a medicinal product of an infectious agent is also considered a 
serious adverse reaction.
B. Non-Serious Adverse Event/Non-Serious Adverse Drug Reaction is an adverse event or
adverse drug reaction that does not meet the criteria listed above for a serious adverse
event/serious adverse drug reaction.’
C. Adverse Events of Special Interest (AESI): Adverse Events of Special Interest (AESI) will
be collected from the time the subject is administered the first dose of study drug on Day 1 until
the withdrawal of study drug (V1), and if spontaneously reported afterwards up until the 30
days ± 2 days follow up assessment
I.
Major bleeding (defined as severe blood loss [>5 mL/kg] or blood loss requiring
transfusion or causing hypotension requiring use of inotropic agents),


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II.
Embolism
III.
Thrombosis
IV.
Catheter related blood stream infections (CRBSI).
10.2 Expectedness
An expected adverse reaction is an adverse reaction, the nature or severity of which is consistent 
with the applicable reference safety information (Investigator’s Brochure for CUSA-081), 
otherwise it is considered unexpected. 
Reports which add significant information on specificity or severity of a known, already 
documented serious adverse drug reaction constitute unexpected events. For example, an event 
more specific or more severe than described in the Investigator’s Brochure would be considered 
as “unexpected”. Examples of such events are: (a) acute renal failure as a labelled ADR with a 
subsequent new report of interstitial nephritis and (b) hepatitis with a first report of fulminant 
hepatitis
In the event an exacerbation is interpreted as due to lack of efficacy, it should not be classified 
as drug related.
10.3 Intensity of Adverse Event
Each Adverse Event must be rated on a 3-point scale of increasing intensity:
Mild: The event causes a minor discomfort or does not interfere with daily activity of
the subject or does not lead to either modification of test treatment dosage or
establishment of a correcting treatment.
Moderate: The event perturbs the usual activity of the subject and is of a sufficient
severity to make the subject uncomfortable. The event leads to a diminution of dosage
of the test treatment, or a temporary interruption of its administration or to the
establishment of a correcting treatment.
Severe: The event prevents any usual routine activity of the subject and causes severe
discomfort. It may be of such severity to cause the definitive interruption of test
treatment.
10.4 Causality Assessment
The following “binary” decision choice will be used by the Investigator to describe the causality 
assessment:
Reasonable possibility of a relatedness
No reasonable possibility of relatedness
The expression “reasonable possibility of relatedness” is meant to convey, in general, that there 
are facts (evidence) or arguments meant to suggest a causal relationship.
The Investigator will be asked to consider the following before reaching a decision on causality 
assessment:
Time relationship between study drug intake and event’s onset;
Dechallenge (did the event abate after stopping drug?);
Rechallenge (did the event reappear after reintroduction?);
Medical history;
Study treatment(s);


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Mechanism of action of the study drug;
Class effects;
Other treatments-concomitant or previous;
Withdrawal of study treatment(s);
Lack of efficacy/worsening of existing condition;
Erroneous treatment with study medication (or concomitant);
Protocol related process.
10.5 Action Taken with the Study Drug Due to an AE
Dose not changed
Drug permanently withdrawn
Not applicable
10.6  Other Actions Taken
Specific therapy/Medication
Concomitant Procedure
Not applicable
10.7 Outcome
Each Adverse Event must be rated by choosing among:
Recovered/resolved
Recovering/resolving
Not recovered/not resolved
Recovered with sequelae/resolved with sequelae
Fatal
Unknown
10.8 Recording Adverse Events
All Adverse Events occurring during the course of the study must be documented in the Adverse 
Event page of the electronic Case Report Form (eCRF). If the Adverse Event is considered 
serious or meets the definition of AESI, the Adverse Event Form in the eCRF must also be 
completed. 
It is responsibility of the Investigator to collect all adverse events (both serious and non-serious) 
derived by spontaneous, unsolicited reports of subjects, by observation and by routine open 
questionings.
The recording period for Adverse Events is the period starting from the Informed Consent 
signature until the subject’s study participation ends.
Clinically significant abnormalities detected at Visit 1 not due to a pre-existing condition or 
clinically significant changes at the following visits in the medical opinion of the investigator 
must be reported as adverse events in the eCRF.
If a clinically significant abnormal laboratory finding or other abnormal assessment meets the 
definition of an AE, then the AE eCRF page must be completed, as appropriate. A diagnosis, if 
known, or clinical signs and symptoms if diagnosis is unknown, rather than the clinically 


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significant abnormal laboratory finding, should be reported on AE eCRF page. If no diagnosis 
is known and clinical signs and symptoms are not present, then the abnormal finding should be 
recorded.
For pharmacovigilance purposes, all SAEs and AESIs should be followed-up in order to 
elucidate as completely and practically as possible their nature and/or causality until resolution 
of all queries, clinical recovery is complete, laboratory results have returned to normal, stable 
condition is reached or until the subject is lost to follow-up. Follow-up may therefore continue 
after the subject has left the study. In this case, the follow-up will continue with no timelines 
for related SAEs and AESIs, while for unrelated SAEs and AESI the type and extent of follow-
up undertaken will be determined for each individual case and will depend upon the nature (e.g. 
events with poor prognosis or which do not resolve), severity and medical significance of the 
event. 
10.9 Reporting Serious Adverse Events and Adverse Events of Special Interest 
The Investigator must report all Serious Adverse Events (SAEs) and all Adverse Events of 
Special Interest (AESIs) to the 
 Safety Contact listed below within 24 hours of 
awareness. The information must be sent by providing the completed SAE/AESI form. The 
 Safety Contact will report all information to Chiesi Global Pharmacovigilance, the 
Clinical Project Manager and the Clinical Research Physician. 
Name and Title
E-mail
 Safety Contact
Chiesi Safety Contact
The reporting period for SAEs from the investigator site is from the time of subject’s
signature of informed consent and until the subject’s study participation ends. After this
date, even if no active monitoring of subjects is required, SAEs occurring to a subject
should be reported if the investigator becomes aware of them.
Reporting of AESI is from the time the subject is administered the first dose of study drug
on Day 1 until the 30 days ± 2 days follow up assessment.
Up to the closure of the site, SAE reports should be reported to the 
 Safety Contact.
New serious adverse events occurring after the site is closed should be reported directly
to the Chiesi Safety Contact.
The SAE and AESI information should also be completed simultaneously in the adverse
event page of the case report form (CRF).
10.10 Reporting Serious Adverse Events to Regulatory Authorities/Ethics 
Committees/IRB
Chiesi or 
 will report serious unexpected adverse reactions to the regulatory authorities 
in compliance with the timelines and standards of reporting according to local regulations as 
well as to the Investigators, ethics committees and Central IRB, if applicable, by 
MedWatch/CIOMS form. The Investigator is responsible for local IRB reporting per Sponsor 


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instruction upon receipt of the SUSAR notification.  Participating Investigators, ECs and IRBs 
will receive a blinded IND Safety Report, unless otherwise specified.  
All SUSARs, which occur with the investigational medicinal products within or outside the 
concerned clinical trial, if required, will be reported in compliance with the timelines and 
standards for reporting SUSARs as set out in FDA 21 CFR part 312.32 and the EU Directive 
2001/20/EC [Directive 2001/20/EC of the European parliament and of the council of 
4/April/2001] and linked guidance [European Commission, Enterprise and Industry Directorate 
General: Detailed guidance on the collection, verification and presentation of adverse 
event/reaction reports arising from clinical trials on medicinal products for human use, latest 
version]. The EMA and the concerned national health authority (if applicable) will be informed 
through Eudravigilance, while the Ethics Committees and the investigators by CIOMS I form 
or by periodic line- listings produced by Chiesi Global Pharmacovigilance. The EMA and the 
concerned national health authority (if applicable) will be informed through Eudravigilance, 
while the Ethics Committees and the investigators by CIOMS I form or by periodic line- listings 
produced by Chiesi Global Pharmacovigilance.
With regard to regulations in force for Pharmacovigilance, the Investigator must fulfill his/her 
obligation according to the law in force in his/her country. 
10.11 General Notes
In case of death, a comprehensive narrative report of the case should be prepared by the
Investigator and sent to the CRO Safety Contact by fax together with the Serious Adverse
Event form, retaining a copy on site.
If an autopsy is performed, copy of autopsy report should be actively sought by the
Investigator and sent to the CRO Safety Contact as soon as available, retaining a copy on
site.
In case of pregnancy, the subject will be followed with due diligence until the outcome
of the pregnancy is known and until the age of one year to detect any congenital anomaly
or birth defect. The pregnancy must be reported by the investigator within 24 hours by
fax/e-mail/via Monitor to the 
 Safety Contact using the paper Pregnancy Report
Form. 
 Safety Contact will inform Chiesi of the pregnancy within one working day
of being notified.
The first two pages of the Pregnancy Report Form should be completed by the investigator with 
all the available information and sent to the CRO Safety Contact. The third page will be 
completed as soon as the investigator has knowledge of the pregnancy outcome, together with 
a follow-up of the first two pages, if necessary (e.g. an update in the medications received 
during pregnancy by the mother). If it meets the criteria for immediate classification of a SAE 
(e.g. spontaneous or therapeutic abortion, stillbirth, neonatal death, congenital anomaly, birth 
defect) the Investigator should follow the procedure for reporting SAEs.
If it is the partner, rather than the subject, who is found to be pregnant, the same procedure
regarding pregnancy reporting is to be followed and the Pregnancy Report Form should
be completed.


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If the pregnancy is discovered before taking any dose either of study drug medication, the
subject will not be eligible for the study and the pregnancy does not need to be reported.
Any ADR occurring with any marketed non-IMP and/or concomitant medication during
the study must be reported by the Investigator to his/her concerned Health Authority
according to the applicable laws. The Investigator is also recommended to report all
ADRs to the relevant Marketing Authorisation Holders of the involved medicinal
products. Additionally, conditions of use outside the marketing authorisation of the
medicinal products (i.e. off label, overdose, misuse, abuse and medication errors) or from
occupational exposure, as well as cases of suspected treatment interaction, pregnancy,
breastfeeding exposure and lack of efficacy should be reported
11.
DATA MANAGEMENT
An electronic CRF (eCRF) will be completed by the Investigator and/or his/her designee for all 
subjects including screen failures. 
Front-end edit checks will run at the time of data collection and back-end edit checks will be 
used by the Data Manager to check for discrepancies and to ensure consistency and 
completeness of the data.
Medical history and Adverse Events will be coded using the MedDRA dictionary; medications 
will be coded using the WHO Drug dictionary and Anatomical Therapeutic Chemical 
classification (ATC).
Access to electronic systems used for data collection will be granted to the study personnel only 
after appropriate training.
After cleaning of the data, review meetings will be held to determine the occurrence of any 
protocol violations and to define the patient populations for the analysis. This process will occur 
for the first set of patients which will be involved in the interim analysis, and then for all 
subsequent patients that will be randomized up to study recruitment closure. Only completed 
and discontinued patients at the time of the cut-off will be included in the interim analysis.
Once all data of patients included in the interim futility analysis have been declared to be 
complete and accurate, the database will be locked (“interim database lock”), the randomization 
codes will be opened (only for the patients involved in the interim analysis) and shared only 
with the Unblinded Statistical/Programming Team and the independent DMC responsible for 
the conduction or interpretation of the interim analysis. Additional details on the interim 
analysis and involved parties are provided in Section 12.3.7.
A final database lock will occur once all data related to the patients enrolled in the study have 
been declared complete and accurate; the randomization list will be shared to the Sponsor and 
the final statistical analysis as planned in the SAP will be performed.
If the database is unlocked after the initial lock, the process must be carefully controlled and 
documented; updates to the study data must be authorized by Chiesi. This applies to the interim 
database lock for patients included in the interim futility analysis as well as for the final database 
lock including all patients.
At the study conclusion, a complete copy of the study data will be created for archival purposes 
at Chiesi. The investigators will receive copies of the patient data for retention at the 
investigational sites.


