Clinical trial NCT04250207
Conditions: Fuchs' Endothelial Corneal Dystrophy
Sponsor: Kowa Research Institute, Inc.
Start date: 2020-06-23; Completion date: 2022-06-27
Enrollment: 65 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objective of this study is to investigate the effect of K-321 in patients with Fuchs endothelial corneal dystrophy (FECD) after descemetorhexis.

Detailed description:

Arms: arm 1: K-321 Ophthalmic Solution Dose A arm 2: K-321 Ophthalmic Solution Dose B arm 3: Vehicle Solution Dose
Interventions: intervention 1: K-321 Solution intervention 2: Placebo Solution
Intervention descriptions: intervention 1: K-321 solution drops intervention 2: Placebo solution drops for K-321

Protocol documents:
SAP_002.pdf: 
 
 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-
Group, 12 Week, Phase 2 Study to Investigate the Safety and 
Efficacy of Ripasudil (K-321) Eye Drops After Descemetorhexis in 
Patients with Fuchs Endothelial Corneal Dystrophy 
 
NCT04250207 
SAP Ver.: 1.0 
Document Date: 09 Feb 2021 




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II
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ............................................................................ IV 
1. 
INTRODUCTION......................................................................................6 
2. 
OBJECTIVES ............................................................................................7 
2.1. PRIMARY OBJECTIVE .................................................................................. 7 
2.2. SECONDARY OBJECTIVE ............................................................................. 7 
2.3. EXPLORATORY OBJECTIVE ......................................................................... 7 
3. 
INVESTIGATIONAL PLAN ...................................................................7 
3.1. OVERALL STUDY DESIGN AND PLAN .......................................................... 7 
3.2. STUDY ENDPOINTS ..................................................................................... 8 
3.2.1. 
Primary Efficacy Endpoint ................................................................. 8 
3.2.2 
Secondary Efficacy Endpoint .............................................................. 8 
3.2.3 
Exploratory Endpoints ........................................................................ 9 
3.2.4 
Safety Endpoints................................................................................ 10 
3.3. STUDY PROCEDURES AND BASELINE ........................................................ 10 
3.4. TREATMENTS ............................................................................................ 11 
4. 
GENERAL STATISTICAL CONSIDERATIONS ..............................12 
4.1. SAMPLE SIZE ............................................................................................ 12 
4.2. RANDOMIZATION, STRATIFICATION AND MASKING .................................. 13 
4.3. ANALYSIS SET .......................................................................................... 13 
4.3.1 
Full-analysis set (FAS) ..................................................................... 13 
4.3.2 
Per-protocol set (PPS) ...................................................................... 13 
4.3.3 
Safety Set (SFS) ................................................................................. 14 
4.4 
ASSESSMENT WINDOWS ........................................................................... 14 
4.4.1 
Study day ........................................................................................... 14 
4.4.2 
Visit Windows for Analysis ............................................................... 14 
5. 
PATIENT DISPOSITION.......................................................................15 
5.1. DISPOSITION ............................................................................................. 15 
5.2. PROTOCOL DEVIATIONS ........................................................................... 15 
6. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ...........16 
6.1 
DEMOGRAPHICS ........................................................................................ 16 
6.2 
BASELINE OCULAR CHARACTERISTICS ..................................................... 17 
6.3 
MEDICAL HISTORY ................................................................................... 17 
6.3.1 
General Medical History .................................................................. 17 
6.4 
INCLUSION AND EXCLUSION CRITERIA ..................................................... 18 
7. 
TREATMENTS AND MEDICATIONS ................................................18 
7.1 
PRIOR AND CONCOMITANT THERAPIES: MEDICATIONS AND SURGICAL 
PROCEDURES ...................................................................................................... 18 
7.2 
STUDY TREATMENTS ................................................................................ 19 
7.2.1 
Extent of Exposure ............................................................................ 19 


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7.2.2 
Treatment Compliance and Modifications........................................ 19 
8. 
EFFICACY ANALYSIS ..........................................................................20 
8.1. PRIMARY EFFICACY ENDPOINT ................................................................. 20 
8.1.1. 
Primary Analysis ............................................................................... 20 
8.1.2. 
Other Analysis ................................................................................... 21 
8.2. SECONDARY EFFICACY ENDPOINT ............................................................ 22 
8.3. EXPLORATORY EFFICACY ENDPOINTS ...................................................... 23 
8.4. EFFICACY ENDPOINTS FOR THE NON-STUDY EYE ..................................... 25 
9. 
SAFETY ANALYSIS...............................................................................25 
9.1. ADVERSE EVENTS ..................................................................................... 25 
9.1.1. 
Incidence of Adverse Events ............................................................. 27 
9.1.2. 
Relationship of Adverse Events to Study Drug ................................. 27 
9.1.3. 
Severity of Adverse Event.................................................................. 27 
9.1.4. 
Treatment-Emergent Adverse Events Leading to Treatment 
Discontinuation .............................................................................................. 28 
9.1.5 
Treatment-Emergent Adverse Events Leading to Study 
Discontinuation .............................................................................................. 28 
9.1.6 
Serious Adverse Events ..................................................................... 28 
9.1.7 
Death ................................................................................................. 28 
9.2 
CLINICAL LABORATORY EVALUATIONS ................................................... 28 
9.2.1 
Hematology ....................................................................................... 29 
9.2.2 
Chemistry .......................................................................................... 29 
9.2.3 
Urinalysis .......................................................................................... 29 
9.3 
VITAL SIGN MEASUREMENTS ................................................................... 29 
9.4 
PHYSICAL EXAMINATION ......................................................................... 30 
9.5 
OPHTHALMOLOGICAL ASSESSMENTS ........................................................ 30 
9.5.1 
Intraocular Pressure ......................................................................... 30 
9.5.2 
Slit-Lamp Biomicroscopy .................................................................. 30 
9.5.3 
Ophthalmoscopy ............................................................................... 31 
10 
INTERIM ANALYSIS ............................................................................31 
11 
REFERENCES .........................................................................................32 
12 
APPENDICES ..........................................................................................33 
12.1 
UNIT CONVERSION ................................................................................ 33 
12.2 
NOTABLE ABNORMAL VITAL SIGNS ...................................................... 33 
12.3 
SCHEDULE OF STUDY PROCEDURES ...................................................... 34 


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IV 
List of Abbreviations 
Abbreviation 
Definition 
AE 
Adverse Event 
ANOVA 
Analysis of Variance 
BCVA 
Best Corrected Visual Acuity 
BID 
Twice Daily 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
 
 
CTMS 
Clinical Trial Management System  
CRO 
Contract Research Organisation 
CS 
Clinically Significant 
CV 
Curriculum Vitae 
ECD 
Endothelial Cell Density 
eCRF 
electronic Case Report Form 
EOS 
End-Of-Study 
EOT 
End-Of-Treatment 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
EU 
European Union 
FAS 
Full-Analysis Set 
FDA 
US Food and Drug Administration 
FECD 
Fuchs endothelial corneal dystrophy 
HRT 
Heidelberg retina tomography 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IOP 
Intra-Ocular Pressure 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
LOCF 
Last-Observation-Carried-Forward 
M/N 
Morning and Night 
MD/E 
Mid-Day and Evening 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHRA 
Medicines and Healthcare Products Regulatory Agency 
NCS 
Not Clinically Significant 
PPS 
Per Protocol Set 


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Abbreviation 
Definition 
Q1 
Quartile 1 
Q3 
Quartile 3 
QID 
4 Times Daily 
ROCK 
Rho-associated Protein Kinase 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SFS 
Safety Set 
SOA 
Schedule Of study site Activities 
TEAE 
Treatment-Emergent Adverse Event 
TESAE 
Treatment-Emergent Serious Adverse Event 
 
 










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3.2.4 Safety Endpoints 
Safety parameters include monitoring of Adverse Events (AEs), treatment 
emergent adverse events (TEAEs), treatment emergent serious adverse events 
(TESAEs), physical examination findings, vital sign measurements, laboratory 
tests, slit-lamp biomicroscopy, measurement of intraocular pressure (IOP), and 
ophthalmoscopy. 
Safety data will be summarized and listed with no statistical hypothesis testing 
performed. Continuous variables will be summarized using the number, mean, the 
standard deviation (SD), median, minimum value, and maximum value. 
Categorical variables will be summarized using frequency counts and percentages. 
3.3. Study Procedures and Baseline 
Before performing any study procedures, all potential patients will sign an informed 
consent form (ICF). Patients will have the opportunity to have any questions 
answered before signing the ICF. The investigator must address all questions raised 
by the patient. The investigator or designee will also sign the ICF.  
After obtaining informed consent at Visit 1, the investigator will select one eye as 
the study eye, and the patient will come back for the descemetorhexis operation and 
random assignment to treatment after 1 to 4 weeks (Visit 2). At Visit 2, each 
patient’s eligibility for study participation will be confirmed, including a successful 
descemetorhexis operation that meets the inclusion criteria. After eligibility is 
confirmed, patients will be randomly assigned to the 3 treatment groups (in a 1:1:1 
ratio). All patients will be instructed to apply the study drug only to the study eye.  
Day 1(Visit 3) is considered to be the day when first dose of study drug is 
administered. Randomization is scheduled to be performed on Day -1 (Visit 2).  
Baseline is defined as the last non-missing measurement including unscheduled 
assessments prior to the administration of first dose of study drug. Change from 
baseline is defined as the post baseline value minus the baseline value for the given 
assessment. 
The EOT (Week 12 or ET [Early Termination]) visit is defined as the visit occurring 
at the end of the 12-week treatment period (Week 12) or the final visit at which the 
patient is prematurely discontinued/withdrawn from study drug administration.  
The EOS (Week 52 or ET) visit is defined as the visit occurring at the end of the 
52-week study period (Week 52) or the final visit at which the patient is 
prematurely discontinued/withdrawn from participation in the study. 
The schedule of study site activities (SOA) is presented in Section 13 (Table 5). 


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3.4. Treatments   
There are 3 treatment groups: K-321 ophthalmic solution 0.4% dosed QID (K-321 
QID), K-321 ophthalmic solution 0.4% dosed BID (K-321 BID); and placebo. To 
preserve masking, all patients will self-administer assigned study drug QID and 
will dose from 
 of assigned 
 one to be used M/N (morning and night) 
and the other to be used MD/E (mid-day and evening), as presented in Table 1.   
Table 1 Vial Contents by Dosing Regimen and Dosing Time for Each 
Treatment Group From Day 1 to Visit 9 (Week 12) 
Treatment  
Morning 
Mid-day 
Evening 
Night 
K-321 QID 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 BID 
K-321 0.4% 
Placebo 
Placebo 
K-321 0.4% 
Placebo 
Placebo 
Placebo 
Placebo 
Placebo 
Except on days of study visits on Day 1 through Week 12 (not including Visit 9) 
during the treatment period, study drug will be dosed by the patient QID: morning 
(9 AM ±1 hour), mid-day (1 PM ±1 hour), evening (5 PM ±1 hour), and night 
(9 PM ±1 hour). Each patient will be provided with 
 of colour-coded 
 
, one set to be applied M/N and the other to be applied MD/E.  
On study visit days Visit 3 through Visit 9, patients will not apply any study drug 
until after all study measurements have been completed; thereafter on those days, 
patients will start dosing with the dose closest in timing to the dosing regimen and 
continue with the remaining doses for the day. Additionally, on Visit 3 they must 
first apply study drug within 
 after the end of the descemetorhexis 
procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient 
applies any study drug on the day of Visit 9 before all study measurements have 
been completed, that patient’s Visit 9 should be rescheduled within the visit 
window allowance (eg, the next day). For visits other than Visit 9 during the 
treatment period, a patient who applies study drug before all study measurements 
have been completed will be assessed as planned, and a protocol deviation must be 
recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up 
period, and new study drug will be dispensed without M/N or MD/E colour coding. 
All patients will self-administer their tapering-phase study drug BID (9 AM 
±1 hour and 9 PM ± 1 hour) for a week, then once daily (9 AM ± 1 hour) for a week, 
and then will discontinue study drug.  




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4.2. Randomization, Stratification and Masking 
Patients will be randomly assigned at Visit 2 to either placebo, K-321 BID, or K-
321 QID treatment (Section 3.1) using a 1:1:1 allocation ratio. There is no 
stratification on the randomization for this study. 
Study drug will be double-masked. All study drug will be supplied in identical 
packaging, colour, smell, and appearance to enable double-masked conditions. K-
321 and matching placebo will be provided in identical packaging so that all 
investigators, study site staff, patients, and clinical monitors will remain masked 
throughout the study. The Interactive Web Response System (IWRS) will assign 
study drug to patients at the time of randomisation. Only personnel in IWRS, the 
independent randomisation team within 
 Biostatistics, and clinical supplies will 
be unmasked and will have access to treatment assignments; all other parties 
involved in the study will be fully masked. 
A patient’s treatment assignment will not be unmasked for the investigator or study 
site staff until the EOS unless medical treatment of the patient depends on knowing 
the study treatment the patient received. In the rare event that unmasking is needed 
because of a medical emergency, the investigator may unmask an individual patient 
through the IWRS. Reasons for treatment unmasking must be clearly explained and 
justified in the electronic case report form (eCRF). The date on which the code was 
broken together with the identity of the person responsible must also be 
documented. 
Patients who are unmasked will be allowed to continue their participation in the 
study; however, their data may be excluded from per-protocol analyses. 
4.3. Analysis Set 
4.3.1 Full-analysis set (FAS) 
The FAS will consist of all participants who have a successful descemetorhexis 
procedure performed, were randomly assigned to receive double-mask study drug, 
received at least one dose of study drug in the study eye, and have at least one 
assessment of central corneal ECD performed after the descemetorhexis procedure 
on the study eye. (An assessment of “cannot perform due to corneal oedema” is 
considered a valid assessment for inclusion in the FAS.) All analyses using the FAS 
will group participants according to randomised treatment.  
4.3.2 Per-protocol set (PPS) 
The PPS will consist of all FAS participants who complete the 12-week dosing 
treatment period for the study eye (for the primary analysis only), have at least 80% 
compliance and at most 125% compliance with study treatment for the study eye, 


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have not taken any prohibited medication and have no major protocol deviations. 
Patients who receive rescue therapy will be considered to have completed study 
treatment for inclusion in the PPS. All analyses using the PPS will group 
participants according to treatment actually received. 
4.3.3 Safety Set (SFS) 
The SFS will consist of all participants who received any study drug. All analyses 
using the safety set will group participants according to treatment actually received. 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy 
analyses will be repeated for the PPS. Safety will be summarised using the SFS.  
4.4 Assessment Windows 
4.4.1 Study day 
When study day is used for display or in comparisons the following algorithm will 
be used: 
• study day = date of assessment - Day 1 +1, if date of assessment ≥ Day 1. 
• study day = date of assessment - Day 1, if date of assessment < Day 1. 
Note that the day before Day 1 is Day -1 (for analysis, there is no Day 0 for study 
day).  
4.4.2 Visit Windows for Analysis 
Visit windows will be defined for by-visit summary and analysis purposes. 
Summary data (such as AEs and concomitant medications) that are not reported by 
visit will not use visit windows. The EOT or EOS assessments will be summarized 
based on visit window of when the visit occurred (e.g., if the patient discontinued 
treatment at Week 7, the EOT assessment will be included in the Week 7 by-visit 
summaries).   
Both scheduled and unscheduled assessments will be considered as valid 
assessments for analysis.  Visit labels will be assigned to each post baseline record 
based on the windows for study day relative to the date of first dose. If an 
assessment at Day 1 is missing, the closest visit with non-missing assessment on or 
before the date of first dose will be used as baseline.  All analyses for FAS, PPS, 
and SFS will be based on the analysis visit windows in Table 2. If there are multiple 
valid assessments within a time window, the assessment which occurs closest to 
the target day will be used in the analysis. If there are multiple valid assessments 
with same difference from the target day, the latest assessment will be used in the 
analysis.    