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12.
STATISTICAL METHODS
12.1 Sample Size
Hypotheses
The primary objective is to demonstrate superiority of CUSA-081 compared to placebo in the 
rate of treatment success (restoration of CVAD function) following a single administration 
with a dwell time up to 90 minutes. The primary hypotheses are as follows:
The null hypothesis H0:
Rc-Rp=0
The alternative hypothesis H1:
Rc-Rp>0
Rc=Rate of success of CUSA-081
Rp=Rate of success of placebo
The first secondary objective is to demonstrate non-inferiority of CUSA-081 vs alteplase in 
the rate of treatment success following a single administration with a dwell time up to 90 
minutes. The hypotheses for the non-inferiority are as follows:
The null hypothesis H0:
Rc-Ra ≤ -10
The alternative hypothesis H1:
Rc-Ra > -10
Rc=Rate of success of CUSA-081
Ra=Rate of success of alteplase
Additional hypotheses are constructed in a similar manner for secondary objectives to 
demonstrate superiority of CUSA-081 vs alteplase following a single administration with a 
dwell time up to 90 minutes, and to demonstrate superiority of CUSA-081 compared with 
placebo in the rate of treatment success at different time points, after one or two 
administrations. The order of the remaining secondary objectives is listed below:
2.
Superiority of CUSA-081 compared to placebo in the rate of treatment success after a
single administration with a dwell time up to 60 minutes;
3.
Superiority of CUSA-081 compared to placebo in the rate of treatment success after up
to two administrations with a dwell time up to 180 minutes;
4.
Superiority of CUSA-081 compared to alteplase in the rate of treatment success after a
single administration with a dwell time up to 90 minutes.
Significance Level
The comparisons of superiority will be performed at the two-sided 5% level of significance. 
The assessment of non-inferiority will be based on the 2.5% level of significance (using one-
sided 97.5% confidence intervals) with a non-inferiority margin of -10%.
Multiplicity Adjustment
Multiplicity adjustment for testing of multiple hypotheses. A closed testing procedure will 
be used to control the overall type I error rate at <5%. The hypothesis testing will start with the 
primary objective, then the secondary efficacy objectives will be tested, in the order listed in 


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the study objectives and shown in the figure below, until a null hypothesis is not rejected (a 
two-sided p-value >0.05 is observed, or lower limit of the two-sided 95% CI ≤ -10% for the 
non-inferiority assessment).
Figure 2: Statistical Testing of Rate of Treatment Success in the READY-1 Trial
Treatment success is defined as restoration of CVAD functionality measured as the ability to 
withdraw 3 mL of blood and infuse 5 mL of saline.
Step 1: Superiority of CUSA-081 vs. placebo; 90 minutes
If significant,
↓
Step 2: Non-inferiority of CUSA-081 vs. Alteplase; 90 minutes
If significant,
↓
Step 3: Superiority of CUSA-081 vs. placebo; 60 minutes;
If significant,
↓
Step 4: Superiority of CUSA-081 vs. placebo; 180 minutes;
If significant,
↓
Step 5: Superiority of CUSA-081 vs. alteplase; 90 minutes
Notes:
1.
All tests are performed at the 2-sided α=0.05 level of significance.
2.
For tests at 60 and 90 minutes, the rate of treatment success is assessed as the percentage
of subjects achieving treatment success following a single instillation of study drug and a
dwell time up to 60 or 90 minutes, respectively.
3.
For the test at 180 minutes, the rate of treatment success is assessed as the percentage of
subjects achieving treatment success following up to two instillations of study drug and a
dwell time up to 180 minutes, respectively.
Multiplicity adjustment not planned for interim analysis futility analysis. No multiplicity 
adjustment will be incorporated for the futility analysis to assess the conditional power for the 
non-inferiority test of CUSA-081 compared to alteplase in the rate of treatment success 
following a single administration of study drug with a dwell time up to 90 minutes (the second 
step in the closed testing procedure). Of note, there will be no stopping of any arm for efficacy 
and no claims of efficacy made on the basis of the futility analysis. The recommendation of a 
threshold to stop the study for futility will be considered as “nonbinding,” meaning the futility 
stopping criteria are guidelines to the DMC that may or may not be followed, depending on 
the totality of the available interim results.


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Required Sample Size
For the primary assessment of the superiority of CUSA-081 vs. placebo, a sample size of 450 
in the active arm (CUSA-081), and 50 in the control arm (placebo), will provide >99% power 
to detect a difference of ≥35% using a two-sided test at significance level of 0.05. The 
expected placebo response rate is approximately 20%.
For the assessment of the non-inferiority of CUSA-081 vs alteplase, a sample size of 450 in 
the active arm (CUSA-081), and 300 in the control arm (alteplase) will provide 87% power at 
the 2.5% significance level (that is by using one-sided 97.5% confidence intervals) with a 
non-inferiority margin of -10%, assuming an equal response rate of 75% in the control arm 
(alteplase), and the active (CUSA-081) arm. A sample size of 450 in the active arm 
(CUSA-081), and 300 in the control arm (alteplase) will also provide 92% power to detect a 
difference of 10% between the success rates using a two-sided test at significance level of 
0.05.
A minimum of 841 subjects will be randomized in order to achieve a minimum of 800 
subjects completing the study.
Statistical Consideration of Non-Inferiority Margin
The selection of the non-inferiority margin was based upon the following reasoning. The 
non-inferiority margin must be less than the smallest treatment difference between standard 
therapy and placebo (M1), and at least part of the treatment effect of the standard therapy 
must be preserved for the test drug (M2).
The NI margin of -10% between reteplase vs. alteplase was selected based on the following 
statistical consideration: 
M1 = the smallest treatment effect of alteplase over placebo
M2 = a fraction 50% of M1 is the most commonly used value
Table 8:
Historical Placebo Response
Study / Time point
Number of 
responders / 
Sample size
% 
Response 
Lower 95% 
CI
Upper 
95% CI
TROPICS 1 
(Gabrail et al, 
2010)
120 minutes
11/47
23%
11%
36%
COOL 1 
(Cathflo PI, 
2017)
120 minutes
12/74
16%
8%
25%
Placebo responses have been reported in hemodialysis setting and in the central venous 
catheter setting. We included the available placebo responses data from 2 studies in the 
central venous access setting (Table 8) with a definition of treatment success similar to the 
one used in this study and with a timepoint assessment of ≥ 90 minutes. Based on the data 
from these two studies, we estimated the placebo response using a weighted analysis based on 


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a random effect model (DerSimonian-Laird). The weighted placebo reponse is 19% with 95% 
CI of 12.9% to 26.8%.
Based on the CATHFLO ACTIVASE product label, the restoration of catheter function was 
assessed at 30 and 120 minutes after the first instillation of alteplase (rtPA). The response at 
90 minutes was estimated based on linear interpolation. Table 9 shows the observed response 
rates at 30 minutes and 120 minutes and the estimated 95% CI at 90 minutes. 
Table 9:
Historical rtPA Response
Time point (COOL 2)
# of responders 
/ Sample size
% 
Response
Lower 
95%CI
Upper 
95%CI
30 minutes
observed
516/995
51.9%
48.8%
55.0%
90 minutes
linear 
interpolation
670/995
67.3%
64.4%
70.3%
120 minutes
observed
747/995
75.1%
72.4%
77.8%
Based on the linear interpolation, the lower bound of the 95% CI of rtPA response at 90 
minutes is 64.4%. The upper bound of the 95% CI for the weighted placebo response is 
estimated to be 26.8%. Therefore, the rtPA margin over placebo is at least 37.6% at 90 
minutes. To preserve 50% of M1 in a NI trial, a NI margin of 18.8% (half of 37.6%) would be 
reasonable.
A more conservative -10% NI margin between reteplase and alteplase will be used for the 90 
minutes endpoint in this trial, which should preserve >70% of M1.
12.2 Populations for Analysis
Full analysis set (FAS): All randomized subjects who receive at least one dose of study
drug, and with at least one available evaluation of efficacy after baseline. Analyses using
the FAS set will be based on the treatment randomized.  The FAS will be the primary
analysis set for all efficacy analyses.
Safety set (SS): All randomized subjects who receive at least one dose of study drug.
Subjects discontinued after dosing will be part of the SS. Analyses based on the SS set
will be based on the actual treatment received.  The SS will be the primary analysis set
for all safety analyses.
ITT analysis set (ITT): All randomized subjects regardless if they received treatment
with study drug.  The ITT will be based on the randomized treatment allocation.  The ITT
will be used for sensitivity analysis for the primary efficacy analyses.
Per-protocol analysis set (PPS): all subjects from the ITT set without any important
protocol deviations. Important protocol deviations are a subset of protocol deviations that
may significantly impact the completeness, accuracy, and/or reliability of key study data
or that may significantly affect a subject's rights, safety, or well-being. Important protocol


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deviations will be assigned in a blinded manner prior to database lock. The PPS will be 
used for sensitivity analysis for the primary efficacy analyses and the non-inferiority 
analyses.
12.3 Statistical Analysis
A detailed statistical analysis plan will be described in a separate document. The plan might be 
reviewed and updated as a result of the blind review of the data and will be finalized before 
breaking the blind. 
12.3.1 Descriptive Statistics 
General descriptive statistics for numeric variables will include the n (number of observed 
values), the mean, the standard deviation, the median, the minimum, and the maximum values.  
For categorical variables, the number and percent of subjects with a specific level of the variable 
will be presented.
12.3.2 Missing Data and Intercurrent Events
By design, all subjects exit the treatment algorithm once patency is achieved and no further 
assessments of catheter clearance are performed. Subjects are considered a treatment success at 
the time point at which patency was achieved and also at all subsequent time points during the 
treatment period.
Otherwise, if missing data is observed for assessment of treatment success and patency was not 
achieved prior to the time point with missing data, then the subject will be considered as a 
failure (worst case scenario) at the time point with missing data. The summary statistics for 
treatment success will display the derived data, including imputation for any missing data 
observed prior to treatment success. This approach will be followed for all the primary and key 
secondary analyses of treatment success. Protocol violations and any other intercurrent events 
will be ignored for analyses performed on the FAS, which target a treatment policy estimand. 
Patients with important protocol deviations will be excluded from analyses based on the PPS.
Further details on dealing with missing data will be described in the SAP.  Other critical missing 
data, if any, will be discussed during the blinded review of the data prior to database lock. 
12.3.3 Subject Demographics and Baseline Characteristics
Demographics and baseline variables will be summarized by treatment arm using descriptive 
statistics for the FAS set.
The following variables will be presented: age, gender, race, height, weight, medical history, 
concomitant diseases, medical history, CVAD history and function, and screening assessments.
12.3.4 Primary Efficacy Variables
The primary efficacy variable is the rate of treatment success following a single administration 
of study drug with a dwell time up to 90 minutes.  The percentage of subjects with treatment 
success and two-sided 95% Wald confidence intervals (CIs) will also be provided by treatment 


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group.  The assessment of superiority vs. placebo will be performed using a 2-sample z test.  A 
two-sided 95% confidence interval on the difference in rates of restoration of CVAD function 
between treatment groups will be constructed. 
Assessment of non-inferiority will be based on the constructed 95% confidence interval for 
the difference in the rate of treatment success between CUSA-081 vs alteplase. The non-
inferiority will be demonstrated (or null hypothesis will be rejected) when the lower limit of 
the 95% confidence interval for the difference in rate of success is greater than -10%. For the 
non-inferiority test, the primary validation of the assay is considered as the demonstration of 
superiority of CUSA-081 versus placebo in the rate of treatment success following a single 
administration of study drug with a dwell time up to 90 minutes, which corresponds to the 
primary objective of the study and the first step in the closed testing procedure (Koch 2004). 
An additional check of assay sensitivity will also be performed as a test of the superiority of 
alteplase versus placebo in the rate of treatment success following a single administration of 
study drug with a dwell time up to 90 minutes. The additional check of assay sensitivity will 
be performed at the same time as the non-inferiority assessment, using the same statistical 
methodology as for the other analyses of superiority, without adjustment for multiplicity.
12.3.5 Secondary Efficacy Variables
The number and percent of subjects with treatment success by treatment and time point will be 
summarized. The rate difference and associated 95% Wald confidence intervals between 
CUSA-081 vs. placebo and alteplase will be estimated. The between treatment comparisons 
will be performed using a 2-sample z test. 
The rate of recurrent catheter dysfunction defined as first re-occlusion within 30 days 
following treatment will be analyzed using the Kaplan-Meier method and summarized with 
median, 25th and 75th percentiles, and 95% confidence intervals. The rate of recurrent catheter 
dysfunction will be estimated. This analysis will be based on all subjects with treatment 
success following up to two administrations of study drug with a total dwell time up to 180 
minutes.
Additional efficacy analyses including demographics and baseline characteristics as 
covariate/subgroup may be explored. 
12.3.6 Safety Variables
Safety analysis will be based on SS set.
Adverse Events
All adverse events starting from the time the subject is administered the first dose of study drug 
on Day 1 until the withdrawal of study drug will be classified as treatment emergent adverse 
event (TEAE).  The number of subjects who experience at least one TEAE, ADR, SAE, serious 
related TEAE, TEAE leading to study withdrawal, TEAE leading to death, and AESI (as 
defined in Section 10.1), will be summarized. Post-treatment adverse events during the 30-day 
follow up period will be summarized.  Summaries will be presented by actual treatment arm 
and by system organ class and preferred term as the number and percentage of subjects having 
at least one event and the total number of events. 


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For AESIs, the percentage of subjects who experienced AESI and associated exact binomial 
95% confidence interval will be provided in the summary. 
All adverse events will be listed.
Vital signs
Vital signs (systolic and diastolic blood pressure, heart rate) will be summarized by means of 
descriptive statistics. Mean, min, max and standard deviation of the observed values will be 
presented, in addition to being listed.
12.3.7 Interim analysis
A single interim analysis for futility will be performed to evaluate if the study should be stopped 
for futility. There will be no stopping rules for efficacy at the time of this interim analysis and 
no efficacy claims on the basis of the interim analysis. Therefore, no statistical adjustment will 
be done for the final analysis. 
The interim futility analysis is planned to be performed when approximately 416 patients have 
completed the study. The timing of the interim futility analysis could be modified based on the 
progress of the recruitment and planning of other studies.
The interim futility analysis will assess the conditional power for the test of non-inferiority of 
CUSA-081 compared to alteplase in the rate of treatment success following a single 
administration of study drug with a dwell time up to 90 minutes.
Number and Timing of Interim Analyses
A single interim futility analysis will be conducted when approximately 416 patients have 
completed the study. 
Statistical Evaluation of Futility
The conditional power for the test of the first secondary objective will be calculated on the basis 
of the observed data at the time of the interim analysis. The study could be terminated early if 
the conditional power at the interim futility analysis is lower than some pre-defined threshold 
(“conditional power threshold”). 
The exact conditional power threshold will be defined in the DMC charter, to be finalized prior 
to the interim database lock and prior to any unblinding for the interim analysis. Of note, the 
futility stopping criteria will be considered as “nonbinding” (i.e., they are guidelines that may 
or may not be followed, depending on the totality of the available interim results). Aside from 
potentially halting the study for futility, there will be no additional changes to the study design 
on the basis of the futility analysis. Specifically, there will be no efficacy stopping rules related 
to the analysis, nor will there be any changes to the planned arms or allocation to the arms, and 
no formal hypothesis testing of the named study objectives. For this reason, the futility analysis 
does not cause inflation of type 1 error and no multiplicity adjustment will be conducted on the 
basis of the futility analysis.
Presentation of Results


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The tables, figures, and listings (TFLs) produced for the interim analysis will include a 
designation as “Interim Analysis” next to the analysis population. 
Roles of Unblinded Teams 
A separate independent and unblinded team (“Unblinded Statistical/Programming Team”) will 
be established to perform the futility analysis and prepare the TFLs. An independent and 
unblinded DMC will be established to review the data and provide a recommendation regarding 
the continuation of the study. The Sponsor’s and CROs’ study teams and the other blinded team 
members will remain blinded. 
The Unblinded Statistical/Programming Team will be responsible for preparing the unblinded 
TFLs to be used for the interim analysis, with further details provided in the SAP. The unblinded 
Statistical/Programming Team may be part of the same CRO who is managing the data 
management and statistical activities for the study. If so, appropriate measures will be put in 
place to ensure that the CRO blinded team and all other members of blinded teams will not have 
access to any unblinded materials or information.
The DMC will be comprised of at minimum 1 statistician and 2 physicians who are experts in 
the field, all completely independent from the day-to-day activities of the study. The DMC will 
review the results of the interim futility analysis and provide a recommendation to Chiesi 
whether the study should continue without modification or whether the study should be 
terminated entirely. The DMC will also review safety data at the same time as the interim futility 
analysis. Review and discussion of unblinded materials will occur in a “closed session” meeting 
including only specifically designated unblinded participants. Aside from the recommendation 
to continue or halt the study, no results of the interim analysis will be shared by the DMC. No 
unblinded materials or information used by the DMC will be shared with blinded study team 
members. A separate DMC charter will be created to describe the specific details of the review 
by the DMC members.
Neither the Unblinded Statistical/Programming Team, nor the DMC will have access to any 
unblinded materials prior to the interim database lock described in Section 11.
13.
ETHICS COMMITTEE/INSTITUTIONAL REVIEW BOARD APPROVAL
The protocol, the written informed consent form and any materials presented to the subject shall 
be submitted for review and approval to the IRB or IEC identified with this responsibility. 
Notification in writing of approval must come from the IRB or IEC chairman or secretary to 
the investigator, either as a letter or as a copy of the appropriate section of the IRB or IEC 
meeting minutes where this protocol and associated informed consent form were discussed, 
before the clinical trial commences at the study site. The study title and number must be clearly 
identified in the IRB approval documentation. If the investigator is an IRB or IEC member, the 
written approval must indicate such non-participation in the voting session.
The investigator will submit status reports to the IRB or IEC annually and as required by the 
governing body. The IRB or IEC must be notified by the investigator in writing of the 
interruption and/or completion of the study; the investigator must promptly report to the IRB 
or IEC all changes in research (protocol amendments) and will not make such changes without 
IRB or IEC approval, except where necessary to eliminate apparent immediate hazards to 


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human subjects. In cases where it is necessary to eliminate immediate hazards to subjects, the 
IRB or IEC must then be notified of the change as per local requirements. 
The investigator is required to maintain an accurate and complete record of all written 
correspondence to and received from the IRB or IEC and must agree to share all such documents 
and reports with the Sponsor.
14.
REGULATORY REQUIREMENTS
The study will be conducted in compliance with the protocol, CRO and/or Chiesi’s standard 
operating procedures and/or guidelines, the United States FDA regulations, the ICH GCP 
guidelines, the Declaration of Helsinki, and other local regulations, as applicable. Selection of 
subjects will not start before the approval of the IRB/IEC.
15.
INFORMED CONSENT
Written informed consent will be obtained from all subjects per IRB/IEC guidelines before any 
study-related procedures (including any pre-treatment procedures) are performed. The 
investigator has both ethical and legal responsibility to ensure that each subject being 
considered for inclusion in this study is given a full explanation of the protocol. This shall be 
documented on a written informed consent form, which shall be approved by the same IRB or 
IEC responsible for approval of this protocol. Each informed consent form shall include the 
elements required by ICH, Part E6, Section 4.8, and any applicable local regulations. The 
investigator agrees to obtain approval from the Sponsor of any written informed consent form 
used in the study, preferably prior to submission to the IRB or IEC.
Once the appropriate essential information has been provided to the subject and fully explained 
by the investigator (or a qualified designee) and it is felt that the subject understands the 
implications of participating, the subject and the investigator (or designee) shall sign and date 
the IRB- or IEC-approved written informed consent form. The subject shall be given a copy of 
the signed informed consent form, and the original shall be filed appropriately, according to the 
institution. A second copy may be filed in the subject's medical record, if allowed by the 
institution.
16.
SOURCE DOCUMENTS/DATA
The Investigators must permit trial-related monitoring, audits, Ethics Committee/Institutional 
Review Board review or regulatory inspection, providing direct access to source 
data/documents.
16.1 Recording of Source Data
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. All source documents should be completed in a 
neat, legible manner to ensure accurate interpretation of data.
Data recorded in the electronic case report form (eCRF) derived from source documents should 
be consistent with the data recorded on the source documents.


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16.2 Direct Access to Source Document/Data
The Investigators or designee (when applicable) must permit trial-related monitoring, audits, 
Ethics Committee/Institutional Review Board review or regulatory inspection, providing direct 
access to source data/documents.
17.
STUDY MONITORING
The Sponsor has ethical, legal and scientific obligations to carefully follow this study in 
accordance with established research principles and applicable regulations. The Investigator, as 
part of his/her responsibilities, is expected to cooperate with the Sponsor in ensuring that the 
study adheres to the protocol and GCP requirements.
As part of a concerted effort to fulfill these obligations, the Sponsor's monitor or designee will 
visit the center(s) during the study in accordance with the Monitoring Plan set forth for this 
trial. The Investigator will permit the Sponsor or designee to monitor the study as frequently as 
is deemed necessary and provide access to medical records/source documents to ensure that 
data are being recorded adequately, that data are verifiable, and that protocol adherence is 
satisfactory.
Monitoring will be performed by the CRO designated by Chiesi.
It is understood that the monitor(s) will contact and visit the Investigator/centre before the 
study, regularly throughout the study and after the study had been completed, and that they will 
be permitted to inspect the various study records: case reports form, Investigator study file and 
source data, provided that subject confidentiality is respected.
The purposes of these visits are:
to assess the progress of the study;
to review the compliance with the study protocol;
to discuss any emergent problem;
to check the CRFs for accuracy and completeness;
to validate the contents of the CRFs against the source documents;
to assess the status of drug storage, dispensing and retrieval.
Prior to each monitoring visit, the Investigator or staff will record all data generated since
the last visit on the case report forms. The Investigator and/or study staff will be expected
to be available for at least a portion of the monitoring visit to answer questions and to
provide any missing information.
18.
QUALITY ASSURANCE
The R&D Quality Assurance Department of Chiesi may perform an audit at any time according 
to the Sponsor’s Standard Operating Procedures, in order to verify whether the study is being 
conducted in agreement with Good Clinical Practices and the protocol.
Regulatory authorities worldwide may also inspect the Investigator/site during or after the 
study. The Investigator should contact the Sponsor immediately if this occurs and must permit 
regulatory authority inspections.
18.1 Protocol Deviations 
This study will be conducted as described in this protocol, except for an emergency situation in 
which the protection, safety, and well-being of the subject requires immediate intervention, 


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based on the judgment of the Investigator (or a responsible, appropriately trained professional 
designated by the investigator). In the event of a significant deviation from the protocol due to 
an emergency, accident, or mistake, the Investigator or designee must contact the CRO at the 
earliest possible time by telephone. This will allow an early joint decision regarding the 
subject’s continuation in the study. The Investigator, Sponsor and the CRO will document this 
decision. The IRB/IEC will be informed of all protocol changes by the Investigator in 
accordance with the IRB/EC established procedure. No deviations from the protocol of any type 
will be made without complying with all the IRB/EC established procedures.  
19.
INSURANCE AND INDEMNITY
Chiesi holds and will maintain an adequate insurance policy covering damages arising out of 
Chiesi’s sponsored clinical research studies.
Chiesi will indemnify the Investigator and hold him/her harmless for claims for damages arising 
out of the investigation, in excess of those covered by his/her own professional liability 
insurance, providing that the drug was administered under his/her or deputy’s supervision and 
in strict accordance with accepted medical practice and with the study protocol.
The Investigator must notify Chiesi immediately upon notice of any claims or lawsuits. 
20.
CONFIDENTIALITY
All study documents are provided by the Sponsor in confidence to the Investigator and his/her 
appointed staff. None of this material may be disclosed to any party not directly involved in the 
study without written permission from Chiesi.
The Investigator must assure the subject’s anonymity will be maintained. The Investigator will 
keep a separate list with at least the initials, the subject’s study numbers, names, and addresses 
and telephone numbers. The Investigator will maintain this for the longest period of time 
allowed by his/her own institution and, in any case, until further communication from Chiesi or 
designee.
21.
PREMATURE TERMINATION OF THE STUDY
Both the Sponsor and the Investigator reserve the right to terminate the study at any time. 
Should this be necessary, the procedures for an early termination or temporary halt will be 
arranged after consultation by all involved parties. The Sponsor should submit a written 
notification to the Regulatory Authority concerned and Ethics Committee/Institutional Review 
Board providing the justification of premature ending or of the temporary halt.
22.
CLINICAL STUDY REPORT
The clinical study report, including the statistical and clinical evaluations, shall be prepared and 
may be sent to the Principal Investigator for agreement and signature.
At the end of the trial a summary of the clinical study report will be provided to all Ethics 
Committees/Institutional Review Boards, to the Competent Authorities and to Investigators.
23.
RECORD RETENTION
After completion of the study, all documents and data relating to the study will be kept in an 
orderly manner by the Investigator in a secure study file.


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Regulations require that essential documents must be retained for at least two years after the 
final marketing approval in an ICH region or until two years have elapsed since the formal 
interruption of the clinical development of the product under study.
It is the responsibility of the Chiesi to inform the Investigator of when these documents can be 
destroyed. The Investigator must contact Chiesi before destroying any trial-related 
documentation. In addition, all subjects’ medical records and other source documentation will 
be kept for the maximum time permitted by the institution. 
24.
PUBLICATION OF RESULTS
Chiesi is entitled to publish and/or present any results of this study at scientific meetings, and 
to submit the clinical trial data to national and international Regulatory Authorities. Chiesi 
furthermore reserves the right to use such data for industrial purposes. Investigators will inform 
Chiesi before using the results of the study for publication or presentation and agree to provide 
the Sponsor with a copy of the proposed presentation. Data from individual study sites must not 
be published separately.
Negative as well as positive results should be published or otherwise made publicly available.