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Table 2 
Analysis Visit Windows 
Analysis Visit 
Analysis Visit Window  
Screening/Washout 
NA 
Baseline/Day 1  
NA 
Week 1 
2 to 10 Days (Target 7) 
Week 2 
11 to 17 Days (Target 14) 
Week 3 
18 to 27 Days (Target 21) 
Week 5 
28 to 41 Days (Target 35) 
Week 7  
42 to 55 Days (Target 49) 
Week 9 
56 to 73 Days (Target 63) 
Week 12 
74 to 97 Days (Target 84) 
Week 16 
98to 125 Days (Target 112) 
Week 20 
126 to 153 Days (Target 140) 
Week 24 
154to 216 Days (Target 168) 
Week 38 
217 to 314 Days (Target 266) 
Week 52  
315 to 413 Days (Target 364) 
5. Patient Disposition 
5.1. Disposition 
The counts of patients who provide informed consent and screen failed will be 
presented. The counts and percentages of patients who are randomized, receive 
study treatment, completed treatment period (12 week) as well as 12 week visit, 
completed treatment period (12 week) as well as 52 week visit, discontinued during 
treatment period (12 week) but completed 12 week visit, discontinued during 
treatment period (12 week) but completed 52 week visit. Withdrawn from the study 
will be presented based on the number of patients in each treatment group and 
overall for all randomized patients. Reasons for discontinuation during treatment 
period and reasons for withdrawn from the study will be summarized for each 
treatment group as treatment disposition and patient disposition, respectively. All 
percentages within the patient disposition summary will be based on the number of 
patients randomized. All percentages within the treatment disposition summary will 
be based on number of patients from SFS. Patient and treatment disposition data 
will be listed. Screen failures will also be listed in the data listing. 
5.2. Protocol Deviations 
Protocol deviations will be tracked by the clinical team on an ongoing basis.  Major 
protocol deviations are the subset of deviations which are considered to have 
potential significant impact on primary analysis. Subjects with major protocol 
deviations will be excluded from the analysis based on PPS. Specific criteria for 


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what constitute a major protocol deviation will be determined by the clinical team 
and the following criteria may be considered: 
• Eligibility criterion violation 
• Not complete the 12-week dosing treatment period for the study eye (for the 
primary analysis only) 
• Less than 80% or more than 125% compliance with study treatment in the 
study eye 
• Taking prohibited medication or treatment listed in protocol section 5.8.3 
Protocol deviations will be recorded within the 
 Clinical Trial Management 
System (CTMS) and will undergo a blinded review prior to database lock and 
unblinding. Significant protocol deviations are defined as the subset of deviations 
which are considered to affect primary efficacy and safety assessments, the safety 
or mental integrity of a subject, or the scientific value of the trial. 
Patients with significant protocol deviations will be tabulated for each treatment 
group, and overall. All deviations, including significant and major protocol 
deviations, will be listed by-patient for all randomized patients. All percentages will 
be based on the number of patients randomized. The total number of significant 
protocol deviations and the number of significant protocol deviations under each 
protocol deviation category will also be presented 
6. Demographics and Baseline Characteristics 
6.1 Demographics 
Demographic information and baseline characteristics collected at Screening will 
be summarized for the FAS and SFS. Continuous variables, including age (years), 
baseline weight (kg), baseline height (cm), and baseline body mass index (BMI) 
(kg/m2) will be summarized using descriptive statistics for each treatment group 
and overall. Continuous variables will be tested with ANOVA. The following 
categorical variables will be summarized by reporting the number and percentage 
of patients in each category for each treatment group and overall. Fisher’s exact test 
will be used for the following categorical variables to test whether there is a 
difference between each treatment group.  
• Sex (Male, Female) 
• Ethnicity (Hispanic or Latino, Not Hispanic or Latino) 
• Race* (White, Black or African American, Asian, American Indian or 
Alaska Native, Native Hawaiian or Other Pacific Islander, Multiple and 
Other) 
• Iris color of study eye (Blue, Brown, Hazel, Green, Grey, Other)  


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• Family History of FECD† (Yes, No) 
o If Yes, Father, Mother, or Both    
*The statistical comparison between the Race categories will not be presented 
because the Race categories are not mutually exclusive. i.e. One subject could be 
marked in multiple race categories. 
†The statistical comparison on Family History of FECD will be between the 
categories of ‘Yes’ and ‘No’.  
Percentages will be based on the total number of patients in the SFS, FAS and PPS. 
All demographic and baseline characteristics will be listed for all randomized 
patients.  
6.2  Baseline Ocular Characteristics 
Descriptive statistics will be provided for patient ocular baseline characteristics on 
study eye and non-study eye by treatment group and overall for ophthalmological 
assessments including Corneal ECD Measurement by specular microscopy 
(including Corneal Morphology Parameters), Corneal ECD Measurement by 
Heidelberg Retina Tomography (HRT) (including Corneal Morphology 
Parameters), BCVA score, Corneal Thickness, presence/absence of Corneal 
Oedema categories of epithelial, stromal, or endothelial, Contrast Sensitivity, 
 domain and total scores, IOP, longest and shortest diameter of descemetorhexis. 
Tests for baseline disease characteristics of study eye will be performed in a 
pairwise manner with each dosing regimen (QID and BID) of K-321 0.4% 
compared to placebo. Numerical results will be tested with ANOVA; Categorical 
results will be tested with Fisher’s Exact Test. Summary and tests will be based on 
the FAS and SFS on study eye, non-study eye and the 
 Score where 
applicable. 
6.3 Medical History 
6.3.1 General Medical History 
Ocular and non-ocular medical histories are captured separately on the eCRF.  For 
non-ocular medical history, the investigator will record the body system, verbatim 
term, start date, and stop date or indication of ongoing.  For ocular medical history, 
the investigator will record the eye (right, left, both), verbatim term, start date, and 
stop date or indication of ongoing. Medical history will be coded using the Medical 
Dictionary for Regulatory Activities (MedDRA) Version 21.1or above.  The 
number and percentage of patients with any medical history (ocular and non-ocular), 
any ocular medical history in the study eye, any ocular medical history in the non-
study eye, an any non-ocular medical history will be summarized by treatment 


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group.  Additionally, non-ocular medical history will be summarized by the number 
and percentage of patients with any non-ocular medical history by reported body 
system and treatment group on SFS.   
Percentages will be calculated based on number of patients in the SFS. Also, by-
patient listings of ocular and non-ocular medical histories will be presented. 
6.4 Inclusion and Exclusion Criteria 
Inclusion and exclusion criteria deviations noted in the eCRF will be presented for 
informed consent patients in a data listing. 
7. Treatments and Medications 
7.1 Prior and Concomitant Therapies: Medications and Surgical 
Procedures 
At screening, the following prior therapies will be recorded in the eCRF: 
• History of prior study eye surgical procedures 
• All medications used within the previous 30 days 
• All surgical procedures within the previous 30 days (any body part) 
At screening, all patients’ prior medications used and surgical procedures 
undergone within the previous 30 days will be recorded in the eCRF.  
Any medicinal product prescribed or over-the-counter (OTC), including herbal 
products, vitamins, and minerals is considered a prior or concomitant medication. 
All patients’ surgical procedures undergone concomitantly during the study must 
be reported and recorded in the eCRF. Prior medication is defined as any 
medication with the stop date prior to the date of the first dose of study drug. 
Concomitant medication is defined as any medication with a stop date after the date 
of the first dose of study drug. Any medications used during the study will be coded 
with the World Health Organization Drug (WHODrug) Dictionary dated March 
2019., which will be updated whenever available throughout the life of the study. 
All surgical procedures will be coded as “All other therapeutic products”. 
All concomitant medications and surgical procedures will be presented in data 
listings with an indicator to identify whether their use is prior and /or concomitant. 
Concomitant medications and surgical procedures will be summarized and listed 
for the SFS.  
Descemetorhexis of the Study Eye will be listed in a separated data listing. Partial 
dates on prior and concomitant medications and surgical procedures start date will 
be handled as follows:  


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• Partial start dates of prior and concomitant medications and surgical 
procedures will be assumed to be the earliest possible date consistent with 
the partial date. 
• If the year is present and the month is missing, then the month is set to 
January. 
• If the month and year are present and the day is missing, then the day is set 
to the 1st day of month. 
• If the year is missing, then the year will be assumed to be the year part of 
the patient’s informed consent date. 
Partial or missing medication stop dates for medications that are not ongoing will 
be handled as follows: 
• Partial stop dates of prior and concomitant medications and procedures will 
be assumed to be the latest possible date consistent with the partial date. 
• If the year is present and the month is missing, then the month is set to 
December. 
• If the month and year are present and the day is missing, then the day is set 
to the last day of month. 
• For patients who are treated, if the year is missing and the month or day are 
not missing, then the year will be assumed to be the year part of the patient’s 
last recorded study visit date.   
• For patients who are not treated, if the year is missing and the month or day 
is not missing, then the year will be assumed to be the year part of the 
patient’s discontinuation date. 
• If the complete stop date is missing, the stop date will be considered to be 
either the patient’s last recorded visit date for treated patients or the patient’s 
discontinuation date for non-treated patients. 
7.2 Study Treatments 
7.2.1 Extent of Exposure  
Descriptive statistics including the number of patients, mean, SD, median, Q1, Q3, 
minimum, and maximum for the study treatment exposure and compliance will be 
provided by treatment group using the SFS.  Duration of exposure will be calculated 
in days as the (date of last dose – date of first dose + 1).  
7.2.2 Treatment Compliance and Modifications 
Percent compliance will be calculated as the total number of study drug 
instillations/planned number of instillations × 100.  
To calculate the planned number of instillations, following scenario are considered: 


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• For all study visits during the treatment period, the planed number of 
instillations will be adjusted based on the time all planned study visit 
assessments are completed on the respective study visit days, as activities 
on these days may reduce the number of doses that can be given. Thus, on 
the study visit days, the number of planned instillations will be based on the 
time of the first dose. (E.g. if the first dose of the day is taken in the morning 
then the planned instillation will be considered to be 4 doses; if the first dose 
of the day is taken at noon then the planned instillation will be considered 
to be 3 doses). 
• For all other study days, the number of planned instillations will be 
considered to be 4 doses.  
• If the first and last doses are administered on the same day, the number of 
planned instillations will be based on the time of the first dose.  
• Patients will be considered compliant with study treatment if the calculated 
percent compliance is between 80% and 125%, inclusive. 
A by-patient listing will be presented for the study treatment exposure and 
compliance, as well as drug accountability. 
8. Efficacy Analysis 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy 
analyses will be repeated for the PPS. Unless otherwise specified, efficacy will be 
analyzed and summarized for the study eye only. 
8.1. Primary Efficacy Endpoint 
The primary efficacy endpoint is the central corneal ECD at Week 12 as assessed 
by the 
 
8.1.1. Primary Analysis 
 
The primary analysis of central corneal ECD will be performed on the study eye 
within the FAS via a Wilcoxon rank sum test and will be based on assessments 
performed by specular microscopy. Testing will be performed in a pairwise manner 
with each dosing regimen (QID and BID) of K-321 0.4% compared to placebo.  
A closed 2-step ordered testing procedure will be used to control overall type I error 
for the primary efficacy endpoint, central corneal ECD at Week 12. In step 1, the 
K-321 0.4% QID group will be statistically compared with the placebo group. If 
the step 1 comparison is significant, step 2 will be performed to statistically 
compare K-321 0.4% BID group with the placebo group. If the statistical 
comparison made during step 1 is not significant, testing will still be performed to 












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TEAEs and TESAEs will be summarized separately by ocular (study eye and fellow 
eye) and non-ocular TEAEs/TESAEs. TEAEs and TESAEs will also be 
summarized for the treatment period, the tapering phase and the 9-month follow-
up period: 
• TEAEs/TESAEs during Treatment Period is defined as TEAEs/TESAEs 
with onsite date on or after the first treatment date (Day 1) until the Week 
12 Visit. For patients who miss Week 12 Visit and/or do not complete 
treatment period, the end date for this definition will be the last dose of 
study treatment. 
• TEAEs/TESAEs during Tapering Phase of Follow up Period is defined as 
TEAEs with the onsite date after the Week 12 Visit date until taper dosing 
is ended, which is defined as the Week 12 Visit date +14 days. For patients 
who do not take any Tapering Phase treatment but are followed-up for 
safety, TEAEs/TESAEs during Tapering Phase should be null.     
• TEAEs/TESAEs during 9-month Follow-up Period is defined as 
TEAEs/TESAEs other than two categories defined above. 
TEAEs/TESAEs will be presented in descending order from the system organ class 
(SOC) code with the highest incidence to the SOC with the lowest incidence from 
K-321 QID group. Within the SOC level, TEAEs/TESAEs will be presented in 
descending order from the MedDRA preferred term (PT) with the highest incidence 
to the PT with the lowest incidence from K-321 QID group. If the incidence for any 
two or more PTs within the SOC from K-321 QID group is equal, the PTs will be 
presented descending order of incidence from K-321 QID group. If the incidence 
for any two or more PTs within the SOC from K-321 QID and K-321 BID groups 
are both equal, the PTs will be presented descending order of incidence from 
Placebo group. If the PTs from all three treatment groups within an SOC are equal, 
the PTs will be presented in alphabetical order. Patients who experienced multiple 
TEAEs/TESAEs for a SOC will be counted once, similarly for patients with 
multiple TEAEs/TESAEs per PT will be counted once. Patients who experienced 
multiple TEAEs/TESAEs for a PT will be counted once.  
Partial dates on AE start dates will be handled as follows:    
• If the onset date is completely missing, onset date is set to the date of first 
dose.  
• If the year is present and the month is missing, then the month is set to 
January. If the year is same as the year of the date of first dose and the AE 
end date is not prior to the date of first dose, then onset date is set to the date 
of first dose. 
• If the month and year are present and the day is missing, then the day is set 
to the 1st day of month. If the month and year are same as the month and 


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K-321-201                                                                                                                                         Date Issued: 28JAN2021 
  
 
27 
year of the date of first dose and the AE end date is not prior to the date of 
first dose, then onset date is set to the date of first dose. 
• If the AE end date is present, then the imputed start date will be no later 
than the end date. 
9.1.1. Incidence of Adverse Events 
Both ocular and non-ocular TEAEs will be summarized by treatment group. The 
total number of TEAEs and the number and percentage of patients with at least one 
TEAE in each SOC and having each individual TEAE based on the PT will be 
presented. The number of events under each SOC and PT will also be presented. 
All AEs will be listed.  
9.1.2. Relationship of Adverse Events to Study Drug 
The relationship or association of the study drug in causing or contributing to the 
TEAE will be characterized as related or not related A summary of TEAEs related 
to study drug will be presented in a table by total number of patients with incidence 
of TEAE occurrence. The investigator will provide an assessment of the 
relationship of the event to the study drug. TEAEs that are missing a relationship 
will be presented in the summary table as “Related” but will be presented in the 
data listing with a missing relationship. Percentages will be calculated out of the 
number of patients in the SS. The number of events under each SOC and PT will 
also be presented. 
The TEAE data will be categorized and presented by SOC, PT, and relationship in 
a manner similar to that described in Section 9.1.1. 
9.1.3. Severity of Adverse Event 
A summary of TEAEs by maximum severity will be presented in a table by total 
number of patients with incidence of TEAE occurrence.  The severity that will be 
presented represents the most extreme severity captured on the eCRF page. The 
possible severities are “Mild,” “Moderate,” and “Severe.”  If a patient reported 
multiple occurrences of the same TEAE, only the most severe will be presented in 
the incidence count. TEAEs that are missing severity will be presented in tables as 
“Severe” but will be presented in the data listing with a missing severity. 
Percentages will be calculated out of the number of patients in the SFS.  
The TEAE data will be categorized and presented by SOC, PT, and severity in a 
manner similar to that described in Section 9.1.1. 