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25.
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Date:  21 FEB 2023
CONFIDENTIAL
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APPENDIX 1 - Approval of the protocol by clinical investigator(s)
Study Title: READY 1: A Phase 3, Randomized, Double-Blind, Active and Placebo-
Controlled Study on the use of CUSA-081 for Dysfunctional Central Venous Access Devices 
(CVADs)
Product: CUSA-081
Approval of Clinical Study Protocol by the Principal Investigator: 
I have carefully read this protocol and I agree that it contains all the necessary information 
required to conduct the study and I agree to conduct it as described.
I understand that this trial will not be initiated without Ethics Committee/Institutional Review 
Board approvals and that the administrative requirements of the governing body of the 
institution will be fully complied with.
Informed written consent will be obtained from all participating subjects and appropriately 
documented, prior to their enrolment in the study. 
The undersigned agrees that the trial will be carried out in conformity with the Declaration of 
Helsinki (as applicable, with attention being drawn to Section concerning freely given consent), 
ICH E6 Good Clinical Practices and with all the other local laws and regulations relevant to the 
use of new and approved therapeutic agents in subjects.
Principal Investigator’s Name:   
Centre No.: 
Signature
Date
Chiesi Farmaceutici S.p.A.
Via Palermo 26/A
43122 Parma - Italy

SAP_001.pdf: 


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Contents 
LIST OF ABBREVIATIONS ................................................................................................. 5 
1 
INTRODUCTION............................................................................................................ 6 
2 
STUDY DESIGN .............................................................................................................. 6 
2.1 
STUDY SCHEDULE ....................................................................................................... 8 
3 
STUDY OBJECTIVES .................................................................................................. 11 
3.1 
PRIMARY OBJECTIVE ................................................................................................ 11 
3.2 
SECONDARY OBJECTIVES .......................................................................................... 11 
4 
STUDY VARIABLES .................................................................................................... 12 
4.1 
EFFICACY VARIABLES............................................................................................... 12 
4.1.1 Primary Efficacy Variable ................................................................................... 12 
4.1.2 Secondary Efficacy Variables .............................................................................. 12 
4.2 
SAFETY VARIABLES .................................................................................................. 12 
5 
SAMPLE SIZE ............................................................................................................... 12 
5.1 
STATISTICAL CONSIDERATION OF NON-INFERIORITY MARGIN ................................. 13 
6 
ANALYSIS SETS........................................................................................................... 14 
6.1 
SAFETY SET (SS) ...................................................................................................... 14 
6.2 
INTENTION-TO-TREAT ANALYSIS SET (ITT) ............................................................. 14 
6.3 
FULL ANALYSIS SET (FAS) ...................................................................................... 14 
6.4 
PER-PROTOCOL ANALYSIS SET (PPS) ....................................................................... 15 
6.5 
OTHER SETS DEFINED FOR TABLES AND LISTINGS.................................................... 15 
7 
GENERAL CONSIDERATIONS FOR STATISTICAL ANALYSIS ...................... 15 
7.1 
STATISTICAL SIGNIFICANCE ...................................................................................... 15 
7.2 
MULTIPLICITY ........................................................................................................... 15 
7.2.1 Multiplicity Adjustment for Testing of Multiple Hypotheses ............................... 15 
7.2.2 Multiplicity Adjustment Not Planned for Interim Analysis Futility Analysis ...... 16 
7.3 
HANDLING OF MISSING DATA ................................................................................... 17 
7.4 
COVARIATES ............................................................................................................. 18 
7.5 
INTERIM ANALYSES .................................................................................................. 18 
7.6 
EXAMINATIONS OF SUBGROUPS ................................................................................ 20 
7.7 
DESCRIPTIVE STATISTICS .......................................................................................... 20 
7.8 
DEFINITIONS ............................................................................................................. 21 
7.8.1 Baseline ................................................................................................................ 21 
7.8.2 Date of First and Last Randomized Study Medication Intake ............................. 21 
7.8.3 Study Day ............................................................................................................. 21 
7.8.4 Visit dates ............................................................................................................. 21 
7.8.5 Duration of Adverse Events or Medications ........................................................ 21 
7.9 
DIARY DATA ............................................................................................................. 22 
7.10 
DATA RE-ALLOCATION ............................................................................................. 22 
7.11 
EXCLUSION OF DATA FROM THE STATISTICAL ANALYSES ........................................ 22 
7.11.1 Exclusion of Data from All Statistical Analyses .................................................. 22 
7.11.2 Exclusion of Data from Per-Protocol Analyses ................................................... 22 
7.12 
LISTINGS ................................................................................................................... 22 


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7.13 
CODING ..................................................................................................................... 22 
8 
STUDY POPULATION ................................................................................................ 23 
8.1 
DISPOSITION OF SUBJECTS AND DISCONTINUATIONS ................................................ 23 
8.1.1 Disposition of Subjects ......................................................................................... 23 
8.1.2 Discontinuation from the Treatment and the Study ............................................. 23 
8.1.3 Protocol Deviations and Analysis Sets ................................................................ 23 
8.2 
DEMOGRAPHIC AND BASELINE CHARACTERISTICS ................................................... 23 
8.2.1 Demographic Characteristics .............................................................................. 23 
8.2.2 CVAD History ...................................................................................................... 24 
8.3 
MEDICAL HISTORY AND CONCOMITANT DISEASES ................................................... 24 
8.4 
MEDICATIONS ........................................................................................................... 24 
8.5 
PROCEDURES ............................................................................................................ 25 
8.6 
COMPLIANCE ............................................................................................................ 25 
9 
EFFICACY ANALYSES .............................................................................................. 25 
9.1 
PRIMARY EFFICACY VARIABLE ................................................................................. 26 
9.2 
SECONDARY EFFICACY VARIABLES .......................................................................... 27 
9.2.1 Percentage of Subjects Who Have Treatment Success Following a Single ......... 27 
Instillation of Study Drug with a Total Dwell Time Up To 30 and 60 Minutes ............... 27 
9.2.2 Percentage of Subjects Who Have Treatment Success After Up to Two 
Instillations of Study Drug with a Total Dwell Time Up To 120, 150 and 180 Minutes . 27 
9.3 
OTHER SECONDARY EFFICACY VARIABLES .............................................................. 27 
9.3.1 Rate of Recurrent Catheter Dysfunction Within 30 Days Following Treatment 
with Study Drug ............................................................................................................... 27 
10 SAFETY ANALYSES ................................................................................................... 28 
10.1 
EXTENT OF EXPOSURE .............................................................................................. 28 
10.2 
ADVERSE EVENTS ..................................................................................................... 28 
10.2.1 Adverse Events of Special Interest (AESI) ........................................................... 30 
10.3 
VITAL SIGNS ............................................................................................................. 30 
11 OTHER ANALYSES ..................................................................................................... 30 
12 CHANGES IN THE PLANNED ANALYSES FROM STUDY PROTOCOL ........ 30 
13 OUTPUT ......................................................................................................................... 31 
13.1 
SOFTWARE ................................................................................................................ 31 
13.2 
REPORTING CONVENTIONS ....................................................................................... 31 
13.2.1 Treatment, Visit and Subgroup Descriptors ........................................................ 31 
13.2.2 Decimal Places .................................................................................................... 32 
13.2.3 Other Reporting Conventions .............................................................................. 34 
13.3 
FORMAT .................................................................................................................... 34 
13.4 
QUALITY CONTROL ................................................................................................... 36 
14 SAS CODE ...................................................................................................................... 37 
14.1 
COMPARISON OF TWO PROPORTIONS USING 2-SAMPLE Z TEST................................ 37 
14.2 
KAPLAN-MEIER ESTIMATES ..................................................................................... 37 
14.3 
COX PROPORTIONAL HAZARDS MODEL .................................................................... 37 
14.4 
LOGISTIC REGRESSION MODEL ................................................................................. 38 
14.5 
CONDITIONAL POWER ............................................................................................... 38 


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15 REFERENCES ............................................................................................................... 40 
16 LIST OF TABLES, LISTINGS AND FIGURES ........................................................ 41 
16.1 
TABLES ..................................................................................................................... 41 
16.2 
LISTINGS ................................................................................................................... 60 
16.3 
FIGURES .................................................................................................................... 67 


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List of Abbreviations 
ADaM 
Analysis Dataset Model 
ADR 
Adverse Drug Reaction 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
BMI 
Body Mass Index 
ATC 
Anatomical Therapeutic Chemical 
CI 
Confidence Interval 
CRBSI 
Catheter Related Bloodstream Infection 
CSR 
Clinical Study Report 
CVAD 
Central Venous Access Device 
DBP 
Diastolic Blood Pressure 
DMC 
Data Monitoring Committee 
eCRF 
Electronic Case Report Form 
FAS 
Full Analysis Set 
HR 
Heart Rate 
ICH 
International Council for Harmonisation 
IRT 
Interactive Response Technology 
ITT 
Intention-to-Treat 
K-M Estimate
Kaplan-Meier Estimate 
PP 
Per-Protocol 
PT 
Preferred Term 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SBP 
Systolic Blood Pressure 
SD 
Standard Deviation 
SDTM 
Study Data Tabulation Model 
SOC 
System Organ Class 
SS 
Safety Set 
TEAE 
Treatment Emergent Adverse Event 
WHO 
World Health Organization 
VERSION HISTORY 
Version 
Date 
Change History 
1.0 
07 Nov 2023 
First Version 


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1 Introduction 
This document presents the Statistical Analysis Plan (SAP) for Chiesi Farmaceutici S.p.A. 
protocol CUSA-081-HEM-01: READY 1: A Phase 3, Randomized, Double-Blind, Active and 
Placebo-Controlled Study on the use of CUSA-081 for Dysfunctional Central Venous Access 
Devices (CVADs). 
This analysis plan is based on the final protocol (version 8.0), dated 21Feb2023 and the final 
electronic case report form (eCRF) dated 05Apr2022.  
The SAP provides the description of the final analyses. In case of deviations from the SAP, 
explanations will be provided in the Clinical Study Report (CSR). 
 is contracted to support statistical analyses and is responsible for the production and 
quality control of all outputs described in this document. 
2 Study Design 
This is a phase 3 multinational, multicenter, prospective, randomized, double-blind, parallel-
group, active and placebo-controlled study examining the effect of instillations of CUSA-081 
versus placebo or alteplase in subjects with dysfunctional non-hemodialysis CVADs.  
Restoration of CVAD function is defined as follows: the ability to both withdraw at least 3 mL 
of blood and infuse 5 mL of saline.  
Subjects will be screened according to the Study Inclusion and Exclusion criteria and will be 
candidates for randomization if all eligibility criteria are met.   
A minimum of 841 subjects, within approximately 100 sites, will be randomized, in a 9:1:6 
ratio of CUSA-081:Placebo:alteplase in order to achieve a minimum of 800 subjects 
completing the study: 
Treatment Arm A (CUSA-081 [reteplase]) 
473 subjects 
Treatment Arm B (Placebo) 
53 subjects 
Treatment Arm C (alteplase) 
315 subjects 
A total of 2-3 visits will be performed during the study, as follows (refer to the Study Treatment 
Schema in Figure 1 for more details): 
•
Screening (Baseline Visit) (Day 0 or Day 1).
•
Treatment Visit 1 (V1) (Day 1):
o
Subjects will receive the first instillation of study drug (CUSA-081, placebo or
alteplase) into the catheter via the single lumen designated for this study.
o
If patency is not restored after 90 minutes following the first instillation, a
second dose of study drug (same drug as at first instillation) will be
administered.
•
Follow-up Visit 2 (V2) (Day 30 ± 2 days) (can be performed by telephone).




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A futility analysis is planned to occur when approximately 416 subjects have completed the 
study (see Section 7.5 for additional details). 
The futility analysis will assess the conditional power for the test of non-inferiority of 
CUSA-081 compared to alteplase in the rate of treatment success following a single 
administration of study drug with a dwell time up to 90 minutes. 
If the futility analysis finds that it is unlikely to demonstrate non-inferiority of CUSA-081 
compared to alteplase, then the study will be terminated early. 
A separate independent and unblinded team will be established to perform the futility analysis 
and prepare the data for review (“Unblinded Statistical/Programming Team”). 
An independent and unblinded Data Monitoring Committee (DMC) will be established to 
review the data and provide a recommendation regarding the continuation of the study. Details 
of the DMC, including roles, responsibilities, communication processes, and frequency of 
meetings will be provided in a separate DMC Charter. 
The DMC will provide a recommendation regarding the continuation as follows: 
•
the study should continue without modification;
•
the study should be terminated prematurely.
The study team and the other blinded team members will remain blinded throughout the futility 
analysis. 
Treatment Allocation 
A balanced block randomization scheme will be prepared via a computerized system. Once the 
subject meets all inclusion and no exclusion criteria and has provided informed consent, the 
investigational pharmacist or designee at the study site will enroll the subject and request the 
study drug assignment using the Interactive Response Technology (IRT) at the time of 
randomization. Randomization of study drug must only occur on the day of study drug 
administration.   
Assignment to double-blind treatment arms will be determined by the randomization schedule 
programmed into the IRT. Once a subject is randomized, the study site unblinded Pharmacist 
will be provided a study drug kit allocation and the appropriate ancillary kit allocation via the 
IRT. The unblinded Pharmacist will prepare the study drug and affix a blinded label to the 
syringes for dispensation to the blinded site personnel for administration to the subject.  
2.1 Study Schedule 
The study schedule (Table 1) summarizes the study assessments by visit and time point. 
Due to the short nature of the treatment period and the ability for the follow up visit to be 
conducted via telephone, no missing data or modifications are anticipated due to COVID-19. 
Additionally, no modifications to study procedures or study drug administration are 
anticipated. In the event that a subject withdraws from the study due to COVID-19, this reason 
will be documented.


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Table 1: Study Schedule 
Assessment or Procedure 
Screening and/or Treatment Visit 1 
(Day 1) 2 
Visit 2 –  
Follow-Up 
Assessment 3 
Screen 
Day 0 
Or 
Day 1 
Syringe 1 First Dose 
(at 0 min) 
Syringe 2 Second Dose 
(at 90 min, if given) 
Day 30      
(±2 days)    
after 
Treatment 4 
0 Min 30 Min 60 Min 90 Min 90 Min 120 Min 150 Min 180 Min 
Informed consent 
X 
Inclusion/exclusion criteria 
X 
Demographic data 
X 
Medical history 
X 
Body weight and height 
X 
Blood pressure measurement and 
heart rate (HR) 
X2 
Urine pregnancy test 5 
X 
Concomitant medications/procedures 
X1 
X6 
CVAD history 
X 
CVAD function assessment 
X1 2  
X7 8 
X7  8 
X7  8 
X7  8 
X7  8 
X7  8 
X 4 9
Enrollment into IRT 
X 
Study drug administration 
X 8 
X 8 
Adverse events 
X1 10 
X10 
X10 
X10 
X10 
X10 
X10 
X10 
X10 
X10 
CVAD = central venous access device; d = day; IRT = interactive response technology, min = minutes. 


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1 If at any time after the first instillation of study drug, a subject is withdrawn from the study (due to subject’s wishes or at the Investigator’s discretion), safety monitoring 
for that subject should still occur for up to 30 days (± 2 days) after the last withdrawal of study drug from the catheter (see adverse event section [AE] collection period in 
protocol Section 10). 
2 Subjects will be screened for eligibility and treated over the course of one or two study visits.  Randomization only occurs on day of treatment and cannot occur on 
screening visit if not the same as the treatment day. Therefore, the Screening Visit may occur on Day 0 or Day 1 (for example Monday and Tuesday; not Friday and 
Monday) and the Treatment Visit will occur on Day 1. Screening assessments will occur prior to the first dose of study medication. Blood pressure, heart rate, CVAD 
assessment and all other eligibility for inclusion criteria must be reassessed if screening and treatment are conducted on separate days.
3 Subjects who are unable to be reached for follow-up assessment after discontinuing study drug will be categorized as lost to follow up from the study. 
4 The Day 30 (± 2 days) follow-up assessment will occur via phone or may occur in person if the subject has a scheduled clinic visit. The follow-up period is calculated from 
the date of the randomization/treatment day. 
5 A urine pregnancy test is required for all females of childbearing potential. Women in natural or surgical post-menopause do not need to be tested for pregnancy. 
6 Only concomitant medications and/or procedures ongoing at the time of consent, or stopped within 30 days prior to informed consent, and only new concomitant 
medications and/or procedures related to spontaneously reported SAEs/AESIs will be collected after randomization. 
7 Note that all catheter assessments are based on the dwell time from the time of study drug instillation (i.e., from “0 min”; NOT from the time of the prior assessment). All 
efforts should be made to conduct the assessment at the required time point. The window of ± 5 minutes is also from the time of study drug instillation and not time of the 
prior assessment. Assessments of catheter dysfunction are described in Sections 7.1.1 and 7.2.3 of the protocol. In the event a 2nd dose of study drug is needed, it should be 
administered within 15 min of withdrawal of the 1st dose from the CVAD.
8 Once CVAD function has been restored (i.e. the ability to both withdraw at least 3 mL of blood and infuse 5 mL of saline solution), the subject will be deemed a Treatment 
Success and will exit the treatment algorithm (Figure 1). Therefore, subsequent dosing and catheter function checks will not be performed during V1. At 180 minutes, or in 
the case of Early Withdrawal, if treatment success is not achieved, any subsequently administered standard of care and related outcome will be documented. 
9 Assessment of recurrent catheter dysfunction as described in Section 7.1.2 of the protocol. Information including, but not limited to, the need for other thrombolytic 
administration and/or catheter removal or replacement will be collected beyond study drug administration and up through Day 30 (± 2 days). Subjects will be requested to 
document the date of re-occlusion and any intervention or the date the catheter was removed in order to provide the data when contacted by site personnel. 
10 Collection of AEs and SAEs will begin from the time of Informed Consent signature. All AEs, TEAEs and SAEs on-going at the time the subject’s study treatment ends 
should be evaluated up to 30 days after last administration of Study Drug. After this period, all unresolved TEAEs will be reported as “ongoing” in the CRF.  


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3 Study Objectives 
3.1 Primary Objective 
•
To demonstrate the superiority of CUSA-081 compared to placebo in the rate of
treatment success following a single administration with a dwell time up to 90 minutes.
Treatment success is defined as restoration of CVAD functionality measured as the
ability to withdraw 3 mL of blood and infuse 5 mL of saline.
3.2 Secondary Objectives 
1. To demonstrate the non-inferiority of CUSA-081 compared to alteplase in the rate of
treatment success following a single administration with a dwell time up to 90 minutes;
2. To demonstrate the superiority of CUSA-081 compared to placebo in the rate of
treatment success following a single administration with a dwell time up to 60 minutes;
3. To demonstrate the superiority of CUSA-081 compared to placebo in the rate of
treatment success following up to two administrations with a dwell time up to 180
minutes;
4. To demonstrate the superiority of CUSA-081 compared to alteplase in the rate of
treatment success following a single administration with a dwell time up to 90 minutes;
5. To evaluate the safety and tolerability of CUSA-081;
6. To evaluate the rate of recurrent catheter dysfunction defined as first re-occlusion
within 30 days following treatment with CUSA-081.


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4 Study Variables 
4.1 Efficacy Variables 
4.1.1 Primary Efficacy Variable 
•
The primary efficacy variable is the percentage of subjects who have treatment success
following a single instillation of study drug (CUSA-081, alteplase, or placebo) with a
dwell time up to 90 minutes. Treatment success is defined as the restoration of CVAD
functionality measured as the ability to withdraw 3 mL of blood and infuse 5 mL of
saline.
4.1.2 Secondary Efficacy Variables 
1. Percentage of subjects who have treatment success following a single instillation of
study drug with a total dwell time up to 30 and 60 minutes;
2. Percentage of subjects who have treatment success after up to two instillations of study
drug with a total dwell time up to 120, 150 and 180 minutes;
3. Rate of recurrent catheter dysfunction (defined as first re-occlusion) within 30 days
following treatment with study drug.
4.2 Safety Variables 
•
Treatment Emergent Adverse Events (TEAE), Adverse Drug Reactions (ADRs),
Serious Treatment Emergent Adverse Events (SAE), serious related TEAEs, TEAEs
leading to study withdrawal, and TEAEs leading to death;
•
Treatment Emergent Adverse Events of Special Interest (AESI) as defined in
Section 10.1 of the protocol.
5 Sample Size 
For the primary assessment of the superiority of CUSA-081 vs. placebo, a sample size of 450 
in the active arm (CUSA-081), and 50 in the control arm (placebo), will provide >99% power 
to detect a difference of ≥35% using a two-sided test at significance level of 0.05. The expected 
placebo response rate is approximately 20%. 
For the assessment of the non-inferiority of CUSA-081 vs alteplase, a sample size of 450 in 
the active arm (CUSA-081), and 300 in the control arm (alteplase) will provide 87% power at 
the 2.5% significance level (that is by using one-sided 97.5% confidence intervals (CI)) with a 
non-inferiority margin of -10%, assuming an equal response rate of 75% in the control arm 
(alteplase), and the active (CUSA-081) arm. A sample size of 450 in the active arm 
(CUSA-081), and 300 in the control arm (alteplase) will also provide 92% power to detect a 
difference of 10% between the success rates using a two-sided test at significance level of 0.05. 
A minimum of 841 subjects will be randomized in order to achieve a minimum of 800 subjects 
completing the study. 


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5.1 Statistical Consideration of Non-Inferiority Margin 
The selection of the non-inferiority margin was based upon the following reasoning. The 
non-inferiority margin must be less than the smallest treatment difference between standard 
therapy and placebo (M1), and at least part of the treatment effect of the standard therapy must 
be preserved for the test drug (M2). 
The NI margin of -10% between reteplase vs. alteplase was selected based on the following 
statistical consideration:  
M1 = the smallest treatment effect of alteplase over placebo   
M2 = a fraction 50% of M1 is the most commonly used value 
Table 2: Historical Placebo Response 
Study / Time point 
Number of 
responders / 
Sample size 
% 
Response 
Lower  
95% CI 
Upper 
95% CI 
TROPICS 1 
(Gabrail et al, 
2010) 
120 minutes 
11/47 
23% 
11% 
36% 
COOL 1 
(Cathflo PI, 
2017) 
120 minutes 
12/74 
16% 
8% 
25% 
Placebo responses in the treatment of thrombotically occluded CVADs have been reported in 
hemodialysis setting and in non-hemodialysis setting. We included the available placebo 
responses data from 2 studies in the non-hemodialysis setting (Table 2) with a definition of 
treatment success similar to the one used in this study and with a timepoint assessment of ≥ 90 
minutes. Based on the data from these two studies, we estimated the placebo response using a 
weighted analysis based on a random effect model (DerSimonian-Laird). The weighted placebo 
response is 19% with 95% CI of 12.9% to 26.8%.  


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Based on the CATHFLO ACTIVASE product label, the restoration of catheter function was 
assessed at 30 and 120 minutes after the first instillation of alteplase. The response at 90 
minutes was estimated based on linear interpolation.  
Table 3 shows the observed response rates at 30 minutes and 120 minutes and the estimated 
95% CI at 90 minutes. 
Table 3: Historical Alteplase Response 
Time point (COOL 2) 
# of responders 
/ Sample size 
% 
Response 
Lower 
95% CI 
Upper 
95% CI 
30 minutes 
observed 
516/995 
51.9% 
48.8% 
55.0% 
90 minutes 
linear interpolation 
670/995 
67.3% 
64.4% 
70.3% 
120 minutes 
observed 
747/995 
75.1% 
72.4% 
77.8% 
Based on the linear interpolation, the lower bound of the 95% CI of Alteplase response at 
90 minutes is 64.4%. The upper bound of the 95% CI for the weighted placebo response is 
estimated to be 26.8%. Therefore, the alteplase margin over placebo is at least 37.6% at 
90 minutes. To preserve 50% of M1 in a NI trial, a NI margin of 18.8% (half of 37.6%) would 
be reasonable.   
A more conservative -10% NI margin between reteplase and alteplase will be used for the 
90 minutes endpoint in this trial, which should preserve >70% of M1.  
6 Analysis Sets 
The definitions of the analysis sets are summarized below. A final agreement on the subjects 
to be included in or excluded from each analysis set will be reached during the blind review of 
the data before breaking the blind. Inclusions and exclusions from analysis sets will be fully 
documented in the Data Review Report.  
6.1 Safety Set (SS) 
All randomized subjects who receive at least one dose of study drug. Subjects discontinued 
after dosing will be part of the SS. Analyses based on the SS will be based on the actual 
treatment received. The SS will be the primary analysis set for all safety analyses. 
6.2 Intention-to-Treat Analysis Set (ITT) 
All randomized subjects regardless if they received treatment with study drug. The ITT Set will 
be based on the randomized treatment allocation. The ITT Set will be used for sensitivity 
analysis for the primary efficacy analyses.  
6.3 Full Analysis Set (FAS) 
All randomized subjects who receive at least one dose of study drug, and with at least one 
available evaluation of efficacy after baseline (i.e. at least one CVAD assessment after study 


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drug administration). Analyses using the FAS will be based on the treatment randomized. The 
FAS will be the primary analysis set for all efficacy analyses involving assessment of 
superiority. 
6.4 Per-Protocol Analysis Set (PPS) 
All subjects from Safety Set without any important protocol deviations. Important protocol 
deviations are a subset of protocol deviations that may significantly impact the completeness, 
accuracy, and/or reliability of key study data or that may significantly affect a subject's rights, 
safety, or well-being. Important protocol deviations will be assigned in a blinded manner prior 
to database lock. The PPS will be used for sensitivity analysis for the primary efficacy analyses 
and the non-inferiority analyses. 
6.5 Other Sets Defined for Tables and Listings 
For the purposes of tables and listings the following set is defined: 
•
Enrolled Set: all subjects who provided informed consent for the study.
7 General Considerations for Statistical Analysis 
7.1 Statistical Significance 
All tests of superiority hypotheses will be two-sided and conducted at the 0.05 significance 
level, and all confidence intervals will be two-sided at the 95% confidence level.  
The assessment of non-inferiority will be based on the 2.5% level of significance (using one-
sided 97.5% confidence intervals) with a non-inferiority margin of -10%. 
7.2 Multiplicity 
7.2.1 Multiplicity Adjustment for Testing of Multiple Hypotheses 
A closed testing procedure will be used to control the overall type I error rate at less than 5% 
across the primary and key secondary variables.  
The hypothesis testing will start with the primary efficacy objective, then the secondary 
efficacy objectives (objectives 1 to 4) will be tested, in the order listed in the study objectives 
(refer to Section 3.2) and shown in the figure below, until a null hypothesis is not rejected (a 
p-value >0.05 is observed for superiority assessment, or lower limit of the two-sided 95% CI
≤-10% for the non-inferiority assessment).
At each step of the procedure, no confirmatory claims will be made unless the objectives are 
met in all the preceding steps. If the two-sided p-values is larger than 0.05 for any of the 
superiority tests or the lower limit of the two-sided 95% CI is less than or equal to -10% for 
the non-inferiority test, then the p-values corresponding to the subsequent tests will be 
considered as exploratory only. 
For all other efficacy and safety analyses, no multiplicity adjustment will be made. 