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9.1.4. Treatment-Emergent Adverse Events Leading to Treatment 
Discontinuation 
TEAEs leading to treatment discontinuation will be identified as TEAEs on the AE 
eCRF page, where the action taken with study drug is “Drug Withdrawn”.  The 
TEAEs leading to treatment discontinuation will be categorized and presented by 
SOC and PT in a manner similar to that described in Section 9.1.1. All TEAEs 
leading to treatment discontinuation will be listed in a data listing. 
9.1.5 Treatment-Emergent Adverse Events Leading to Study 
Discontinuation 
TEAEs leading to study discontinuation will be identified as TEAEs on the AE 
eCRF page, where the “Caused Study Discontinuation?” is “Yes”.  The TEAEs 
leading to study discontinuation will be categorized and presented by SOC and PT 
in a manner similar to that described in Section 9.1.1. All TEAEs leading to study 
discontinuation will be listed in a data listing. 
9.1.6 Serious Adverse Events 
The TESAE data will be categorized and presented by SOC and PT in a manner 
similar to that described in Section 9.1.1. The TESAE leading to treatment 
discontinuation and study discontinuation will be categorized and presented by 
SOC and PT in a manner similar to that described in Section 9.1.4 and 9.1.5. For 
All TESAE summaries, the number of events under each SOC and PT will also be 
presented. All SAE data will be listed in a data listing. 
9.1.7 Death 
Deaths will be identified as AEs on the AE eCRF page. Patient deaths will be 
identified as AEs where the outcome is “Death”.  Deaths will be categorized and 
presented by SOC and PT in a manner similar to that described in Section 9.1.1. 
All deaths will be listed in a data listing. 
9.2 Clinical Laboratory Evaluations 
Laboratory assessments will be performed by a central laboratory. All summaries 
will be based on the units provided by the central laboratory and no conversion will 
be performed. Lab test names listed in the statistical analysis plan are the same as 
indicated in the protocol; however, the tables and listings will report results using 
the test names based on Clinical Data Interchange Standards Consortium 
(CDISC)-controlled terminologies.  


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9.2.1 Hematology 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All hematology parameters will be summarized using descriptive 
statistics for each treatment group at Screening and EOT (Week 12 or ET). 
Additionally, shift tables providing the number of patients with indicated shifts 
(low, normal, high) in their results from Screening and EOT (Week 12 or ET) will 
also be presented for all hematology parameters.   
All summaries will be done for the SFS and all hematology data will be listed. 
9.2.2 Chemistry 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All chemistry parameters will be summarized using descriptive statistics 
for each treatment group at Screening and EOT (Week 12 or ET). Additionally, 
shift tables providing the number of patients with indicated shifts (low, normal, 
high) in their results from Screening and EOT (Week 12 or ET) will also be 
presented for all chemistry parameters.   
All summaries will be done for the SFS and all chemistry data will be listed. 
9.2.3 Urinalysis 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All urinalysis parameters will be summarized using descriptive statistics 
for each treatment group at Screening and EOT (Week 12 or ET).  
All summaries will be done for the SFS and all urinalysis data will be listed. 
9.3 Vital Sign Measurements 
The vital sign variables including blood pressure (systolic and diastolic), pulse rate, 
body temperature, height, and body weight. Notable abnormal values will be 
flagged in the data listings. The conversion between conventional unit to SI unit for 
temperature and weight and definitions for notable abnormal values are listed in the 
appendix (Table 3 and Table 4). Observed values for vital sign measurements at 
Baseline and EOT (Week 12 or ET)⸷ — as well as changes from Baseline to 
postbaseline values in vital sign measurements — will be summarized descriptively 
by treatment group. 
All summaries will be done for the SFS and all vital sign data will be listed.  
⸷ Week 12 vital sign measurements will only be done in EU (European 
Union) countries according to protocol version 2.0.  


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9.4 Physical Examination 
A limited physical examination will be performed at Screening and EOT (Week 12 
or ET) ⸷. The limited physical examination includes a review of the patient’s 
medical history, medication history, and evaluation of relevant body systems 
complaints. In case of relevant findings, full evaluation of the condition needs to be 
conducted.  Physical examination findings will be listed. 
⸷ Week 12 vital sign measurements will only be done in EU countries 
according to protocol version 2.0.  
9.5 Ophthalmological assessments 
Ophthalmological assessments will be performed at the indicated visits in the SOA 
(Table 5).   
9.5.1 Intraocular Pressure 
Intra-ocular pressure (IOP) may be measured by either contact or non-contact 
tonometry, but the same method must be used for all study visits for a given patient 
throughout the study and all reasonable efforts must be made to have the same 
examiner obtain all IOP measurements for a given patient. At each study visit, a 
single IOP measurement will be made for each eye, with pressure recorded in 
millimetres of mercury (mm Hg).  
Observed values at Baseline and each subsequent study visit, as well as changes 
from Baseline in IOP for each subsequent study visit, will be summarized 
descriptively by treatment group for the study eye and non-study eye. All IOP data 
will be listed. 
9.5.2 Slit-Lamp Biomicroscopy  
Slit-lamp biomicroscopy will be performed at every visit using slit-lamp 
examination without pupil dilation to evaluate the condition of the lids, conjunctiva, 
anterior chamber, cornea, and lens.  
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal with not clinical significance (NCS) 
• Abnormal with clinical significance (CS) 
The number and percentage of patients with at least 1 treatment-emergent CS 
abnormality by ocular assessment will be summarised for each treatment group for 
the study eye and non-study eye. An abnormality will be deemed treatment-


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emergent if the onset date is on or after the date of first treatment. A patient will be 
counted once even if the patient experiences more than 1 treatment-emergent CS 
abnormality.  
All slit-lamp biomicroscopy and ophthalmoscopy assessments will be listed. 
9.5.3 Ophthalmoscopy 
Ocular examination will be performed with pupil dilation using an indirect 
ophthalmoscope according to the current standard of practice to evaluate the 
condition of the vitreous, macula, retina, optic nerve, choroid, and retinal periphery. 
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal (NCS) 
• Abnormal with clinical significance (CS) 
The number and percentage of patients with at least 1 treatment-emergent CS 
abnormality by ocular assessment will be summarised for each treatment group for 
the study eye and non-study eye. An abnormality will be deemed treatment-
emergent if the onset date is on or after the date of first treatment. A patient will be 
counted once even if the patient experiences more than 1 treatment-emergent CS 
abnormality.  
All ophthalmoscopy assessments will be listed. 
10 Interim Analysis 
No interim analyses will be performed. The analysis for primary, secondary, 
exploratory, and safety will be conducted at the Week 12 database lock ie, after all 
patients have completed dose tapering at the Week 16 visit or have been 
discontinued from the study and/or treatment before this visit. Analysis of the 
complete study data (through Week 52) will be performed after all patients have 
completed the Week 52 visits or have been discontinued from the study before the 
Week 52 visit. As the primary efficacy endpoint is at the Week 12 visit, no alpha 
adjustments will be required. No changes to study conduct (eg, change to sample 
size or early stopping) are planned.  
 
 


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11 References 
 
Hodges JL Jr, Lehmann EL. Estimates of location based on rank test. Ann. Math 
Statist. 1963;34(2):598-611. 
 
 
 
Kowa Company Ltd. Ripasudil hydrochloride hydrate ophthalmic solution. 
Investigator’s Brochure, version 00.01. Tokyo (Japan); 2019. 115 p.  
 
 
 
Moloney G, Petsoglou C, Ball M, et al. Descemetorhexis without grafting for 
Fuchs endothelial dystrophy-supplementation with topical ripasudil. Cornea. 
2017;36(6):642-8. 
 
 
 
Moses LE. Query 10: Confidence limits from rank tests. Technometrics. 
1965;7(2):257-60. 
 
 
 


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12 Appendices 
12.1 Unit Conversion 
Table 3 
Unit Conversion 
Quantity 
From SI to Conventional Unit 
From Conventional Unit to SI 
Height 
0.394 x [cm] = [in] 
2.540 [in]. = [cm] 
Weight 
2.205 x [kg] = [lb] 
0.454 x [lb] = [kg] 
Temperature 
([°C] x 9/5) + 32 = [°F] 
([°F] – 32) 5/9 = [°C] 
12.2 Notable Abnormal Vital Signs  
Table 4 
Notable Abnormal Vital Signs 
Parameter 
Abnormality 
Change from Baseline 
Pulse 
<40 bpm 
>120 bpm 
>=15 bpm increase from baseline 
>=15 bpm decrease from baseline 
Systolic blood 
pressure 
<80 mmHg 
>180 mmHg 
>=20 mmHg increase from baseline 
>=20 mmHg decrease from baseline 
Diastolic blood 
pressure 
<50 mmHg 
>105 mmHg 
>=15 mmHg increase from baseline 
>=15 mmHg decrease from baseline 
Temperature 
NA 
>= 1 C increase from baseline 
>=1 C decrease from baseline 


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12.3 Schedule of Study Procedures 
Table 5 
Schedule of Study Site Activities 
 Activity 
Screening 
Treatment Period 
Follow-Up Period 
Time Point Day -28 
Day –1 
Day 1 
Week 1 Week2 Week 3 Week 5 Week 7 Week 9 Week 12 Week 16 Week 20 Week 24 Week 38 Week 52 
Visit Window (Day) -28 to -7 
- 
- 
5-9 
12-16 
18-24 
32-38 
46-52 
60-66 
81-87 
105-119 133-147 161-175 259-273 357-371 
Visit No  Visit 1 
Visit 2 
Visit 3 
Visit 4 Optional 
Visit 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and 
concomitant disease 
X 
X a 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination and 
vital sign measurements 
X 
 
 
 
 
 
 
 
 
Xh 
 
 
 
 
 
Urine drug screen 
X 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Urine pregnancy test (if 
applicable) 
 
X a 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Laboratory sampling  
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Eligibility evaluation 
X 
X b 
 
 
 
 
 
 
 
 
 
 
 
 
 
Descemetorhexis with photo 
or video documentation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense study drug 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 study drug c 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Study drug administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Slit-lamp evaluation 
X 
X a 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 




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 36 
Abbreviation: HRT, Heidelberg retina tomography; 
 
 
a 
Pre-operative. 
b 
Post-operative. 
c 
Both 
 study drug 
 will 
 and 
 will be recorded in the eCRF. Morning/night and mid-
day/evening drug will 
 separately. 
d 
Week 2 is an optional visit (at the discretion of the investigator), but if this visit is applied, all marked assessment items must be conducted. 
e 
The study eye must be measured at all indicated visits, whereas the non-study eye must be measured only at Visit 1 and Visit 14.  
f 
Only at sites where equipment is available. 
g 
If investigator judges it necessary.  
h 
Assessment only performed in EU countries 
i 
This will be done according to current standard of care to evaluate the condition of the vitreous, macula, retina, optic nerve, choroid, and 
retinal periphery. 
 

SAP_004.pdf: 
 
 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-
Group, 12 Week, Phase 2 Study to Investigate the Safety and 
Efficacy of Ripasudil (K-321) Eye Drops After Descemetorhexis in 
Patients with Fuchs Endothelial Corneal Dystrophy 
 
NCT04250207 
SAP Ver.: 2.0 Addendum 
Document Date: 08 Jun 2022 




Kowa Research                                                                                             Statistical Analysis Plan, Version 2.0 Addendum   
K-321-201                                                                                                                                      Date Issued: 08JUN2022   
  
 
 
 
II
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ....................................................................................................................... III 
1. 
INTRODUCTION ............................................................................................................................ 4 
2. 
ADDITIONAL ANALYSES ............................................................................................................ 4 
3. 
APPENDIX 1 – TLF SHELLS ........................................................................................................ 7 
TABLE 14.2.1.42 TIME TO ACHIEVE INTERVENTION OF RESCUE THERAPY  (FULL 
ANALYSIS SET) ............................................................................................................................. 8 
TABLE 14.2.1.45 PROPORTION OF PATIENTS WHO ACHIEVE NO CORNEAL OEDEMA OF 
STUDY EYE BY VISIT (LOCF) (FULL ANALYSIS SET) ............................................... 12 
TABLE 14.2.1.48 SUMMARY OF BCVA BY ETDRS BY CENTRAL ECD VALUE BY VISIT  
(FULL ANALYSIS SET) ............................................................................................................ 16 
TABLE 14.2.1.49 C
TION BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL ECD 
(INCLUDE 
 DATA) (PEARSON AND SPEARMAN) (FULL ANALYSIS SET)
 .......................
.................................................................................................................. 18 
TABLE 14.2.1.50 CO
TION BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL ECD 
(INCLUDE 
 DATA) (NO CORNEAL OEDEMA) (PEARSON AND SPEARMAN) 
(FULL ANAL
ET) ............................................................................................................ 19 
TABLE 14.2.1.52 C
TION BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL ECD 
(EXCLUDE 
 DATA) (NO CORNEAL OEDEMA) (PEARSON AND SPEARMAN) 
(FULL ANAL
ET) ............................................................................................................ 21 
FIGURE 14.2.1.42 TIME TO ACHIEVE INTERVENTION OF RESCUE THERAPY (KAPLAN-
MEIER PLOT) (FULL ANALYSIS SET) ................................................................................ 22 
FIGURE 14.2.1.43 CENTRAL CORNEAL ECD (CELLS/MM2) OF STUDY EYE PERFORMED 
BY SPECULAR MICROSCOPY (LOCF) (TIME SERIES PLOT) (FULL ANALYSIS 
SET) ................................................................................................................................................ 24 
FIGURE 14.2.1.45 PROPORTION OF PATIENTS WHO ACHIEVE NO CORNEAL OEDEMA OF 
STUDY EYE (LOCF) (BAR CHART) (FULL ANALYSIS SET) ........................................ 27 
FIGURE 14.2.1.48 BCVA BY ETDRS BY CENTRAL ECD VALUE BY VISIT (SCATTER 
PLOT) (FULL ANALYSIS SET) .............................................................................................. 31 
FIGURE 14.2.1.49 CO
ON BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL 
ECD (INCLUDE 
 DATA) (PEARSON AND SPEARMAN) (FULL ANALYSIS 
SET) ........................
........................................................................................................ 33 
FIGURE 14.2.1.50 CORRE
BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL 
ECD (INCLUDE 
 DATA) (NO CORNEAL OEDEMA) (PEARSON AND 
SPEARMAN) (FULL A
S SET) ..................................................................................... 34 
FIGURE 14.2.1.52 CORRE
BETWEEN BCVA BY ETDRS AND CENTRAL CORNEAL 
ECD (EXCLUDE 
 DATA) (NO CORNEAL OEDEMA) (PEARSON AND 
SPEARMAN) (FULL A
S SET) ..................................................................................... 36 
  
 


Kowa Research                                                                                             Statistical Analysis Plan, Version 2.0 Addendum   
K-321-201                                                                                                                                      Date Issued: 08JUN2022   
  
  
 
III 
List of Abbreviations 
 
Abbreviation 
Definition 
BCVA 
Best Corrected Visual Acuity 
BID 
Twice Daily 
CI 
Confidence Interval 
CSR 
Clinical Study Report 
ECD 
Endothelial Cell Density 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FAS 
Full-Analysis Set 
LOCF 
Last-Observation-Carried-Forward 
QID 
4 Times Daily 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 








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3. Appendix 1 – TLF shells




















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<DIRECTORY PATH>PROGRAM NAME  Executed: DDMMMYYYY hh:mm  
Prog Notes: Continue with ‘Central ECD Value > Mean’ and 
. 
 
 












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DIRECTORY PATH>PROGRAM NAME  Executed: DDMMMYYYY hh:mm 
Programming note: Please show x-axis as Day 1 to Day 371. Please display y-axis label as ‘Probability 
of Achieving Intervention of Rescue Keratoplasty (%)’. 
Please display all treatment groups: K-321 QID, K-321 BID, Placebo 
 
 




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K-321-201 
                                                                                                                                                                         Date Issued: 08JUN2022   
  
 
 
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DIRECTORY PATH>PROGRAM NAME  Executed: DDMMMYYYY hh:mm 
 
Programming note: Please show x-axis according to the analysis visit. Please display y-axis label as 
‘Mean(+/-SD) Central Corneal ECD (cells/mm2) by Specular Microscopy’. 
Please display all treatment groups: K-321 QID, K-321 BID, Placebo 
Below the figure please include the N counts at each visit. 
Please include N counts for each treatment arm at the annotation. 
 
 
 














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Source Data: Table 14.2.1.48 
DIRECTORY PATH>PROGRAM NAME  Executed: DDMMMYYYY hh:mm  
Programming note: Please show x-axis according to the subgroups ‘1 < Central ECD Value <= Mean’, ‘Central 
ECD Value > Mean’ ‘and 
. Please display y-axis label as ‘BCVA by ETDRS Letter Score’. 
Please display ‘All Post-Baseline Visits’ in the first page, then start the second page with 
‘Baseline’, ‘Week X’… Each visit should be on a separate page.  
In each page please display all treatment groups: Total, K-321 QID, K-321 BID, Placebo 
In each page please include number of subjects under each treatment arm and subgroups under the plot. 
Please display the spearman correlation and p-value under the figure for each treatment group and 
total. 
 
 









SAP_003.pdf: 
 
 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-
Group, 12 Week, Phase 2 Study to Investigate the Safety and 
Efficacy of Ripasudil (K-321) Eye Drops After Descemetorhexis in 
Patients with Fuchs Endothelial Corneal Dystrophy 
 
NCT04250207 
SAP Ver.: 2.0 
Document Date: 25 Aug 2021 




Kowa Research Institute, Inc.                                                                                           Statistical Analysis Plan, Version 2.0 
K-321-201 
 
Date Issued: 25AUG2021 
 
  
 
 
 
II
TABLE OF CONTENTS 
MODIFICATION HISTORY ............................................................................ IV 
LIST OF ABBREVIATIONS .............................................................................. V 
1. 
INTRODUCTION......................................................................................7 
2. 
OBJECTIVES ............................................................................................8 
2.1. PRIMARY OBJECTIVE .................................................................................. 8 
2.2. SECONDARY OBJECTIVE ............................................................................. 8 
2.3. EXPLORATORY OBJECTIVE ......................................................................... 8 
3. 
INVESTIGATIONAL PLAN ...................................................................8 
3.1. OVERALL STUDY DESIGN AND PLAN .......................................................... 8 
3.2. STUDY ENDPOINTS ..................................................................................... 9 
3.2.1. 
Primary Efficacy Endpoint ................................................................. 9 
3.2.2 
Secondary Efficacy Endpoint .............................................................. 9 
3.2.3 
Exploratory Endpoints ...................................................................... 10 
3.2.4 
Safety Endpoints................................................................................ 11 
3.3. STUDY PROCEDURES AND BASELINE ........................................................ 11 
3.4. TREATMENTS ............................................................................................ 12 
4. 
GENERAL STATISTICAL CONSIDERATIONS ..............................13 
4.1. SAMPLE SIZE ............................................................................................ 13 
4.2. RANDOMIZATION, STRATIFICATION AND MASKING .................................. 14 
4.3. ANALYSIS SET .......................................................................................... 14 
4.3.1 
Full-analysis set (FAS) ..................................................................... 14 
4.3.2 
Per-protocol set (PPS) ...................................................................... 14 
4.3.3 
Safety Set (SFS) ................................................................................. 15 
4.4 
ASSESSMENT WINDOWS ........................................................................... 15 
4.4.1 
Study day ........................................................................................... 15 
4.4.2 
Visit Windows for Analysis ............................................................... 15 
5. 
PATIENT DISPOSITION.......................................................................16 
5.1. DISPOSITION ............................................................................................. 16 
5.2. PROTOCOL DEVIATIONS ........................................................................... 16 
6. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ...........17 
6.1 
DEMOGRAPHICS ........................................................................................ 17 
6.2 
BASELINE OCULAR CHARACTERISTICS ..................................................... 18 
6.3 
MEDICAL HISTORY ................................................................................... 18 
6.3.1 
General Medical History .................................................................. 18 
6.4 
INCLUSION AND EXCLUSION CRITERIA ..................................................... 19 
7. 
TREATMENTS AND MEDICATIONS ................................................19 
7.1 
PRIOR AND CONCOMITANT THERAPIES: MEDICATIONS AND SURGICAL 
PROCEDURES ...................................................................................................... 19 
7.2 
STUDY TREATMENTS ................................................................................ 20 


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7.2.1 
Extent of Exposure ............................................................................ 20 
7.2.2 
Treatment Compliance and Modifications........................................ 20 
8. 
EFFICACY ANALYSIS ..........................................................................21 
8.1. PRIMARY EFFICACY ENDPOINT ................................................................. 21 
8.1.1. 
Primary Analysis ............................................................................... 21 
8.1.2. 
Sensitivity Analysis............................................................................ 23 
8.1.3 
Exploratory Analysis ......................................................................... 23 
8.2 
SECONDARY EFFICACY ENDPOINT ............................................................ 25 
8.3 
EXPLORATORY EFFICACY ENDPOINTS ...................................................... 26 
8.4 
EFFICACY ENDPOINTS FOR THE NON-STUDY EYE ..................................... 27 
9 
SAFETY ANALYSIS...............................................................................27 
9.1 
ADVERSE EVENTS ..................................................................................... 28 
9.1.3 
Incidence of Adverse Events ............................................................. 29 
9.1.4 
Relationship of Adverse Events to Study Drug ................................. 29 
9.1.5 
Severity of Adverse Event.................................................................. 30 
9.1.6 
Treatment-Emergent Adverse Events Leading to Treatment 
Discontinuation .............................................................................................. 30 
9.1.5 
Treatment-Emergent Adverse Events Leading to Study 
Discontinuation .............................................................................................. 30 
9.1.6 
Serious Adverse Events ..................................................................... 30 
9.1.7 
Death ................................................................................................. 31 
9.2 
CLINICAL LABORATORY EVALUATIONS ................................................... 31 
9.2.1 
Hematology ....................................................................................... 31 
9.2.2 
Chemistry .......................................................................................... 31 
9.2.3 
Urinalysis .......................................................................................... 31 
9.3 
VITAL SIGN MEASUREMENTS ................................................................... 32 
9.4 
PHYSICAL EXAMINATION ......................................................................... 32 
9.5 
OPHTHALMOLOGICAL ASSESSMENTS ........................................................ 32 
9.5.1 
Intraocular Pressure ......................................................................... 32 
9.5.2 
Slit-Lamp Biomicroscopy .................................................................. 33 
9.5.3 
Ophthalmoscopy ............................................................................... 33 
10 
INTERIM ANALYSIS ............................................................................34 
11 
REFERENCES .........................................................................................35 
12 
APPENDICES ..........................................................................................36 
12.1 
UNIT CONVERSION ................................................................................ 36 
12.2 
NOTABLE ABNORMAL VITAL SIGNS ...................................................... 36 
12.3 
SCHEDULE OF STUDY PROCEDURES ...................................................... 37 




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List of Abbreviations 
Abbreviation 
Definition 
AE 
Adverse Event 
ANOVA 
Analysis of Variance 
BCVA 
Best Corrected Visual Acuity 
BID 
Twice Daily 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
 
 
CTMS 
Clinical Trial Management System  
CRO 
Contract Research Organisation 
CS 
Clinically Significant 
CV 
Curriculum Vitae 
ECD 
Endothelial Cell Density 
eCRF 
electronic Case Report Form 
EOS 
End-Of-Study 
EOT 
End-Of-Treatment 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
EU 
European Union 
FAS 
Full-Analysis Set 
FDA 
US Food and Drug Administration 
FECD 
Fuchs endothelial corneal dystrophy 
HRT 
Heidelberg retina tomography 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IOP 
Intra-Ocular Pressure 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
LOCF 
Last-Observation-Carried-Forward 
M/N 
Morning and Night 
MD/E 
Mid-Day and Evening 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHRA 
Medicines and Healthcare Products Regulatory Agency 
NCS 
Not Clinically Significant 
PPS 
Per Protocol Set 


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Abbreviation 
Definition 
Q1 
Quartile 1 
Q3 
Quartile 3 
QID 
4 Times Daily 
ROCK 
Rho-associated Protein Kinase 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SFS 
Safety Set 
SOA 
Schedule Of study site Activities 
TEAE 
Treatment-Emergent Adverse Event 
TESAE 
Treatment-Emergent Serious Adverse Event 
 
 






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immediately following the EOT visit, each enrolled patient will taper dosing of 
study drug to zero (Section 3.4).  
The second period is a follow-up observation period of 40 weeks, including the 
tapering phase and containing 4 interim visits and an end-of-study (EOS) visit. 
Patients are not to self-administer study drug after approximately Week 14. The 
maximum total study period for each enrolled patient is 56 weeks, with a maximum 
of 15 visits (including the 1 optional visit). Patients will be considered to have 
completed the study with the completion of their EOS visit (scheduled for Week 
52). The duration of the study is defined for each patient as the date signed written 
informed consent is provided through the last follow-up visit. 
 
3.2. Study Endpoints 
3.2.1. Primary Efficacy Endpoint 
The primary efficacy endpoint is the central corneal ECD at Week 12 as assessed 
by the Central 
 
3.2.2 Secondary Efficacy Endpoint 
Secondary efficacy endpoints include: 




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3.2.4 Safety Endpoints 
Safety parameters include monitoring of Adverse Events (AEs), treatment 
emergent adverse events (TEAEs), treatment emergent serious adverse events 
(TESAEs), physical examination findings, vital sign measurements, laboratory 
tests, slit-lamp biomicroscopy, measurement of intraocular pressure (IOP), and 
ophthalmoscopy. 
Safety data will be summarized and listed with no statistical hypothesis testing 
performed. Continuous variables will be summarized using the number, mean, the 
standard deviation (SD), median, minimum value, and maximum value. 
Categorical variables will be summarized using frequency counts and percentages. 
3.3. Study Procedures and Baseline 
Before performing any study procedures, all potential patients will sign an informed 
consent form (ICF). Patients will have the opportunity to have any questions 
answered before signing the ICF. The investigator must address all questions raised 
by the patient. The investigator or designee will also sign the ICF.  
After obtaining informed consent at Visit 1, the investigator will select one eye as 
the study eye, and the patient will come back for the descemetorhexis operation and 
random assignment to treatment after 1 to 4 weeks (Visit 2). At Visit 2, each 
patient’s eligibility for study participation will be confirmed, including a successful 
descemetorhexis operation that meets the inclusion criteria. After eligibility is 
confirmed, patients will be randomly assigned to the 3 treatment groups (in a 1:1:1 
ratio). All patients will be instructed to apply the study drug only to the study eye.  
Day 1(Visit 3) is considered to be the day when first dose of study drug is 
administered. Randomization is scheduled to be performed on Day -1 (Visit 2).  
Baseline is defined as the last non-missing measurement including unscheduled 
assessments prior to the administration of first dose of study drug. Change from 
baseline is defined as the post baseline value minus the baseline value for the given 
assessment. 
The EOT (Week 12 or ET [Early Termination]) visit is defined as the visit occurring 
at the end of the 12-week treatment period (Week 12) or the final visit at which the 
patient is prematurely discontinued/withdrawn from study drug administration.  
The EOS (Week 52 or ET) visit is defined as the visit occurring at the end of the 
52-week study period (Week 52) or the final visit at which the patient is 
prematurely discontinued/withdrawn from participation in the study. 
The schedule of study site activities (SOA) is presented in Section 13 (Table 5). 