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Figure: Statistical Testing of Rate of Treatment Success in the READY-1 Trial 
Treatment success is defined as restoration of CVAD functionality measured as the ability to 
withdraw 3 mL of blood and infuse 5 mL of saline. 
Step 1: Superiority of CUSA-081 vs. Placebo; 90 minutes 
If significant, 
↓
Step 2: Non-inferiority of CUSA-081 vs. Alteplase; 90 
minutes 
If significant, 
↓
Step 3: Superiority of CUSA-081 vs. placebo; 60 minutes; 
If significant, 
↓
Step 4: Superiority of CUSA-081 vs. placebo; 180 minutes; 
If significant, 
↓
Step 5: Superiority of CUSA-081 vs. alteplase; 90 minutes 
Notes: 
1.
All tests are performed at the 2-sided α=0.05 level of significance.
2.
For tests at 60 and 90 minutes, the rate of treatment success is assessed as the percentage
of subjects achieving treatment success following a single instillation of study drug and a
dwell time up to 60 or 90 minutes, respectively.
3.
For the test at 180 minutes, the rate of treatment success is assessed as the percentage of
subjects achieving treatment success following up to two instillations of study drug and a
dwell time up to 180 minutes, respectively.
7.2.2 Multiplicity Adjustment Not Planned for Interim Analysis Futility Analysis 
Of note, the interim analysis will not be conducted as a result of the early stopping of the study. 
The below text in this section is maintained for consistency with the protocol and to maintain 
a record of the original plan. See Section 12 for additional details. 
No multiplicity adjustment will be incorporated for the separate interim futility analysis to 
assess the conditional power for the non-inferiority test of CUSA-081 compared to alteplase in 
the rate of treatment success following a single administration of study drug with a dwell time 
up to 90 minutes (the second step in the closed testing procedure). Of note, there will be no 
stopping of any arm for efficacy and no claims of efficacy made on the basis of the futility 
analysis. The recommendation of a threshold to stop the study for futility will be considered as 
“nonbinding,” meaning the futility stopping criteria are guidelines to the DMC that may or may 
not be followed, depending on the totality of the available interim results. 






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𝑛𝑛𝐼𝐼1 = number of subjects in CUSA-081 group at interim 
𝑛𝑛𝐼𝐼2 = number of subjects in alteplase group at interim 
𝑁𝑁1 = number of subjects in CUSA-081 group planned at the end of the trial (i.e. 473 subjects) 
𝑁𝑁2 = number of subjects in alteplase group planned at the end of the trial (i.e. 315 subjects) 
𝑧𝑧α = the standard normal critical value for a test with a type I error rate of α (α = one-sided 
0.025)  
𝑧𝑧𝐼𝐼= 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝐼𝐼−∆
𝑆𝑆𝑆𝑆𝐼𝐼
𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝐼𝐼= 𝑝𝑝𝐼𝐼1 −𝑝𝑝𝐼𝐼2 
𝑝𝑝𝐼𝐼1 = proportion of successes in CUSA-081 group at interim 
𝑝𝑝𝐼𝐼2 = proportion of successes in alteplase group at interim 
∆ = non-inferiority margin = -0.1 (based on the non-inferiority hypotheses presented in 
section 9.1) 
𝑆𝑆𝑆𝑆𝐼𝐼= ඨ𝑝𝑝𝐼𝐼1(1 −𝑝𝑝𝐼𝐼1)
𝑛𝑛𝐼𝐼1
+ 𝑝𝑝𝐼𝐼2(1 −𝑝𝑝𝐼𝐼2)
𝑛𝑛𝐼𝐼2
Number and Timing of Interim Analyses 
A single interim analysis will be conducted when approximately 416 subjects have completed 
the study.  
Statistical Evaluation of Futility 
The conditional power for the test of the first secondary objective will be calculated on the 
basis of the observed data at the time of the interim analysis. The study could be terminated 
early in case the conditional power at the interim analysis is lower than some pre-defined 
threshold (“conditional power threshold”).  
The exact conditional power threshold will be defined in the DMC charter, to be finalized prior 
to the interim database lock and prior to any unblinding for the interim analysis. Of note, the 
futility stopping criteria will be considered as “nonbinding” (i.e., they are guidelines that may 
or may not be followed, depending on the totality of the available interim results). Aside from 
potentially halting the study for futility, there will be no additional changes to the study design 
on the basis of the futility analysis. Specifically, there will be no efficacy stopping rules related 
to the analysis, nor will there be any changes to the planned arms or allocation to the arms, and 
no formal hypothesis testing of the named study objectives. For this reason, the futility analysis 
does not cause inflation of type 1 error and no multiplicity adjustment will be conducted on the 
basis of the futility analysis. 
Interim Analysis Results 
A separate independent and unblinded team (“Unblinded Statistical/Programming Team”) will 
be established to perform the analysis and prepare the TFLs. An independent and unblinded 


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DMC will be established to review the data and provide a recommendation regarding the 
continuation of the study. The Sponsor’s and CROs’ study teams and the other blinded team 
members will remain blinded. The outputs produced for the interim analysis will include a 
designation as “Interim Analysis” next to the analysis population.  
7.6 Examinations of Subgroups 
The analyses on the percentage of subjects who have treatment success following a single 
and/or two installations of study drug will be also analyzed for each of the below subgroups: 
•
Age (<18, ≥18-<65, ≥65-<85, ≥85 years).
•
Sex (Female, Male).
•
BMI (<18.5 kg/m², ≥18.5–<25 kg/m², ≥25.0–<30 kg/m², ≥30.0 kg/m²).
•
Country.
•
Region (US vs Non-US).
•
Type of CVAD (Non-tunneled or Tunneled Central Venous Catheter (CVC), Implanted
Ports, Peripherally Inserted Central Catheters (PICCs)).
Other category is excluded from the analyses.
•
Number of lumens (single lumen, multi-lumen: 2, multi-lumen: 3, multi-lumen: 4).
•
Where and by whom was the CVAD identified as dysfunctional (Patient/caregiver
identified, Healthcare provider not at study site, Healthcare provider at study site)
•
Duration of CVAD dysfunction (0-5 hours, >5–48 hours)
•
Race (American Indian or Alaska Native, Asian, Black or African American, Native
Hawaiian or Other Pacific Islander, White, Other, Not Reported).
In case of multiple races for a same subject, the combination of the races will be
displayed as a new category.
•
Ethnicity (Hispanic or Latino, Not Hispanic or Latino, Not Reported, Unknown).
For all subgroup analyses, any category with fewer than 1 subject will be excluded from the 
analyses.  
For treatment comparisons, if one of the 2 treatment groups compared has fewer than 3 
subjects, the comparison will not be performed. 
7.7 Descriptive Statistics 
General descriptive statistics for quantitative variables will include the n (the number of non-
missing values), the mean, the standard deviation (SD), the median, the minimum (min) and 
the maximum values (max).  
For categorical variables, the number (n) and percentage (%) of subjects with a specific level 
of the variable will be presented. The number of missing values will be displayed as a 
“Missing” category, where appropriate. Unless otherwise specified, the denominator for each 
percentage will be the number of non-missing observations within the analysis set and 
treatment group. 


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7.8 Definitions 
7.8.1 Baseline 
Baseline is defined as the last non-missing value available before the first study drug 
administration. 
7.8.2 Date of First and Last Randomized Study Medication Intake 
The date of first randomized study medication intake is the earliest date of randomized study 
medication intake considering the eCRF data, corresponding to the date part of the variable 
RFXSTDTC in the study data tabulation model (SDTM) dataset DM. 
The date of last randomized study medication intake is the one recorded in the Treatment 
Termination form of the eCRF, corresponding to the date part of the variable RFXENDTC in 
the SDTM dataset DM. 
7.8.3 Study Day 
If not otherwise specified, all assessment days will be related to the first day of study drug 
administration (CUSA-081, placebo or alteplase).  
The first day of study medication administration is referred to as Day 1. Day -1 is the day that 
precedes Day 1.  
•
If the date of the assessment is on or after the first study drug administration date,
then
Study Day = (date of assessment – first study drug administration date) + 1.
•
If the date of the assessment is prior to the first study drug administration date, then
Study Day = (date of assessment – first study drug administration date).
7.8.4 Visit dates 
For each visit, the variable SVSTDTC (minimum start date for each of the subject’s 
assessments performed during a specific visit) in the SDTM SV dataset will be considered as 
the visit date in all the algorithms and the listings.  
--TPT variable defined in each relevant SDTM will be used as the timepoint in all the 
algorithms and the listings. 
7.8.5 Duration of Adverse Events or Medications  
The duration of an AE or medication will be calculated as follows: 
•
End date – Start date + 1 (when both dates are completely known);
•
Date of study completion/discontinuation – Start date + 1 (when the start date is fully
known but the AE or medication still ongoing at the end of the trial): in this case the
duration will be presented as “>x” days in the listing rather than “x” days;


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•
Missing (when the start date is incomplete or unknown, or when the AE or medication
ended but with an incomplete or unknown end date, or when the start date is greater
than date of study completion/discontinuation).
7.9 Diary Data 
Not applicable. Diary data are not collected in this study. 
7.10 Data Re-allocation 
In general, for by-visit (or timepoint) summaries, data recorded at the nominal visit (or nominal 
timepoint) will be presented. Data collected at unscheduled time points will be reviewed at the 
Data Review Meeting prior to database lock. Any re-allocation of unscheduled data will be 
described and justified in the Data Review Report, which will be finalized prior to database 
lock. In any case, listings will include scheduled, unscheduled and early discontinuation data. 
7.11 Exclusion of Data from the Statistical Analyses 
7.11.1 Exclusion of Data from All Statistical Analyses 
All data collected in the database will be used in all statistical analyses. 
7.11.2 Exclusion of Data from Per-Protocol Analyses 
In case of important protocol deviation (e.g., wrong inclusions, incomplete dosing, non-
permitted concomitant medications), the subject will be excluded from the PP Set and therefore 
from all PP analyses. 
In case of important protocol deviation impacting specific datapoint, only the specific datapoint 
will be excluded from the PP analyses.  
Protocol deviations will be reviewed during the DRM. Important protocol deviations leading 
to the exclusion of the subject from the PP analysis, or to the exclusion of isolated data from 
the PP analysis will be agreed by the study team during the Data Review Meeting and 
documented in the Data Review Report prior to database lock and unblinding. 
7.12 Listings 
All data collected in the eCRF will be presented in the individual listings. All listings will be 
presented for the ITT Set, unless specified otherwise. 
7.13 Coding 
Medical and surgical history, concomitant diseases, procedures and adverse events will be 
coded according to Medical Dictionary for Regulatory Activities (MedDRA) version 22.0 or 
higher. 
Medications will be coded using the World Health Organization Drug Dictionary (WHO-DD) 
version March 2019 or later. 


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8 Study Population 
8.1 Disposition of Subjects and Discontinuations 
8.1.1 Disposition of Subjects 
The number of subjects screened, the number screen failures and the reasons for screen failure 
will be presented (overall) based on Enrolled Set. 
The number of subjects screened, randomized and completed will be presented by country and 
by site using ITT Set. 
The number of subjects who performed each study visit will be summarized for the ITT Set. 
8.1.2 Discontinuation from the Treatment and the Study 
The number of subjects who completed the treatment, withdrew from the treatment, the reasons 
for withdrawal from the treatment, completed the study, withdrew from the study and the 
reasons for withdrawal from the study will be presented by treatment group using the ITT Set. 
The number of subjects who discontinued the treatment and the study will be also presented by 
country and by site using ITT Set. 
An individual subject listing will be provided for the disposition data. 
8.1.3 Protocol Deviations and Analysis Sets 
Deviations will be classified according to the categories defined in the Data Review Report. 
Important protocol deviations affecting the efficacy analyses will be summarized by treatment 
group using the FAS. The ones excluding from PPS will be also summarized. 
The number of subjects included in each of the FAS, Safety and PP Sets will be summarized 
for each treatment group and overall using the ITT Set. 
8.2 Demographic and Baseline Characteristics 
No formal comparison between treatment groups on demographic and baseline characteristics 
will be performed.  
8.2.1 Demographic Characteristics 
Demographic and baseline characteristics will be summarized by treatment arm and overall 
using descriptive statistics for the FAS. These will include age, age category, gender, race, 
ethnicity, height, weight at baseline, body mass index (BMI) at baseline, systolic blood pressure 
(SBP) at baseline, diastolic blood pressure (DBP) at baseline, heart rate (HR) at baseline and 
urine pregnancy test results. Separate summaries for demographic and baseline characteristics 
will also be produced using the Safety, ITT and PP Sets.  