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3.4. Treatments   
There are 3 treatment groups: K-321 ophthalmic solution 0.4% dosed QID (K-321 
QID), K-321 ophthalmic solution 0.4% dosed BID (K-321 BID); and placebo. To 
preserve masking, all patients will self-administer assigned study drug QID and 
will dose from 
 of assigned 
 one to be used M/N (morning and night) 
and the other to be used MD/E (mid-day and evening), as presented in Table 1.   
Table 1 Vial Contents by Dosing Regimen and Dosing Time for Each 
Treatment Group From Day 1 to Visit 9 (Week 12) 
Treatment  
Morning 
Mid-day 
Evening 
Night 
K-321 QID 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 BID 
K-321 0.4% 
Placebo 
Placebo 
K-321 0.4% 
Placebo 
Placebo 
Placebo 
Placebo 
Placebo 
Except on days of study visits on Day 1 through Week 12 (not including Visit 9) 
during the treatment period, study drug will be dosed by the patient QID: morning 
(9 AM ±1 hour), mid-day (1 PM ±1 hour), evening (5 PM ±1 hour), and night 
(9 PM ±1 hour). Each patient will be provided with 
 of colour-coded 
 
, one set to be applied M/N and the other to be applied MD/E.  
On study visit days Visit 3 through Visit 9, patients will not apply any study drug 
until after all study measurements have been completed; thereafter on those days, 
patients will start dosing with the dose closest in timing to the dosing regimen and 
continue with the remaining doses for the day. Additionally, on Visit 3 they must 
first apply study drug within 
 after the end of the descemetorhexis 
procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient 
applies any study drug on the day of Visit 9 before all study measurements have 
been completed, that patient’s Visit 9 should be rescheduled within the visit 
window allowance (eg, the next day). For visits other than Visit 9 during the 
treatment period, a patient who applies study drug before all study measurements 
have been completed will be assessed as planned, and a protocol deviation must be 
recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up 
period, and new study drug will be dispensed without M/N or MD/E colour coding. 
All patients will self-administer their tapering-phase study drug BID (9 AM 
±1 hour and 9 PM ± 1 hour) for a week, then once daily (9 AM ± 1 hour) for a week, 
and then will discontinue study drug.  




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4.2. Randomization, Stratification and Masking 
Patients will be randomly assigned at Visit 2 to either placebo, K-321 BID, or K-
321 QID treatment (Section 3.1) using a 1:1:1 allocation ratio. There is no 
stratification on the randomization for this study. 
Study drug will be double-masked. All study drug will be supplied in identical 
packaging, colour, smell, and appearance to enable double-masked conditions. K-
321 and matching placebo will be provided in identical packaging so that all 
investigators, study site staff, patients, and clinical monitors will remain masked 
throughout the study. The Interactive Web Response System (IWRS) will assign 
study drug to patients at the time of randomisation. Only personnel in IWRS, the 
independent randomisation team within 
 Biostatistics, and clinical supplies will 
be unmasked and will have access to treatment assignments; all other parties 
involved in the study will be fully masked. 
A patient’s treatment assignment will not be unmasked for the investigator or study 
site staff until the EOS unless medical treatment of the patient depends on knowing 
the study treatment the patient received. In the rare event that unmasking is needed 
because of a medical emergency, the investigator may unmask an individual patient 
through the IWRS. Reasons for treatment unmasking must be clearly explained and 
justified in the electronic case report form (eCRF). The date on which the code was 
broken together with the identity of the person responsible must also be 
documented. 
Patients who are unmasked will be allowed to continue their participation in the 
study; however, their data may be excluded from per-protocol analyses. 
4.3. Analysis Set 
4.3.1 Full-analysis set (FAS) 
The FAS will consist of all participants who have a successful descemetorhexis 
procedure performed, were randomly assigned to receive double-mask study drug, 
received at least one dose of study drug in the study eye, and have at least one 
assessment of central corneal ECD performed after the descemetorhexis procedure 
on the study eye. (An assessment of “cannot perform due to corneal oedema” is 
considered a valid assessment for inclusion in the FAS.) All analyses using the FAS 
will group participants according to randomised treatment.  
4.3.2 Per-protocol set (PPS) 
The PPS will consist of all FAS participants who complete the 12-week dosing 
treatment period for the study eye (for the primary analysis only), have at least 80% 
compliance and at most 125% compliance with study treatment for the study eye, 


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have not taken any prohibited medication and have no major protocol deviations. 
Patients who receive rescue therapy will be considered to have completed study 
treatment for inclusion in the PPS. All analyses using the PPS will group 
participants according to treatment actually received. 
4.3.3 Safety Set (SFS) 
The SFS will consist of all participants who received any study drug. All analyses 
using the safety set will group participants according to treatment actually received. 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy 
analyses will be repeated for the PPS. Safety will be summarised using the SFS.  
4.4 Assessment Windows 
4.4.1 Study day 
When study day is used for display or in comparisons the following algorithm will 
be used: 
• study day = date of assessment - Day 1 +1, if date of assessment ≥ Day 1. 
• study day = date of assessment - Day 1, if date of assessment < Day 1. 
Note that the day before Day 1 is Day -1 (for analysis, there is no Day 0 for study 
day).  
4.4.2 Visit Windows for Analysis 
Visit windows will be defined for by-visit summary and analysis purposes. 
Summary data (such as AEs and concomitant medications) that are not reported by 
visit will not use visit windows. The EOT or EOS assessments will be summarized 
based on visit window of when the visit occurred (e.g., if the patient discontinued 
treatment at Week 7, the EOT assessment will be included in the Week 7 by-visit 
summaries).   
Both scheduled and unscheduled assessments will be considered as valid 
assessments for analysis.  Visit labels will be assigned to each post baseline record 
based on the windows for study day relative to the date of first dose. If an 
assessment at Day 1 is missing, the closest visit with non-missing assessment on or 
before the date of first dose will be used as baseline.  All analyses for FAS and PPS 
be based on the analysis visit windows in Table 2. If there are multiple valid 
assessments within a time window, the assessment which occurs closest to the 
target day will be used in the analysis. If there are multiple valid assessments with 
same difference from the target day, the latest assessment will be used in the 
analysis.    


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Table 2 
Analysis Visit Windows 
Analysis Visit 
Analysis Visit Window  
Screening/Washout 
NA 
Baseline/Day 1  
NA 
Week 1 
2 to 10 Days (Target 7) 
Week 2 
11 to 17 Days (Target 14) 
Week 3 
18 to 27 Days (Target 21) 
Week 5 
28 to 41 Days (Target 35) 
Week 7  
42 to 55 Days (Target 49) 
Week 9 
56 to 73 Days (Target 63) 
Week 12 
74 to 97 Days (Target 84) 
Week 16 
98to 125 Days (Target 112) 
Week 20 
126 to 153 Days (Target 140) 
Week 24 
154to 216 Days (Target 168) 
Week 38 
217 to 314 Days (Target 266) 
Week 52  
315 to 413 Days (Target 364) 
5. Patient Disposition 
5.1. Disposition 
The counts of patients who screened and screen failed will be presented. The counts 
and percentages of patients who are randomized, receive study treatment, 
completed treatment period (12 week) as well as 12 week visit, completed treatment 
period (12 week) as well as 52 week visit, discontinued during treatment period (12 
week) but completed 12 week visit, discontinued during treatment period (12 week) 
but completed 52 week visit. Withdrawn from the study will be presented based on 
the number of patients in each treatment group and overall for all randomized 
patients. Reasons for discontinuation during treatment period and reasons for 
withdrawn from the study will be summarized for each treatment group as treatment 
disposition and patient disposition, respectively. All percentages within the patient 
disposition summary will be based on the number of patients randomized. All 
percentages within the treatment disposition summary will be based on number of 
patients from SFS. Patient and treatment disposition data will be listed. Screen 
failures will also be listed in the data listing. 
5.2. Protocol Deviations 
Protocol deviations will be tracked by the clinical team on an ongoing basis.  Major 
protocol deviations are the subset of deviations which are considered to have 
potential significant impact on primary analysis. Subjects with major protocol 
deviations will be excluded from the analysis based on PPS. Specific criteria for 


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what constitute a major protocol deviation will be determined by the clinical team 
and the following criteria may be considered: 
• Eligibility criterion violation 
• Not complete the 12-week dosing treatment period for the study eye (for the 
primary analysis only) 
• Less than 80% or more than 125% compliance with study treatment in the 
study eye 
• Taking prohibited medication or treatment listed in protocol section 5.8.3 
Protocol deviations will be recorded within the 
 Clinical Trial Management 
System (CTMS) and will undergo a blinded review prior to database lock and 
unblinding. Significant protocol deviations are defined as the subset of deviations 
which are considered to affect primary efficacy and safety assessments, the safety 
or mental integrity of a subject, or the scientific value of the trial. 
Patients with significant protocol deviations will be tabulated for each treatment 
group, and overall. All deviations, including significant and major protocol 
deviations, will be listed by-patient for all randomized patients. All percentages will 
be based on the number of patients randomized. The total number of significant 
protocol deviations and the number of significant protocol deviations under each 
protocol deviation category will also be presented 
6. Demographics and Baseline Characteristics 
6.1 Demographics 
Demographic information and baseline characteristics collected at Screening will 
be summarized for the FAS and SFS. Continuous variables, including age (years), 
baseline weight (kg), baseline height (cm), and baseline body mass index (BMI) 
(kg/m2) will be summarized using descriptive statistics for each treatment group 
and overall. Continuous variables will be tested with ANOVA. The following 
categorical variables will be summarized by reporting the number and percentage 
of patients in each category for each treatment group and overall. Fisher’s exact test 
will be used for the following categorical variables to test whether there is a 
difference between each treatment group.  
• Sex (Male, Female) 
• Ethnicity (Hispanic or Latino, Not Hispanic or Latino) 
• Race* (White, Black or African American, Asian, American Indian or 
Alaska Native, Native Hawaiian or Other Pacific Islander, Multiple and 
Other) 
• Iris color of study eye (Blue, Brown, Hazel, Green, Grey, Other)  


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• Family History of FECD† (Yes, No) 
o If Yes, Father, Mother, or Both    
*The statistical comparison between the Race categories will not be presented 
because the Race categories are not mutually exclusive. i.e. One subject could be 
marked in multiple race categories. 
†The statistical comparison on Family History of FECD will be between the 
categories of ‘Yes’ and ‘No’.  
Percentages will be based on the total number of patients in the SFS, FAS and PPS. 
All demographic and baseline characteristics will be listed for all randomized 
patients.  
6.2  Baseline Ocular Characteristics 
Descriptive statistics will be provided for patient ocular baseline characteristics on 
study eye and non-study eye by treatment group and overall for ophthalmological 
assessments including Corneal ECD Measurement by specular microscopy 
(including Corneal Morphology Parameters), Corneal ECD Measurement by 
Heidelberg Retina Tomography (HRT) (including Corneal Morphology 
Parameters), BCVA score, Corneal Thickness, presence/absence of Corneal 
Oedema categories of epithelial, stromal, or endothelial, Contrast Sensitivity, 
 domain and total scores, IOP, longest and shortest diameter of descemetorhexis. 
Tests for baseline disease characteristics of study eye will be performed in a 
pairwise manner with each dosing regimen (QID and BID) of K-321 0.4% 
compared to placebo. Numerical results will be tested with ANOVA; Categorical 
results will be tested with Fisher’s Exact Test. Summary and tests will be based on 
the FAS and SFS on study eye, non-study eye and the 
 Score where 
applicable. 
6.3 Medical History 
6.3.1 General Medical History 
Ocular and non-ocular medical histories are captured separately on the eCRF.  For 
non-ocular medical history, the investigator will record the body system, verbatim 
term, start date, and stop date or indication of ongoing.  For ocular medical history, 
the investigator will record the eye (right, left, both), verbatim term, start date, and 
stop date or indication of ongoing. Medical history will be coded using the Medical 
Dictionary for Regulatory Activities (MedDRA) Version 21.1or above.  The 
number and percentage of patients with any medical history (ocular and non-ocular), 
any ocular medical history in the study eye, any ocular medical history in the non-
study eye, an any non-ocular medical history will be summarized by treatment 


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group.  Additionally, non-ocular medical history will be summarized by the number 
and percentage of patients with any non-ocular medical history by reported body 
system and treatment group on SFS.   
Percentages will be calculated based on number of patients in the SFS. Also, by-
patient listings of ocular and non-ocular medical histories will be presented. 
6.4 Inclusion and Exclusion Criteria 
Inclusion and exclusion criteria deviations noted in the eCRF will be presented for 
randomized patients in a data listing. 
7. Treatments and Medications 
7.1 Prior and Concomitant Therapies: Medications and Surgical 
Procedures 
At screening, the following prior therapies will be recorded in the eCRF: 
• History of prior study eye surgical procedures 
• All medications used within the previous 30 days 
• All surgical procedures within the previous 30 days (any body part) 
At screening, all patients’ prior medications used and surgical procedures 
undergone within the previous 30 days will be recorded in the eCRF.  
Any medicinal product prescribed or over-the-counter (OTC), including herbal 
products, vitamins, and minerals is considered a prior or concomitant medication. 
All patients’ surgical procedures undergone concomitantly during the study must 
be reported and recorded in the eCRF. Prior medication is defined as any 
medication with the stop date prior to the date of the first dose of study drug. 
Concomitant medication is defined as any medication with a stop date after the date 
of the first dose of study drug. Any medications used during the study will be coded 
with the World Health Organization Drug (WHODrug) Dictionary dated March 
2019., which will be updated whenever available throughout the life of the study. 
All surgical procedures will be coded as “All other therapeutic products”. 
All concomitant medications and surgical procedures will be presented in data 
listings with an indicator to identify whether their use is prior and /or concomitant. 
Concomitant medications and surgical procedures will be summarized and listed 
for the SFS.  
Descemetorhexis of the Study Eye will be listed in a separated data listing. Partial 
dates on prior and concomitant medications and surgical procedures start date will 
be handled as follows:  


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• Partial start dates of prior and concomitant medications and surgical 
procedures will be assumed to be the earliest possible date consistent with 
the partial date. 
• If the year is present and the month is missing, then the month is set to 
January. 
• If the month and year are present and the day is missing, then the day is set 
to the 1st day of month. 
• If the year is missing, then the year will be assumed to be the year part of 
the patient’s informed consent date. 
Partial or missing medication stop dates for medications that are not ongoing will 
be handled as follows: 
• Partial stop dates of prior and concomitant medications and procedures will 
be assumed to be the latest possible date consistent with the partial date. 
• If the year is present and the month is missing, then the month is set to 
December. 
• If the month and year are present and the day is missing, then the day is set 
to the last day of month. 
• For patients who are treated, if the year is missing and the month or day are 
not missing, then the year will be assumed to be the year part of the patient’s 
last recorded study visit date.   
• For patients who are not treated, if the year is missing and the month or day 
is not missing, then the year will be assumed to be the year part of the 
patient’s discontinuation date. 
• If the complete stop date is missing, the stop date will be considered to be 
either the patient’s last recorded visit date for treated patients or the patient’s 
discontinuation date for non-treated patients. 
7.2 Study Treatments 
7.2.1 Extent of Exposure  
Descriptive statistics including the number of patients, mean, SD, median, Q1, Q3, 
minimum, and maximum for the study treatment exposure and compliance will be 
provided by treatment group using the SFS.  Duration of exposure will be calculated 
in days as the (date of last dose – date of first dose + 1).  
7.2.2 Treatment Compliance and Modifications 
Percent compliance will be calculated as the total number of study drug 
instillations/planned number of instillations × 100.  
To calculate the planned number of instillations, following scenario are considered: 


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• For all study visits during the treatment period, the planed number of 
instillations will be adjusted based on the time all planned study visit 
assessments are completed on the respective study visit days, as activities 
on these days may reduce the number of doses that can be given. Thus, on 
the study visit days, the number of planned instillations will be based on the 
time of the first dose. (E.g. if the first dose of the day is taken in the morning 
then the planned instillation will be considered to be 4 doses; if the first dose 
of the day is taken at noon then the planned instillation will be considered 
to be 3 doses). 
• For all other study days, the number of planned instillations will be 
considered to be 4 doses.  
• If the first and last doses are administered on the same day, the number of 
planned instillations will be based on the time of the first dose.  
• Patients will be considered compliant with study treatment if the calculated 
percent compliance is between 80% and 125%, inclusive. 
A by-patient listing will be presented for the study treatment exposure and 
compliance, as well as drug accountability. 
8. Efficacy Analysis 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy 
analyses will be repeated for the PPS. Unless otherwise specified, efficacy will be 
analysed and summarized for the study eye only. 
8.1. Primary Efficacy Endpoint 
The primary efficacy endpoint is the central corneal ECD at Week 12 as assessed 
by the 
 
8.1.1. Primary Analysis 
 
The primary analysis of central corneal ECD will be performed on the study eye 
within the FAS via a Wilcoxon rank sum test and will be based on assessments 
performed by specular microscopy. Testing will be performed in a pairwise manner 
with each dosing regimen (QID and BID) of K-321 0.4% compared to placebo. The 
ranks are based on the observations of the two groups that are compared. P values 
from the Wilcoxon rank sum test will be presented. 
A closed 2-step ordered testing procedure will be used to control overall type I error 
for the primary efficacy endpoint, central corneal ECD at Week 12. In step 1, the 
K-321 0.4% QID group will be statistically compared with the placebo group. If 
the step 1 comparison is significant, step 2 will be performed to statistically 
compare K-321 0.4% BID group with the placebo group. If the statistical 














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median, Q1, Q3, minimum value, and maximum value. Categorical variables will 
be summarised using frequency counts and percentages. 
9.1 Adverse Events  
AEs will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA) which will be updated whenever available throughout the life of the 
study.  
In general, all AEs will be listed and all TEAEs and TESAEs will be summarized. 
AE will be deemed treatment-emergent if the onset date is on or after the date of 
first treatment.  
An overall summary of TEAEs and TESAEs will be provided, with number 
(percentage) of patients and number of events for all TEAEs/TESAEs by the 
following categories: any TEAEs, any TESAEs, Study Drug-Related 
TEAEs/TESAEs, TEAEs/TESAEs by Severity, TEAEs/TESAEs leading to 
treatment discontinuation, TEAEs/TESAEs leading to study discontinuation, 
TEAEs leading to death).  
TEAEs and TESAEs will be summarized separately by ocular (study eye and fellow 
eye) and non-ocular TEAEs/TESAEs. TEAEs and TESAEs will also be 
summarized for the treatment period, the tapering phase and the 9-month follow-
up period: 
• TEAEs/TESAEs during Treatment Period is defined as TEAEs/TESAEs 
with onsite date on or after the first treatment date (Day 1) until the Week 
12 Visit. For patients who miss Week 12 Visit and/or do not complete 
treatment period, the end date for this definition will be the last dose of 
study treatment. 
• TEAEs/TESAEs during Tapering Phase of Follow up Period is defined as 
TEAEs with the onsite date after the Week 12 Visit date until taper dosing 
is ended, which is defined as the Week 12 Visit date +14 days. For patients 
who do not take any Tapering Phase treatment but are followed-up for 
safety, TEAEs/TESAEs during Tapering Phase should be null.     
• TEAEs/TESAEs during 9-month Follow-up Period is defined as 
TEAEs/TESAEs other than two categories defined above. 
TEAEs/TESAEs will be presented in descending order from the system organ class 
(SOC) code with the highest incidence to the SOC with the lowest incidence from 
K-321 QID group. Within the SOC level, TEAEs/TESAEs will be presented in 
descending order from the MedDRA preferred term (PT) with the highest incidence 
to the PT with the lowest incidence from K-321 QID group. If the incidence for any 


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two or more PTs within the SOC from K-321 QID group is equal, the PTs will be 
presented descending order of incidence from K-321 QID group. If the incidence 
for any two or more PTs within the SOC from K-321 QID and K-321 BID groups 
are both equal, the PTs will be presented descending order of incidence from 
Placebo group. If the PTs from all three treatment groups within an SOC are equal, 
the PTs will be presented in alphabetical order. Patients who experienced multiple 
TEAEs/TESAEs for a SOC will be counted once, similarly for patients with 
multiple TEAEs/TESAEs per PT will be counted once. Patients who experienced 
multiple TEAEs/TESAEs for a PT will be counted once.  
Partial dates on AE start dates will be handled as follows:    
• If the onset date is completely missing, onset date is set to the date of first 
dose.  
• If the year is present and the month is missing, then the month is set to 
January. If the year is same as the year of the date of first dose and the AE 
end date is not prior to the date of first dose, then onset date is set to the date 
of first dose. 
• If the month and year are present and the day is missing, then the day is set 
to the 1st day of month. If the month and year are same as the month and 
year of the date of first dose and the AE end date is not prior to the date of 
first dose, then onset date is set to the date of first dose. 
• If the AE end date is present, then the imputed start date will be no later 
than the end date. 
9.1.3 Incidence of Adverse Events 
Both ocular and non-ocular TEAEs will be summarized by treatment group. The 
total number of TEAEs and the number and percentage of patients with at least one 
TEAE in each SOC and having each individual TEAE based on the PT will be 
presented. The number of events under each SOC and PT will also be presented. 
All AEs will be listed.  
9.1.4 Relationship of Adverse Events to Study Drug 
The relationship or association of the study drug in causing or contributing to the 
TEAE will be characterized as related or not related A summary of TEAEs related 
to study drug will be presented in a table by total number of patients with incidence 
of TEAE occurrence. The investigator will provide an assessment of the 
relationship of the event to the study drug. TEAEs that are missing a relationship 
will be presented in the summary table as “Related” but will be presented in the 
data listing with a missing relationship. Percentages will be calculated out of the 
number of patients in the SS. The number of events under each SOC and PT will 
also be presented. 


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The TEAE data will be categorized and presented by SOC, PT, and relationship in 
a manner similar to that described in Section 9.1.1. 
9.1.5 Severity of Adverse Event 
A summary of TEAEs by maximum severity will be presented in a table by total 
number of patients with incidence of TEAE occurrence.  The severity that will be 
presented represents the most extreme severity captured on the eCRF page. The 
possible severities are “Mild,” “Moderate,” and “Severe.”  If a patient reported 
multiple occurrences of the same TEAE, only the most severe will be presented in 
the incidence count. TEAEs that are missing severity will be presented in tables as 
“Severe” but will be presented in the data listing with a missing severity. 
Percentages will be calculated out of the number of patients in the SFS.  
The TEAE data will be categorized and presented by SOC, PT, and severity in a 
manner similar to that described in Section 9.1.1. 
9.1.6 Treatment-Emergent Adverse Events Leading to Treatment 
Discontinuation 
TEAEs leading to treatment discontinuation will be identified as TEAEs on the AE 
eCRF page, where the action taken with study drug is “Drug Withdrawn”.  The 
TEAEs leading to treatment discontinuation will be categorized and presented by 
SOC and PT in a manner similar to that described in Section 9.1.1. All TEAEs 
leading to treatment discontinuation will be listed in a data listing. 
9.1.5 Treatment-Emergent Adverse Events Leading to Study 
Discontinuation 
TEAEs leading to study discontinuation will be identified as TEAEs on the AE 
eCRF page, where the “Caused Study Discontinuation?” is “Yes”.  The TEAEs 
leading to study discontinuation will be categorized and presented by SOC and PT 
in a manner similar to that described in Section 9.1.1. All TEAEs leading to study 
discontinuation will be listed in a data listing. 
9.1.6 Serious Adverse Events 
The TESAE data will be categorized and presented by SOC and PT in a manner 
similar to that described in Section 9.1.1. The TESAE leading to treatment 
discontinuation and study discontinuation will be categorized and presented by 
SOC and PT in a manner similar to that described in Section 9.1.4 and 9.1.5. For 
All TESAE summaries, the number of events under each SOC and PT will also be 
presented. All SAE data will be listed in a data listing. 


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9.1.7 Death 
Deaths will be identified as AEs on the AE eCRF page. Patient deaths will be 
identified as AEs where the outcome is “Death”.  Deaths will be categorized and 
presented by SOC and PT in a manner similar to that described in Section 9.1.1. 
All deaths will be listed in a data listing. 
9.2 Clinical Laboratory Evaluations 
Laboratory assessments will be performed by a central laboratory. All summaries 
will be based on the units provided by the central laboratory and no conversion will 
be performed. Lab test names listed in the statistical analysis plan are the same as 
indicated in the protocol; however, the tables and listings will report results using 
the test names based on Clinical Data Interchange Standards Consortium 
(CDISC)-controlled terminologies.  
9.2.1 Hematology 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All hematology parameters will be summarized using descriptive 
statistics for each treatment group at Screening and EOT (Week 12 or ET). 
Additionally, shift tables providing the number of patients with indicated shifts 
(low, normal, high) in their results from Screening and EOT (Week 12 or ET) will 
also be presented for all hematology parameters.   
All summaries will be done for the SFS and all hematology data will be listed. 
9.2.2 Chemistry 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All chemistry parameters will be summarized using descriptive statistics 
for each treatment group at Screening and EOT (Week 12 or ET). Additionally, 
shift tables providing the number of patients with indicated shifts (low, normal, 
high) in their results from Screening and EOT (Week 12 or ET) will also be 
presented for all chemistry parameters.   
All summaries will be done for the SFS and all chemistry data will be listed. 
9.2.3 Urinalysis 
Routine laboratory testing will be performed at Screening and EOT (Week 12 or 
ET) visits. All urinalysis parameters will be summarized using descriptive statistics 
for each treatment group at Screening and EOT (Week 12 or ET).  
All summaries will be done for the SFS and all urinalysis data will be listed. 


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9.3 Vital Sign Measurements 
The vital sign variables including blood pressure (systolic and diastolic), pulse rate, 
body temperature, height, and body weight. Notable abnormal values will be 
flagged in the data listings. The conversion between conventional unit to SI unit for 
temperature and weight and definitions for notable abnormal values are listed in the 
appendix (Table 3 and Table 4). Observed values for vital sign measurements at 
Baseline and EOT (Week 12 or ET)⸷ — as well as changes from Baseline to 
postbaseline values in vital sign measurements — will be summarized descriptively 
by treatment group. 
All summaries will be done for the SFS and all vital sign data will be listed.  
⸷ Week 12 vital sign measurements will only be done in EU (European 
Union) countries according to protocol version 2.0.  
9.4 Physical Examination 
A limited physical examination will be performed at Screening and EOT (Week 12 
or ET) ⸷. The limited physical examination includes a review of the patient’s 
medical history, medication history, and evaluation of relevant body systems 
complaints. In case of relevant findings, full evaluation of the condition needs to be 
conducted.  Physical examination findings will be listed. 
⸷ Week 12 vital sign measurements will only be done in EU countries 
according to protocol version 2.0.  
9.5 Ophthalmological assessments 
Ophthalmological assessments will be performed at the indicated visits in the SOA 
(Table 5).   
9.5.1 Intraocular Pressure 
Intra-ocular pressure (IOP) may be measured by either contact or non-contact 
tonometry, but the same method must be used for all study visits for a given patient 
throughout the study and all reasonable efforts must be made to have the same 
examiner obtain all IOP measurements for a given patient. At each study visit, a 
single IOP measurement will be made for each eye, with pressure recorded in 
millimetres of mercury (mm Hg).  
Observed values at Baseline and each subsequent study visit, as well as changes 
from Baseline in IOP for each subsequent study visit, will be summarized 
descriptively by treatment group for the study eye and non-study eye. All IOP data 
will be listed. 


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9.5.2 Slit-Lamp Biomicroscopy  
Slit-lamp biomicroscopy will be performed at every visit using slit-lamp 
examination without pupil dilation to evaluate the condition of the lids, conjunctiva, 
anterior chamber, cornea, and lens.  
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal with not clinical significance (NCS) 
• Abnormal with clinical significance (CS) 
The number and percentage of patients with at least 1 treatment-emergent CS 
abnormality by ocular assessment will be summarised for each treatment group for 
the study eye and non-study eye. An abnormality will be deemed treatment-
emergent if the onset date is on or after the date of first treatment. A patient will be 
counted once even if the patient experiences more than 1 treatment-emergent CS 
abnormality.  
All slit-lamp biomicroscopy and ophthalmoscopy assessments will be listed. 
9.5.3 Ophthalmoscopy 
Ocular examination will be performed with pupil dilation using an indirect 
ophthalmoscope according to the current standard of practice to evaluate the 
condition of the vitreous, macula, retina, optic nerve, choroid, and retinal periphery. 
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal (NCS) 
• Abnormal with clinical significance (CS) 
The number and percentage of patients with at least 1 treatment-emergent CS 
abnormality by ocular assessment will be summarised for each treatment group for 
the study eye and non-study eye. An abnormality will be deemed treatment-
emergent if the onset date is on or after the date of first treatment. A patient will be 
counted once even if the patient experiences more than 1 treatment-emergent CS 
abnormality.  
All ophthalmoscopy assessments will be listed. 


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10 Interim Analysis 
No interim analyses will be performed. The analysis for primary, secondary, 
exploratory, and safety will be conducted at the Week 12 database lock ie, after all 
patients have completed dose tapering at the Week 16 visit or have been 
discontinued from the study and/or treatment before this visit. Analysis of the 
complete study data (through Week 52) will be performed after all patients have 
completed the Week 52 visits or have been discontinued from the study before the 
Week 52 visit. As the primary efficacy endpoint is at the Week 12 visit, no alpha 
adjustments will be required. No changes to study conduct (eg, change to sample 
size or early stopping) are planned.  
 
 


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11 References 
 
Hodges JL Jr, Lehmann EL. Estimates of location based on rank test. Ann. Math 
Statist. 1963;34(2):598-611. 
 
 
 
Kowa Company Ltd. Ripasudil hydrochloride hydrate ophthalmic solution. 
Investigator’s Brochure, version 00.01. Tokyo (Japan); 2019. 115 p.  
 
 
 
Moloney G, Petsoglou C, Ball M, et al. Descemetorhexis without grafting for 
Fuchs endothelial dystrophy-supplementation with topical ripasudil. Cornea. 
2017;36(6):642-8. 
 
 
 
Moses LE. Query 10: Confidence limits from rank tests. Technometrics. 
1965;7(2):257-60. 
 