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Notes: 
•
BMI will be calculated as: weight at the visit (kg) / height at Visit 1 (m²).
•
Age categories are: <18 years, ≥18-<65 years, ≥65-<85 years and ≥85 years. All
categories must be reported even if there are no subjects under a particular category.
An individual subject listing will be provided for demographic and baseline characteristics 
data. 
8.2.2 CVAD History 
Time between Screening CVAD assessment and first drug administration (hours), CVAD type, 
CVAD lumen, number of lumens, indications for CVAD insertion, duration of CVAD 
dysfunctional (hours) and when and by whom the CVAD was identified as dysfunctional 
(Patient/caregiver identified, Healthcare provider not at study site, Healthcare provider at study 
site) recorded at Screening Visit will be presented by treatment group and overall using the 
FAS. Summaries will be repeated using the Safety, ITT and PP Sets. 
Notes: 
•
Time between Screening CVAD assessment and first drug administration (hours) will
be calculated as: Date/Time of first study drug administration – Date/Time of Screening
CVAD assessment.
In addition, CVAD history data will be presented in an individual listing. 
8.3 Medical History and Concomitant Diseases 
Medical/surgical history and concomitant diseases will be summarized by System Organ Class 
(SOC) and Preferred Term (PT), by treatment group and overall. Summaries will be produced 
using the Safety Set.  
Notes: 
•
Medical/surgical history is defined as conditions in the medical/surgical history and
concomitant diseases eCRF form which are not ongoing at Screening Visit (even if end
date is missing);
•
Concomitant diseases are defined as conditions in the medical/surgical history and
concomitant diseases eCRF form which are ongoing at Screening Visit (“ongoing”
tickbox is ticked or end date > Screening Visit date).
In addition, medical, surgical and concomitant diseases data will be listed. 
8.4 Medications 
Prior medications, medications maintained during the randomized treatment period, and 
concomitant medications will be summarized by treatment group and overall (except for 
concomitant medications) for the Safety Set through frequency distributions and percentages 
by Anatomical Main Group [1st level of the Anatomical Therapeutic Chemical (ATC) 
classification], Therapeutic Subgroup (2nd level of the ATC classification), Chemical 
Subgroup (4th level of the ATC classification) and Preferred Name. In addition, maintained 


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medications and concomitant medications will be summarized using the FAS. Post-treatment 
medications will only be presented in a listing.  
The medications will be classified according to the following rules (See also Section 7.3): 
•
Prior medication: start date/time < date/time of first randomized study medication
intake and stop date ≤ date/time of first randomized study medication intake;
•
Medication maintained during the randomized treatment period: start date/time
< date/time of first randomized study medication intake and stop date/time > date/time
of first randomized study medication intake or ongoing;
•
Concomitant medication: date/time of first randomized study medication intake ≤ start
date/time < date/time of last randomized study medication intake;
•
Post-treatment medication: date/time of last randomized study medication intake ≤ start
date/time.
8.5 Procedures 
Prior procedures, procedures maintained during the randomized treatment period and 
concomitant procedures will be summarized by treatment group for the Safety Set through 
frequency distributions and percentages by SOC and PT. Post-treatment procedures will only 
be presented in a listing. 
The procedures will be classified according to the following rules (See also Section 7.3): 
•
Prior procedure: start date/time < date/time of first randomized study medication intake
and stop date ≤ date/time of first randomized study medication intake;
•
Procedure maintained during the randomized treatment period: start date/time
< date/time of first randomized study medication intake and stop date/time > date/time
of first randomized study medication intake or ongoing;
•
Concomitant procedure: date/time of first randomized study medication intake ≤ start
date/time < date/time of last randomized study medication intake;
•
Post-treatment procedure: date/time of last randomized study medication intake ≤ start
date/time.
8.6 Compliance 
No treatment compliance will be derived. 
9 Efficacy Analyses 
All the efficacy comparisons between the treatment arms including the primary will be based 
on the FAS and the ITT Set will be used for sensitivity analysis for the primary efficacy 
analysis. The PP Set will be used for sensitivity analysis for the primary efficacy analysis and 
also as the primary analysis for the non-inferiority analyses.  


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9.1 Primary Efficacy Variable 
The primary efficacy variable is the percentage of subjects who have treatment success 
following a single instillation of study drug (CUSA-081, alteplase, or placebo) with a dwell 
time up to 90 minutes. Treatment success will be derived as mentioned in Section 7.3.  
Protocol violations and any other intercurrent events will be ignored for analyses performed on 
the FAS, which target a treatment policy estimand. Subjects with important protocol deviations 
will be excluded from analyses based on the PPS. 
Superiority Hypotheses 
The hypothesis corresponding to the primary efficacy analysis is as follows: 
The null hypothesis H0:  
Rc-Rp=0 
The alternative hypothesis H1:  
Rc-Rp>0 
Where Rc=Rate of success of CUSA-081 
            Rp=Rate of success of placebo 
Additional hypotheses are constructed in a similar manner for secondary study objectives to 
demonstrate superiority. 
The number of subjects will be presented by treatment group. The percentage of subjects with 
treatment success and two-sided 95% Wald CI will be presented by treatment group. Treatment 
effects will also be presented as the mean difference in proportions (CUSA-081 versus placebo 
or alteplase) together with the 95% Wald CI and p-value based on a 2-sample Z test for 
proportions. 
Superiority of CUSA-081 over placebo or alteplase will be demonstrated by a statistically 
significant difference (p-value < 0.05) between treatments with a dwell time up to 90 minutes 
favoring CUSA-081 using a 2-sample Z test for proportions.  
A two-sided 95% confidence interval on the difference in rates of restoration of CVAD 
function between treatment groups will be constructed from SAS Proc Freq, option Riskdiff 
for treatment difference (See Section 14.1). 
A sensitivity analysis will also be performed as a test of the superiority of alteplase versus 
placebo in the rate of treatment success following a single administration of study drug with a 
dwell time up to 90 minutes. This sensitivity analysis will be performed at the same time as the 
non-inferiority assessment, using the same statistical methodology as for the other analyses of 
superiority, without adjustment for multiplicity. 
Non-Inferiority Hypotheses 
The hypotheses for the non-inferiority are as follows: 
The null hypothesis H0:  
Rc-Ra≤-10 
The alternative hypothesis H1:  
Rc-Ra>-10 


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Where Rc=Rate of success of CUSA-081 
            Ra=Rate of success of alteplase 
Assessment of non-inferiority will be based on the 95% confidence interval for the difference 
in the rate of treatment success between CUSA-081 vs alteplase following a single 
administration with a dwell time up to 90 minutes.  
Non-inferiority will be demonstrated (or null hypothesis will be rejected) when the lower limit 
of the 95% confidence interval for the difference in rate of success is greater than -10% (See 
Section 14.1). 
The number and percent of subjects with treatment success by treatment group and time point 
will be summarized. The rate difference and associated 95% Wald confidence intervals 
between CUSA-081 and placebo or alteplase will be estimated. The between treatment 
comparisons will be performed using a 2-sample z test for proportions.  
For superiority hypotheses, the primary analysis will be the analysis performed on the FAS. 
Analyses will be repeated based on the ITT and PP Sets as sensitivity analyses. For non-
inferiority hypotheses, the analysis based on the PP Set will be considered as the primary 
analysis. 
Sensitivity Analysis 
A sensitivity analysis will be performed on the primary efficacy endpoint using a logistic 
regression model with age (<18, ≥18-<65, ≥65-<85, ≥85 years) and region (US vs Non-US) as 
factors. The odds-ratio, 95% confidence interval for the odds-ratio and the p-value will be 
displayed for each treatment group comparison (See Section 14.4). 
9.2 Secondary Efficacy Variables 
All the below efficacy endpoints will be analyzed based on FAS, ITT and PP Set. 
9.2.1 Percentage of Subjects Who Have Treatment Success Following a Single 
Instillation of Study Drug with a Total Dwell Time Up To 30 and 60 Minutes 
The rate of treatment success following a single administration with a dwell time up to 30 and 
60 minutes will be analyzed using the same method as the primary efficacy variable. 
9.2.2 Percentage of Subjects Who Have Treatment Success After Up to Two Instillations 
of Study Drug with a Total Dwell Time Up To 120, 150 and 180 Minutes 
The rate of treatment success following up to two administrations with a dwell time up to 120, 
150 and 180 minutes will be analyzed using the same method as the primary efficacy variable. 
9.3 Other Secondary Efficacy Variables 
9.3.1 Rate of Recurrent Catheter Dysfunction Within 30 Days Following Treatment 
with Study Drug 
The rate of recurrent catheter dysfunction defined as first re-occlusion within 30 days following 
treatment (CUSA-081, placebo or alteplase) will be displayed by treatment group.  


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Time to first re-occlusion will be derived only for subjects with treatment success as follows: 
•
Time to first re-occlusion (days) = (date of start of first re-occlusion – date of first
treatment success assessment date + 1), if subject has a re-occlusion.
•
Time to first re-occlusion (days) = (date of CVAD – first treatment success assessment
date + 1), if the subject removed the CVAD before end of study (censored).
•
Time to first re-occlusion (days) = (date of study completion/discontinuation – first
treatment success assessment date + 1), if the subject is censored (i.e. no re-occlusion
before end of study and no CVAD removal).
The time to first re-occlusion will be analyzed using the Kaplan-Meier method with median, 
25th and 75th percentiles and 95% CI (See Section 14.2). The estimated probability of re-
occlusion free at day 30 and its 95% CI will be presented by treatment group. A Kaplan-Meier 
plot will also be presented.  
In addition, time to first re-occlusion will be analyzed using a Cox proportional hazards model 
including treatment as factor. Treatment effects will be presented as a hazard ratio with the 
associated 95% Wald CI (See Section 14.3). 
These analyses will be based on all subjects with treatment success following up to two 
administrations of study drug with a total dwell time up to 180 minutes within FAS. 
Additional efficacy analyses including demographics and baseline characteristics as 
covariate/subgroup may be explored, if needed.  
10 Safety Analyses 
Safety endpoints will be presented using descriptive statistics (Section 7.7). Safety analysis 
will be based on the Safety Set using actual treatment received. 
10.1 Extent of Exposure 
The number and percentage of subjects will be summarized by the number of doses received. 
The number and percentage of subjects assessed at each timepoint for CVAD will be also 
summarized by number of doses received.  
10.2 Adverse Events 
An AE will be classified as follow (See also Section 7.3): 
•
Pre-treatment AE: if it starts before the first study drug intake (AE onset date/time <
date/time of first study drug intake);
•
Treatment emergent AE (TEAE): if it starts on or after the first dose of study drug intake
up to the end of the treatment period (date/time of first study drug intake ≤ AE onset
date/time ≤ end date/time of the treatment period);
•
Post-treatment AE: if the onset date is after the end of the treatment period (AE onset
date/time > end date/time of the treatment period).


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The treatment period is defined as the period between first study drug administration and the 
following end: 
•
For subjects who completed the treatment period without achieving treatment success,
the end of the treatment period will be the date/time of the 180-minutes CVAD
assessment;
•
For subjects who achieving treatment success, the end of the treatment period will be
the date/time of the CVAD assessment where patency is achieved;
•
For subjects who discontinued the treatment due to AE or death, the end of the treatment
period will be the start date/time of the relevant event and the event will be considered
as treatment emergent;
•
For subjects who discontinued the treatment due to other reasons, the end of the
treatment period will be the date/time the last CVAD assessment performed.
An ADR is an AE related to study medication. A serious ADR is a SAE related to study 
medication. 
A severe AE is an AE with severe intensity. Refer to Section 7.3 for missing severity. 
An AE leading to discontinuation is an AE with action taken with study drug equal to “Drug 
Permanently Withdrawn”. 
An AE leading to death is an AE with outcome equal to “Fatal”. 
Two AEs with the same PT and classified in the same category (pre-treatment AE, TEAE or 
post-treatment AE) will be considered as two different events when calculating the “number of 
events” in the tables.  
Pre-treatment AEs, TEAEs and post-treatment AEs will be presented separately. Pre-treatment 
AEs will be presented in the listings only. 
A summary table displaying the number and the percentage of subjects experiencing at least 
one TEAE, serious TEAEs, ADRs/drug-related TEAE, serious ADRs, severe TEAEs, TEAEs 
leading to study drug discontinuation, TEAE of special interest (See Section 10.2.1) and 
TEAEs leading to death, as well as the number of events, will be presented by actual treatment 
group. 
The SOC and PT will also be used for tabulation using the MedDRA dictionary. The number 
and percentage of subjects with at least one AE and the number of events will be presented by 
SOC and PT by actual treatment group for treatment emergent AEs, non-serious TEAEs, 
serious TEAEs, ADRs, serious ADRs, severe AEs, AEs leading to treatment discontinuation 
and AEs leading to death.  
The number of subjects with the most common non-serious TEAEs (reported in >=5% of 
subjects in any treatment arm) will be also displayed by SOC and PT. The number of events 
will be also included in this analysis. The number of subjects with TEAEs will be also displayed 
by SOC and PT and maximum severity.  
Tables will be presented by SOC and then by PT, alphabetically sorted within each SOC and 
PT.  
These tables will be repeated for the post-treatment emergent AEs. 


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10.2.1 Adverse Events of Special Interest (AESI) 
AESI include the following:  
•
Major bleeding (defined as severe blood loss [>5 mL/kg] or blood loss requiring
transfusion or causing hypotension requiring use of inotropic agents);
•
Embolism;
•
Thrombosis;
•
Catheter related blood stream infections (CRBSI).
The number and percentage of subjects who experienced treatment emergent AESI and 
associated exact binomial 95% confidence interval for proportion will be provided in the 
summary. The number and percentage of subjects who experienced treatment emergent AESI 
by AESI category will be also summarized. 
All AESI will be listed. 
10.3 Vital Signs 
Vital signs collected at Screening will be presented as baseline characteristics using descriptive 
statistics. All vital signs results will be listed. 
11 Other Analyses 
No other analyses planned. All other data collected in the eCRF will be only listed. 
12 Changes in the Planned Analyses from Study 
Protocol 
•
The protocol Section 12.2 describes the FAS as “the primary analysis set for all efficacy
analyses”. The first secondary objective is “to demonstrate the non-inferiority of
CUSA-081 compared to alteplase in the rate of treatment success following a single
administration with a dwell time up to 90 minutes”. The primary analysis corresponding
to this objective, which is included within the hierarchical closed testing procedure, will
be based on the Per-Protocol Set, since this approach is considered as more conservative
as per Section 5.2.3 of ICH E9. The FAS will be the primary analysis set for all efficacy
analyses involving assessment of superiority.
•
The protocol Section 12.3.7 describes an interim futility analysis. Since the study has
been terminated by the Sponsor prematurely for other reasons, the futility analysis will
not be conducted. The analysis to be conducted following the premature closure of the
study will be exactly the analysis which is planned for end of study. The database lock
and unblinding will be conducted according to the relevant SOPs of the Sponsor and
CRO and the analysis conducted will be considered as the final study analysis.
•
The definition of the Per-Protocol Set has been modified to use Safety Set instead of
ITT Set in order to include in the Per-Protocol Set only treated subjects.


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13 Output 
13.1 Software 
SAS version 9.4 will be used to perform all the statistical analyses. 
13.2 Reporting Conventions 
13.2.1 Treatment, Visit and Subgroup Descriptors 
In the tables, listings and figures, the treatments, visits and timepoints will be identified as 
described below. 
Treatment group 
(as displayed in outputs) 
Descriptor for treatment 
CUSA-081 
One or two doses of “CUSA-081” (reteplase) 
Placebo 
One or two doses of “Placebo” (normal saline) 
Alteplase 
One or two doses of “alteplase” 
Output 
Descriptor for visits 
Tables 
Baseline Visit (Day 0 or Day 1) 
Visit 1 (Day 1) 
Follow-up Visit 2 (Day 30) 
Timepoints will be presented as 30 mins, 60 mins, 90 mins, 120 mins, 
150 mins and 180 mins. 
Listings 
Baseline, Visit 1 and Visit 2 will be presented in the “Visit” column. 
Timepoints will be presented as 30 mins, 60 mins, 90 mins, 120 mins, 
150 mins and 180 mins. 
Figures 
Baseline, Visit 1 and Visit 2 
Timepoints will be presented as 30 mins, 60 mins, 90 mins, 120 mins, 
150 mins and 180 mins. 


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Subgroup 
Category descriptor 
Age 
•
<18 years
•
≥18-<65 years
•
≥65-<85 years
•
≥85 years
Sex 
•
Male
•
Female
BMI 
•
<18.5 kg/m²
•
≥18.5–<25 kg/m²
•
≥25.0–<30 kg/m²
•
≥30.0 kg/m²
Country 
•
All countries to be included in full text.
Region 
•
US
•
Non-US
Type of CVAD 
•
Non-tunneled or Tunneled Central Venous Catheter (CVC)
•
Implanted Ports
•
Peripherally Inserted Central Catheters (PICCs)
Number of lumens 
•
Single lumen
•
Multi-lumen: 2
•
Multi-lumen: 3
•
Multi-lumen: 4
Where and by 
whom was the 
CVAD identified as 
dysfunctional 
•
Patient/caregiver identified
•
Healthcare provider not at study site
•
Healthcare provider at study site
Duration of CVAD 
dysfunction 
•
0-5 hours
•
>5–48 hours
Race 
•
American Indian or Alaska Native
•
Asian
•
Black or African American
•
Native Hawaiian or Other Pacific Islander
•
White
•
Other
•
Not Reported
In case of multiple races for a same subject, the combination of the 
races will be displayed as a new category. 
Ethnicity 
•
Hispanic or Latino
•
Not Hispanic or Latino
•
Not Reported
•
Unknown
13.2.2 Decimal Places 
Quantitative variables will be listed with the same number of decimal places as in the actual 
data. 


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The following rules on decimal places will be considered in the listings for the derived variables 
(in the analyses rounding will not be performed): 
Variables 
Decimal Places 
•
Duration of AE, medication (days)
•
Time to first re-occlusion (days)
0 decimal 
•
BMI (kg/m2)
•
Time between screening CVAD assessment
and first drug administration (hours) 
•
Duration of CVAD dysfunctional (hours)
1 decimal 
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): 
All summary statistics will be rounded (using the SAS® function ROUND) and wherever 
possible data will be decimal aligned. 
Statistic 
Number of decimal places for reporting 
Counts (n) 
None 
Percentages (%)/Proportion 
1 decimal place 
Note: 
If the calculated percentage is >0.0% but <0.1% 
then <0.1% is to be presented in the relevant table 
and/or listing. 
If the calculated percentage is >99.9% but <100.0% 
then >99.9% is to be presented in the relevant table 
and/or listing. 
Mean, Median, SD, 25th percentile, 75th 
percentile, Confidence intervals, Kaplan-
Meier estimate 
Actual data + 1 decimal place 
Probability of re-occlusion free 
3 decimals 
Difference between percentages (%)/ 
Proportions  
Actual data + 1 decimal place 
Min, Max 
Same as actual data 
Odds-ratio and its confidence limits 
3 decimals 
p-values
3 decimal places. 
Note: 
If the p-value is less than 0.001, it will be presented 
as <0.001.  
If the rounded result is a value of 1.000, it will be 
displayed as >0.999.  


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13.2.3 Other Reporting Conventions 
Treatments will be presented with the following order in the tables: CUSA-081, Placebo, 
Alteplase. 
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 
replaced by dashes. 
In the listings, a unit associated with a variable will be presented only once within parentheses 
either below or next to that variable in the heading portion. If a parameter has multiple units, 
each unit will be displayed only once, as applicable. 
All tables will have their source listing referenced in a footnote. All figures will include the 
source table in a footnote. Listings should be sorted by subject ID and visit (unless otherwise 
specified) and have the SDTM and/or ADaM source data referenced in a footnote. The columns 
of each listing should fit into one page and should not be split into different pages.  
When an output is split in multiple pages, page-break should be adequately controlled. 
When a table is split in multiple pages, breaking of a block information in different pages is 
not allowed.   
In a listing, in the case that a subject’s record has been continued to the next page, an 
appropriate identification (e.g., the subject ID number and any other relevant information 
which is split between 2 pages) must be presented at the beginning of that page. 
When a listing contains a lot of information, in order to optimize space on the page, some 
columns can be merged (e.g. “reported term” and “indication” may be presented in the same 
column. 
13.3 Format 
The following information should always be presented: 
•
‘Clinical Study Code No.:<Study Code No.>’ followed by Chiesi denomination in the
top portion of each page. Chiesi denomination is ‘Chiesi Farmaceutici S.p.A’.
•
The table/listing/figure number followed by the title, the analysis set used and the
output page number in the format of ‘Page x of Y’ in the top portion of each page of
any table/listing/figure.
•
The SAS program name followed by the datetime of the output production and the
analysis type (e.g. Dry Run; Draft Version; Final Version) in the bottom portion of each
page of any table/listing/figure. The source listing/table/dataset will appear bottom left
for every table/figure/listing.
•
Tables and listings will be produced in rich text format (i.e., they will be tabular in
format). Individual outputs must be provided in both portable format document (.pdf)
and rich text format (.rtf).
•
Combined PDF and RTF documents must also be provided, including a table of
contents with hyperlinks. The combined documents should be divided by document
type (tables, figures, listings).


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•
SAS outputs will be provided to the Sponsor in a similar manner (PDF and RTF;
combined and with hyperlinked table of contents). The SAS outputs will be a separate
deliverable to the Sponsor and are not intended for inclusion within the CSR.
•
The combined documents page number in the format of ‘Page n of N’ will be presented
bottom right corner.
The following should be followed for the tables: 
•
A landscape layout and Letter size will be used.
•
A 9-point font size will be used using Courier New font.
•
Horizontal lines will appear before and after the column heading of the output.
•
Additional footnotes may be included if strictly necessary for clarification. Footnotes
will be put under the main body of text at the bottom left of the page and will be
displayed on each page of the output and not only on the last one.
•
The left and right margins will be a minimum of 2.1 cm from the left and 1.9 cm from
the right. The top and bottom margins will be a minimum 2.92 cm. Header and footer
will be both 1.27 cm.
The following should be followed for the listings: 
•
A landscape layout and Letter size will be used.
•
An 8-point font size will be used using Courier New font.
•
Horizontal lines will appear before and after the column heading of the output.
•
Additional footnotes may be included if strictly necessary for clarification. Footnotes
will be put under the main body of text at the bottom left of the page and will be
displayed on each page of the output and not only on the last one.
•
The left and right margins will be a minimum of 2.1 cm from the left and 1.9 cm from
the right. The top and bottom margins will be a minimum 2.92 cm. Header and footer
will be both 1.27 cm.
The following should be followed for the figures: 
•
A portrait layout and Letter size will be used.
•
A 9-point font size will be used using Courier New font.
•
Figures will be produced in RTF and PDF formats (as described above), including
relevant titles and footnotes as separate elements on the page (not within the body of
the figure).
•
Additional footnotes may be included if strictly necessary for clarification. Footnotes
will be put under the main body of text at the bottom left of the page and will be
displayed on each page of the output and not only on the last one.


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•
The size of the figures will be: width=16.3 cm, height=12.2 cm. The resolution will be
set using the option IMAGE_DPI=400. Figures will have a footer specifying the source
table or listing. Figures should clearly identify each treatment arm and require care of
colors/symbols.
•
The left margin will be a minimum of 2.5 cm, the right margin will be a minimum of
2 cm. The top and bottom margins will be a minimum of 0.8 cm.
13.4 Quality Control 
The Quality Control steps will be defined in the Datasets, Tables, Listings, Figures QC Plan. 


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14 SAS Code 
14.1 Comparison of Two Proportions Using 2-Sample Z Test 
PROC FREQ DATA = <dataset>; 
TABLES trt01pn*Response / RISKDIFF (CL=(WALD) COLUMN=2); 
WHERE <population> = "Y" and ATPT = <timepoint>;  
RUN; 
Notes: 
–
TRT01PN represents the treatment arm, incorporating the following ordering:
1=CUSA-081, 2=Placebo and 3=Alteplase;
–
Response represents the treatment success indicator (1=Yes, 0=No).
–
The Chi-square statistic corresponds to the square of the Z-statistics (so the p-value
for Table 14.2.1.1.1 corresponds to the Chi-square p-value).
14.2 Kaplan-Meier Estimates 
PROC LIFETEST data=<dataset> timelist=(0 5 10 15 20 25 30) alpha=0.05; 
TIME time*censor(1); 
STRATA trt01pn; 
RUN; 
Notes: 
–
Time represents the time to event or time to censoring variable;
–
Censor represents the censoring indicator (0=event, 1=censored);
–
TRT01PN represents the treatment arm, incorporating the following ordering:
1=CUSA-081, 2=Placebo and 3=Alteplase;
14.3 Cox Proportional Hazards Model 
PROC PHREG data=<dataset>; 
CLASS trt01pn / param=glm order=internal; 
MODEL time*censor(1) = trt01pn; 
CONTRAST ‘CUSA-081 vs Placebo' 
trt01pn 1 -1 0 / estimate=exp e; 
CONTRAST 'CUSA-081 vs Alteplase' 
trt01pn 1 0 -1 / estimate=exp e; 
CONTRAST 'Alteplase vs Placebo’ 
trt01pn 0 -1 1 / estimate=exp e; 
RUN; 
Notes: 
–
Time represents the time to event or time to censoring variable;
–
Censor represents the censoring indicator (0=event, 1=censored);
–
TRT01PN represents the treatment arm, incorporating the following ordering:
1=CUSA-081, 2=Placebo and 3=Alteplase;


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14.4 Logistic Regression Model 
PROC LOGISTIC data=<dataset>; 
CLASS trt01pn (ref=”2”) agec region / param=glm; 
MODEL resp = trt01pn agec region; 
       LSMEANS trt01pn / e diff oddsratio cl; 
RUN; 
Notes: 
–
TRT01PN represents the treatment arm, incorporating the following ordering:
1=CUSA-081, 2=Placebo and 3=Alteplase;
–
AGEC represents the age in category (<18, ≥18-<65, ≥65-<85, ≥85 years).
–
REGION represents the country in category (US vs Non-US).
14.5 Conditional Power 
Of note, the interim analysis will not be conducted as a result of the early stopping of the study. 
The below text in this section is maintained for consistency with the protocol and to maintain 
a record of the original plan.  See Section 12 for additional details. 
Create a dataset with a single record and the following variables included as defined below: 
•
n_i1 = number of subjects in CUSA-081 group at interim
•
n_i2 = number of subjects in alteplase group at interim
•
n1 = 473 (This is the total number of subjects in CUSA-081 group planned at the end
of the trial)
•
n2 = 315 (This is the number of subjects in alteplase group planned at the end of the
trial)
•
e_i1 = number of successes in CUSA-081 group at interim
•
e_i2 = number of successes in alteplase group at interim
Next, create a new dataset, based on the prior dataset: 
DATA <dataset name>; 
   SET <name of prior dataset>; 
   alpha = 0.025; 
   NIMarg = -0.1; 
* Information fraction;
i=(n_i1+n_i2)/(n1+n2);
* Interim proportions;
p_i1=e_i1/n_i1;
p_i2=e_i2/n_i2;
* Interim difference between proportions, SE and test statistic (H0: diff <= NI Margin;
* H1: diff > NI Margin);
diff_i=p_i1-p_i2;


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   se_i=sqrt( (p_i1*(1-p_i1)/n_i1) + (p_i2*(1-p_i2)/n_i2) ); 
   z_i=(diff_i-(NIMarg))/se_i; 
* Conditional power CP(d) formula in Gallo, Mao, Shih, Alternative views on;
* setting clinical trial futility criteria, Journal of Biopharmaceutical Statistics 2014;
cp=probnorm(-1/sqrt(1-i)*(probit(1-alpha) - z_i/sqrt(i)));
RUN;