 
 


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12 Appendices 
12.1 Unit Conversion 
Table 3 
Unit Conversion 
Quantity 
From SI to Conventional Unit 
From Conventional Unit to SI 
Height 
0.394 x [cm] = [in] 
2.540 [in]. = [cm] 
Weight 
2.205 x [kg] = [lb] 
0.454 x [lb] = [kg] 
Temperature 
([°C] x 9/5) + 32 = [°F] 
([°F] – 32) 5/9 = [°C] 
12.2 Notable Abnormal Vital Signs  
Table 4 
Notable Abnormal Vital Signs 
Parameter 
Abnormality 
Change from Baseline 
Pulse 
<40 bpm 
>120 bpm 
>=15 bpm increase from baseline 
>=15 bpm decrease from baseline 
Systolic blood 
pressure 
<80 mmHg 
>180 mmHg 
>=20 mmHg increase from baseline 
>=20 mmHg decrease from baseline 
Diastolic blood 
pressure 
<50 mmHg 
>105 mmHg 
>=15 mmHg increase from baseline 
>=15 mmHg decrease from baseline 
Temperature 
NA 
>= 1 C increase from baseline 
>=1 C decrease from baseline 


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12.3 Schedule of Study Procedures 
Table 5 
Schedule of Study Site Activities 
 Activity 
Screening 
Treatment Period 
Follow-Up Period 
Time Point Day -28 
Day –1 
Day 1 
Week 1 Week2 Week 3 Week 5 Week 7 Week 9 Week 12 Week 16 Week 20 Week 24 Week 38 Week 52 
Visit Window (Day) -28 to -7 
- 
- 
5-9 
12-16 
18-24 
32-38 
46-52 
60-66 
81-87 
105-119 133-147 161-175 259-273 357-371 
Visit No  Visit 1 
Visit 2 
Visit 3 
Visit 4 Optional 
Visit 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and 
concomitant disease 
X 
X a 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination and 
vital sign measurements 
X 
 
 
 
 
 
 
 
 
Xh 
 
 
 
 
 
Urine drug screen 
X 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Urine pregnancy test (if 
applicable) 
 
X a 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Laboratory sampling  
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Eligibility evaluation 
X 
X b 
 
 
 
 
 
 
 
 
 
 
 
 
 
Descemetorhexis with photo 
or video documentation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense study drug 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 study drug c 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Study drug administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Slit-lamp evaluation 
X 
X a 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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 Activity 
Screening 
Treatment Period 
Follow-Up Period 
Time Point Day -28 
Day –1 
Day 1 
Week 1 Week2 Week 3 Week 5 Week 7 Week 9 Week 12 Week 16 Week 20 Week 24 Week 38 Week 52 
Visit Window (Day) -28 to -7 
- 
- 
5-9 
12-16 
18-24 
32-38 
46-52 
60-66 
81-87 
105-119 133-147 161-175 259-273 357-371 
Visit No  Visit 1 
Visit 2 
Visit 3 
Visit 4 Optional 
Visit 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 
Corneal ECD measurement e 
(specular microscopy, 
non-contact type) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal ECD measurement 
by HRT f 
(X) 
 
 
(X) 
(X) d 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
BCVA (ETDRS scale) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal thickness  
(ultrasound pachymeters) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal Oedema 
(slit-lamp evaluation) 
X 
Xa 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal morphology 
(Pentacam or Orbscan) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Contrast sensitivity f 
(CSV-1000 chart) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Ocular examination with 
pupil dilation using an 
indirect ophthalmoscope i 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
Intra-ocular pressure 
(contact or non-contact type 
tonometer)  
X 
 
(X) g 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
 
X 
 
X 
Photo documentation of 
ocular findings 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant therapy  
X 
X 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse event documentation 
 
X 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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Abbreviation: HRT, Heidelberg retina tomography; 
 
 
a 
Pre-operative. 
b 
Post-operative. 
c 
Both 
 study drug 
 will 
 and 
 will be recorded in the eCRF. Morning/night and mid-
day/evening drug will 
 separately. 
d 
Week 2 is an optional visit (at the discretion of the investigator), but if this visit is applied, all marked assessment items must be conducted. 
e 
The study eye must be measured at all indicated visits, whereas the non-study eye must be measured only at Visit 1 and Visit 14.  
f 
Only at sites where equipment is available. 
g 
If investigator judges it necessary.  
h 
Assessment only performed in EU countries 
i 
This will be done according to current standard of care to evaluate the condition of the vitreous, macula, retina, optic nerve, choroid, and 
retinal periphery. 
 

Prot_001.pdf: 
 
 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-
Group, 12 Week, Phase 2 Study to Investigate the Safety and 
Efficacy of Ripasudil (K-321) Eye Drops After Descemetorhexis in 
Patients with Fuchs Endothelial Corneal Dystrophy 
 
NCT04250207 
Clinical Study Protocol Ver.: 2.0 
Document Date: 31 December 2019 






Kowa Research Institute, Inc. 
K-321 
Protocol: K-321-201, version 2.0 
31 December 2019 
 
 
Declaration of Investigator 
I have read and understood all sections of the protocol entitled “A double-masked, 
randomised, placebo-controlled, parallel-group, 12-week, Phase 2 study to investigate the 
safety and efficacy of ripasudil (K-321) eye drops after descemetorhexis in patients with 
Fuchs endothelial corneal dystrophy” and the accompanying investigator’s brochure. 
I agree to supervise all aspects of the protocol and to conduct the clinical investigation in 
accordance with the Protocol Version 2.0, dated 31 December 2019, the International Council 
for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), 
Integrated Addendum to E6 (R1): Guideline For Good Clinical Practice E6 (R2), the 
principles of the Declaration of Helsinki, and applicable regulations in the countries where 
the study will be conducted. I will not make changes to the protocol before consulting with 
Kowa Research Institute, Inc. or implement protocol changes without independent ethics 
committee approval except to eliminate an immediate risk to patients. I agree to administer 
study treatment only to patients under my personal supervision or the supervision of a 
sub-investigator. 
I will not supply the investigational drug to any person not authorised to receive it. 
Confidentiality will be protected. Patient identity will not be disclosed to third parties or 
appear in any study reports or publications. 
I will not disclose information regarding this clinical investigation or publish results of the 
investigation without authorisation from Kowa Research Institute, Inc.  
 
 
 
Signature of Principal Investigator 
 
Date 
 
 
 
Printed Name of Principal Investigator 
 
 
 


Kowa Research Institute, Inc. 
K-321 
Protocol: K-321-201, version 2.0 
31 December 2019 
 
 
Page 4 
Table of Contents 
Table of Contents .......................................................................................................................4 
List of Tables ..............................................................................................................................8 
List of Figures ............................................................................................................................8 
Protocol Synopsis.......................................................................................................................9 
List of Abbreviations................................................................................................................17 
List of Terms ............................................................................................................................18 
1 
Introduction ......................................................................................................................19 
2 
Study Objectives...............................................................................................................23 
2.1 
Primary Objective ...................................................................................................23 
2.2 
Secondary Objectives..............................................................................................23 
2.3 
Exploratory Objectives ...........................................................................................23 
3 
Investigational Plan ..........................................................................................................24 
3.1 
Study Design ...........................................................................................................24 
4 
Patient Selection and Discontinuation/Withdrawal Criteria .............................................27 
4.1 
Selection of Study Population .................................................................................27 
4.1.1 
Inclusion Criteria .....................................................................................27 
4.1.2 
Exclusion Criteria ....................................................................................28 
4.2 
Discontinuation of Treatment and Withdrawal of Patients From the Study ...........30 
4.2.1 
Reasons for Discontinuation or Withdrawal ............................................31 
4.2.2 
Handling of Withdrawals .........................................................................32 
4.2.3 
Replacements ...........................................................................................32 
5 
Study Drug Treatments .....................................................................................................33 
5.1 
Method of Assigning Patients to Treatment Groups ...............................................33 
5.2 
Treatments Administered ........................................................................................33 
5.3 
Identity of Study Drug ............................................................................................34 
5.4 
Management of Clinical Supplies ...........................................................................35 
5.4.1 
Study Drug Packaging and Storage .........................................................35 
5.4.2 
Study Drug Accountability ......................................................................36 


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K-321 
Protocol: K-321-201, version 2.0 
31 December 2019 
 
 
Page 5 
5.5 
Medication Errors ...................................................................................................36 
5.5.1 
Treatment of Medication Errors ...............................................................36 
5.5.2 
Overdose Management ............................................................................37 
5.5.3 
Missed Doses ...........................................................................................37 
5.6 
Masking...................................................................................................................37 
5.6.1 
Unmasking Treatment ..............................................................................37 
5.7 
Treatment Compliance ............................................................................................38 
5.8 
Prior and Concomitant Therapy ..............................................................................38 
5.8.1 
Descemetorhexis of the Study Eye ..........................................................39 
5.8.2 
Rescue Surgical Procedures .....................................................................39 
5.8.3 
Prohibited and Restricted Concomitant Therapy .....................................40 
5.8.3.1 
Prohibited and Restricted Surgical Intervention ......................................40 
5.8.3.2 
Prohibited and Restricted Medication......................................................40 
6 
Study Assessments and Procedures ..................................................................................42 
6.1 
Eligibility Assessments and Procedures..................................................................45 
6.1.1 
Medical History, Physical Examination, and Laboratory Assessments ...45 
6.1.2 
Descemetorhexis of the Study Eye ..........................................................45 
6.2 
Efficacy Assessments ..............................................................................................45 
6.2.1 
Corneal Endothelial Cell Density Measurement .....................................45 
6.2.1.1 
Non-Contact Specular Microscopy ..........................................................45 
6.2.1.2 
Heidelberg Retina Tomography ...............................................................46 
6.2.2 
Corneal Thickness ....................................................................................46 
6.2.3 
Corneal Oedema ......................................................................................46 
6.2.4 
Corneal Morphology ................................................................................46 
6.2.5 
Best Corrected Visual Acuity ...................................................................46 
6.2.6 
Contrast Sensitivity ..................................................................................47 
6.2.7 
 ...........................................................47 
6.3 
Safety Assessments .................................................................................................47 
6.3.1 
Ocular Safety Assessments ......................................................................47 
6.3.2 
Adverse Events ........................................................................................48 
6.3.2.1 
Definitions of Adverse Events .................................................................48 
6.3.2.2 
Eliciting and Documenting Adverse Events ............................................50 
6.3.2.3 
Reporting Adverse Events .......................................................................51 


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6.3.2.4 
Suspected Unexpected Serious Adverse Reactions .................................53 
6.3.2.5 
Follow-Up of Adverse Events..................................................................53 
6.3.3 
Vital Sign Measurements .........................................................................53 
6.3.4 
Eligibility .................................................................................................54 
6.4 
Safety Monitoring Committee ................................................................................54 
6.5 
Pregnancy ................................................................................................................54 
6.6 
Laboratory Analyses ...............................................................................................55 
6.6.1 
Future Genetic Testing .............................................................................55 
6.7 
Sample Collections .................................................................................................56 
7 
Statistical and Analytical Plan ..........................................................................................57 
7.1 
Primary Efficacy Endpoint .....................................................................................57 
7.2 
Secondary Efficacy Endpoints ................................................................................57 
7.3 
Exploratory Endpoints ............................................................................................58 
7.4 
Sample Size Calculations ........................................................................................58 
7.5 
Analysis Sets ...........................................................................................................59 
7.6 
Description of Subgroups to be Analysed ...............................................................59 
7.7 
Statistical Analysis Methodology ...........................................................................60 
7.7.1 
Analysis of Primary Efficacy Endpoint ...................................................60 
7.7.2 
Analysis of Secondary Efficacy Endpoints .............................................61 
7.7.3 
Analyses of Exploratory Efficacy Endpoints ...........................................62 
7.7.4 
Safety Analyses ........................................................................................63 
7.7.4.1 
Adverse Events ........................................................................................63 
7.7.4.2 
Vital Signs ................................................................................................64 
7.7.4.3 
Clinical Laboratory ..................................................................................64 
7.7.4.4 
Intra-Ocular Pressure ...............................................................................64 
7.7.4.5 
Slit-Lamp Biomicroscopy and Ophthalmoscopy .....................................64 
7.7.5 
Other Analyses .........................................................................................65 
7.7.5.1 
Patient Demographics and Other Baseline Characteristics ......................65 
7.7.5.2 
Treatment Exposure and Compliance ......................................................65 
7.7.6 
Interim Analyses ......................................................................................66 
8 
Data Quality Assurance ....................................................................................................67 
8.1 
Data Management ...................................................................................................67 


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9 
Ethics ................................................................................................................................69 
9.1 
Independent Ethics Committee or Institutional Review Board ..............................69 
9.2 
Ethical Conduct of the Study ..................................................................................69 
9.3 
Patient Information and Consent ............................................................................69 
10 Investigator’s Obligations ................................................................................................71 
10.1 Confidentiality ........................................................................................................71 
10.2 Financial Disclosure and Obligations .....................................................................71 
10.3 Investigator Documentation ....................................................................................72 
10.4 Study Conduct .........................................................................................................72 
10.5 Adherence to Protocol.............................................................................................73 
10.6 Adverse Events and Study Report Requirements ...................................................73 
10.7 Investigator’s Final Report .....................................................................................73 
10.8 Records Retention ...................................................................................................73 
10.9 Publications .............................................................................................................73 
11 Study Management ...........................................................................................................75 
11.1 Monitoring ..............................................................................................................75 
11.1.1 
External Data Monitoring Committee .....................................................75 
11.1.2 
Monitoring of the Study ...........................................................................76 
11.1.3 
Inspection of Records ..............................................................................76 
11.2 Management of Protocol Amendments and Deviations..........................................76 
11.2.1 
Modification of the Protocol ....................................................................76 
11.2.2 
Protocol Deviations..................................................................................77 
11.3 Study Termination ...................................................................................................77 
11.4 Final Report ............................................................................................................77 
12 Reference List...................................................................................................................79 
 
 


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List of Tables 
Table 1 
Dosing Regimen for Each Treatment Group From Day 1 to Visit 9............... 14 
Table 5-1 
Vial Contents by Dosing Regimen and Dosing Time for Each Treatment 
Group From Day 1 to Visit 9 (Week 12) ......................................................... 33 
Table 6-1 
Schedule of Study Site Activities .................................................................... 43 
Table 11-1 
Study Administration ...................................................................................... 75 
 
List of Figures 
Figure 3-1 
Study Design ................................................................................................... 25 
 


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Protocol Synopsis 
Protocol Number: 
K-321-201 
Title: 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-Group, 12-Week, Phase 2 
Study to Investigate the Safety and Efficacy of Ripasudil (K-321) Eye Drops After 
Descemetorhexis in Patients with Fuchs Endothelial Corneal Dystrophy 
Sponsor:  
Kowa Research Institute, Inc. 
430 Davis Drive, Suite 200 
Morrisville, NC 27560 
Study Phase: 
Phase 2 
Study Sites: 
Approximately 30 sites in the United States, Europe, and Australia 
Indication: 
The treatment of Fuchs endothelial corneal dystrophy after descemetorhexis 
Rationale: 
In Fuchs endothelial corneal dystrophy (FECD), there is an increased rate of loss of 
endothelial cells, starting in the centre of Descemet’s membrane and spreading to the 
periphery. There is an increased deposition of extracellular matrix on Descemet’s 
membrane, resulting in excrescences known as guttae, which are a marker of the 
condition. Guttae may coalesce and inhibit the migration of endothelial cells. Eventually 
the corneal endothelium ceases to function effectively, and the cornea begins to cloud, 
leading eventually to blindness.  
Currently, most patients undergoing keratoplasty for FECD have Stage 2 disease and the 
procedure starts with descemetorhexis, removing an area of the roughened and irregular 
Descemet’s membrane (including the central guttae) from the diseased cornea. The 
excised area is replaced with a graft of endothelial cells: either donor Descemet’s 
membrane with a thin layer of corneal stroma (Descemet stripping endothelial 
keratoplasty [DSEK]), or, increasingly commonly, donor Descemet’s membrane alone 
(Descemet’s membrane endothelial keratoplasty [DMEK]). 
Experience with failed grafts has indicated that even though there is an underlying 
abnormality of endothelial cells in FECD, areas of stroma exposed by descemetorhexis 
can be re-endothelialised and the migrated endothelial cells can maintain corneal 
transparency. Mathematical modelling of the healing of an inadvertent 
 diameter 
descemetorhexis in an elderly man without FECD suggested that normal healing is 
achieved by a redistribution of peripheral endothelial cells into the central defect without 
any increase in the number of endothelial cells. This led to the proposal that some cases 








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A female of childbearing potential is defined as a woman who has experienced 
menarche and has not undergone successful surgical sterilisation or is not post-
menopausal, and a post-menopausal woman is defined as having had no menses for 
the previous 12 months (without a known cause). 
2. Has a study eye with confluent guttae in the periphery or confluent guttae outside the 
stripped area (individual guttae are allowed). 
Note that patients may have individual or small number of guttae remaining outside 
the stripped area, but there should be no areas of confluent guttae remaining after the 
descemetorhexis. For example, a patient who had 5 to 10 guttae remaining in a few 
spots around the circumference of the descemetorhexis would not be excluded by this 
criterion.  
3. Has a study eye with a history of cataract surgery within 90 days of Visit 1. 
4. Has a study eye with a history of any previous ocular surgery other than for cataract.  
5. Has a non-study eye with a history of any previous ocular surgery within 30 days of 
Visit 1. 
6. Plans to receive any surgical treatment on the study eye during any study period, other 
than the study descemetorhexis.  
7. Plans to receive any surgical treatment for FECD or cataract on the non-study eye in 
the screening or treatment period. 
8. Has advanced corneal stromal oedema defined as the presence of widespread haze or 
bullae on slit-lamp examination. 
9. Has a study eye with central corneal thickness ≥ 670 μm. 
10. Has known severe comorbidities that may interfere with descemetorhexis (eg, a 
bacterial, viral, or fungal ophthalmic infection). 
11. Has any clinically significant ocular condition other than FECD or cataract that 
requires medication or ocular surgery. 
12. Has diabetes with poor blood sugar control (eg, HbA1c value > 8.5%). 
13. Has used contact lenses within 7 days of Visit 1. 
14. Has hypersensitivity to any ophthalmic medication used for diagnosis or treatment, 
including eye drops containing antibiotic(s) or glucocorticoid(s).  
15. Has known hypersensitivity to any component of the study drugs. 
16. Has previously used ripasudil, netarsudil, or other ROCK inhibitors. 
17. Has used any investigational medications within 30 days of Visit 1. 
18. Has a positive urine test result for drugs of abuse (opiates, methadone, cocaine, 
amphetamines, barbiturates, or benzodiazepines) or alcohol at screening. 
19. Has donated more than 450 mL of blood within 60 days of Visit 1. 
20. Is a member or a family member of the professional or ancillary personnel working at 
the study site or the sponsor involved in the study. 
21. Has a concomitant medical or psychological condition that could interfere with study 


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participation or is otherwise not suitable for entry into the study in the opinion of the 
investigator.  
Estimated Study Duration: The maximum total study period for each enrolled patient is 
56 weeks, with a maximum of 15 visits (including 1 optional visit at Week 2). 
Efficacy Assessments: 
Efficacy assessments include corneal ECD, corneal thickness, corneal oedema, corneal 
morphology, BCVA, contrast sensitivity, and the 
 
  
Safety Assessments: 
Safety assessments include the identification of AEs; ocular safety assessments of both 
eyes (including: slit-lamp examination without pupil dilation to evaluate the condition of 
the lids, conjunctiva, anterior chamber, and cornea; intra-ocular pressure measurement; 
and ocular examination with pupil dilation using an indirect ophthalmoscope according to 
the current standard of practice to evaluate the condition of the vitreous, macula, retina, 
optic nerve, choroid, and retinal periphery); vital sign measurements; and laboratory 
examinations.  
Study Drug, Dosage, and Route of Administration: 
Study drug is either K-321 ophthalmic solution containing ripasudil 0.4% (K-321 0.4%) 
or matching placebo. The K-321 treatments will comprise either BID or QID dosing of 
K-321, as presented in Table 1. 
Table 1 
Dosing Regimen for Each Treatment Group From Day 1 to Visit 9 
Treatment  
Morning 
Mid-day 
Evening 
Night 
K-321 QID 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 BID 
K-321 0.4% 
Placebo 
Placebo 
K-321 0.4% 
Placebo 
Placebo 
Placebo 
Placebo 
Placebo 
 
For each dose, patients must instil 1 drop to the study eye only and then close the eyelids, 
pressing on the lacrimal punctum. Patients will be advised to keep the eye closed for 1 to 
5 minutes before opening the eye. Any other concurrent ocular medication(s) may be 
applied at least 5 minutes following instillation of study drug. 
Sample Size: 
Approximately 60 patients will be enrolled in the study. A sample size of 20 patients in 
each group will be needed to detect a difference of 
 in the measurement in the corneal 
ECD at Week 12 between K-321 0.4% QID/BID and placebo groups with a power of 
 for Step 1 and a power of 
 for Step 2 using a Wilcoxon rank sum test with a 
0.050 two-sided significance level with closed testing procedures. Sample size was 
calculated by simulation. For the simulation, based on the existing literature (Borkar DS, 
et al. 2016, Moloney G, et al. 2017, Iovieno A, et al. 2017), placebo was assumed to be 






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List of Abbreviations 
Abbreviation 
Definition 
AE 
adverse event 
BCVA 
best controlled visual acuity 
BID 
twice daily 
CFR 
Code of Federal Regulations 
CI 
confidence interval 
 
 
CRO 
contract research organisation 
CS 
clinically significant 
CV 
curriculum vitae 
DMEK 
Descemet membrane endothelial keratoplasty 
DSEK 
Descemet stripping endothelial keratoplasty 
DSO 
Descemet stripping only 
ECD 
endothelial cell density 
eCRF 
electronic case report form 
EDC 
electronic data capture 
EOS 
end-of-study 
EOT 
end-of-treatment 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FAS 
full-analysis set 
FDA 
US Food and Drug Administration 
FECD 
Fuchs endothelial corneal dystrophy 
GCP 
Good Clinical Practice 
HbA1c 
haemoglobin A1c (glycated haemoglobin) 
HRT 
Heidelberg retina tomography 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
IEC 
independent ethics committee 
IOP 
intra-ocular pressure 
IRB 
institutional review board 
IWRS 
interactive web response system 


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Abbreviation 
Definition 
LOCF 
last-observation-carried-forward 
M/N 
morning and night 
MD/E 
mid-day and evening 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHRA 
Medicines and Healthcare Products Regulatory Agency 
NCS 
not clinically significant 
PPS 
per-protocol set 
QID 
4 times daily 
ROCK 
Rho-associated protein kinase 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SFS 
safety set 
SOA 
schedule of study site activities 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE 
treatment-emergent adverse event 
UK 
United Kingdom 
US 
United States 
 
 
 
List of Terms 
Term 
Definition 
Descemetorhexis 
Removal of an area of roughened and irregular Descemet membrane 
along with central guttae (also referred to herein as Descemet 
membrane stripping) 
 


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1 Introduction 
In the early 20th century, Ernst Fuchs described a bilateral corneal dystrophy now referred to 
as Fuchs endothelial corneal dystrophy (FECD). The primary defect in FECD seems to be 
related to the endothelial cells that normally sit on a modified basement membrane 
(Descemet’s membrane) that lines the inner surface of the cornea.  
In the normal adult human cornea, the endothelial cell density (ECD) on Descemet’s 
membrane is in the range of 2,000 to 3,500 cells/mm2 (Ianchulev et al 2019) and the 
endothelial cells maintain the cornea in a state of relative dehydration, which preserves 
corneal transparency. During adulthood there is continuous loss of endothelial cells at an 
approximate rate of 0.6% per year. Since the number of endothelial cells cannot increase 
because of a state of mitotic arrest, gaps caused by apoptosis are filled by the migration of 
adjacent cells that increase in size and change shape. The function of the corneal endothelium 
can be maintained while the ECD is maintained above approximately 400 cells/mm2. Below 
that threshold (due to disease, trauma or old age), there is decompensation of the endothelial 
pumping mechanism and corneal oedema produces a visual haze (Van den Bogerd et al 
2018).  
In FECD, there is an increased rate of loss of endothelial cells, starting in the centre of 
Descemet’s membrane and spreading to the periphery. There is an increased deposition of 
extracellular matrix on Descemet’s membrane, resulting in excrescences known as guttae, 
which are a marker of the condition. Guttae may coalesce and inhibit the migration of 
endothelial cells. Eventually the corneal endothelium ceases to function effectively, and the 
cornea begins to cloud, eventually leading to blindness (Elhalis et al. 2010).  
There are clinical staging systems for FECD that describe the progression of the disease 
(Elhalis et al. 2010). Stage 1 is characterised by the presence of corneal guttae that are 
centrally located, non-confluent, and asymptomatic. Guttae may also form in response to 
trauma, toxins, or infection and may not be indicative of FECD. Guttae appearing with age 
are considered a normal finding in the elderly if they appear in the periphery. In Stage 2, 
central guttae start to coalesce and spread to the periphery and there is loss of endothelial 
cells, with ECD being inversely proportional to the number of guttae. The cells become much 
more variable in size and lose their regular shape (pleomorphism), and painless visual 
symptoms start to appear. The irregularity of the Descemet’s membrane, and particularly the 


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presence of central confluent guttae, may lead to forward scatter of light in the eye, loss of 
visual contrast sensitivity, and impairment of visual acuity, even in the absence of oedema 
(Watanabe et al 2015). In the early stages of the disease, oedema of the stroma causes 
blurriness of vision in the morning that clears during the day, but this progresses to 
permanent loss of visual acuity with glare. In Stage 3, there is more marked corneal oedema 
and painful sub-epithelial and epithelial bullae may form. Rupture of the bullae disrupts the 
outer surface of the cornea and exposes the patient to risk of infection. Finally, in Stage 4, the 
cornea becomes opaque and new vessel growth may occur. In addition, there may be 
sub-epithelial fibrous tissue formed due to long-standing oedema. In this stage, the eye 
becomes blind, but the pain may subside (Elhalis et al. 2010).  
Fuchs endothelial corneal dystrophy is currently treated by a variety of keratoplasty 
techniques, including full-thickness cornea transplantation (for Stages 3 and 4) and 
2 techniques involving transplantation of a graft of donor endothelial cells on donor 
Descemet’s membrane. Currently, most patients undergoing keratoplasty for FECD have 
Stage 2 disease and the procedure starts with descemetorhexis, removing an area of the 
roughened and irregular Descemet’s membrane (including the central guttae) from the 
diseased cornea. The excised area is replaced with a graft of endothelial cells: either donor 
Descemet’s membrane with a thin layer of corneal stroma (Descemet stripping endothelial 
keratoplasty [DSEK]), or, increasingly commonly, donor Descemet’s membrane alone 
(Descemet’s membrane endothelial keratoplasty [DMEK]). 
Experience with failed grafts has indicated that even though there is an underlying 
abnormality of endothelial cells in FECD, areas of stroma exposed by descemetorhexis can 
be re-endothelialised and the migrated endothelial cells can maintain corneal transparency. 
Mathematical modelling of the healing of an inadvertent 5-mm diameter descemetorhexis in 
an elderly man without FECD suggested that normal healing is achieved by redistribution of 
peripheral endothelial cells into the central defect without any increase in the number of 
endothelial cells (Jullienne et al 2015). This led to the proposal that some cases of FECD, 
particularly those with relatively preserved peripheral endothelial cells, can be treated by 
descemetorhexis alone (Garcerant et al 2019). 
There are limits to the ability of the endothelium to heal. In a recent analysis of cases of 
patients who underwent descemetorhexis without grafting as a possible treatment for FECD 


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(compiled from multiple papers), 31 (65%) out of 47 cases eventually achieved a clear 
cornea, with healing taking place as early as 1 month after the procedure (Van den Bogerd et 
al 2018). If there was persistent oedema at 6 months, then it was unlikely to resolve. If 
corneal oedema did not clear, some patients’ corneas cleared without residual stromal 
damage after DMEK performed as rescue therapy between 4 and 8.5 months after 
Descemet’s membrane stripping (Van den Bogerd et al 2018). All keratoplasty procedures, 
including DMEK, are associated with risks of graft detachment, graft failure or immune 
rejection. There is a medical need for medication that can speed healing after 
descemetorhexis and that can help establish a high, functional ECD to maintain corneal 
transparency. 
Ripasudil ophthalmic solution 0.4% (K-321) is a Rho-associated protein kinase (ROCK) 
inhibitor that has been marketed in Japan since December 2014 as Glanatec®, with twice 
daily (BID) dosing for glaucoma and ocular hypertension. Studies in endothelial cells have 
indicated that K-321 can lead to reduction of apoptosis, enhancement of cell proliferation, 
and increased rates of migration, all of which may support endothelial healing (Kowa 
Company Ltd 2019). Moloney et al (2017) first reported use of a ROCK inhibitor to promote 
healing in patients with FECD treated with Descemet stripping only (DSO). More recently, 
Macsai et al (2019) reported results from a small study of 18 patients with FECD and central 
confluent guttae of up to 5 mm diameter who underwent DSO and were subsequently treated 
with either ripasudil ophthalmic solution 0.4% four times daily (QID) or no ripasudil for 
2 months. Overall, patients who underwent DSO with ripasudil recovered vision more 
quickly 
 
 The patients in the no-ripasudil 
group had 
 
 while in the ripasudil group there was 
 
. In the short-term 
studies (8 weeks duration and less) for the development of Glanatec, there were 
 
 
 
 (Kowa Company Ltd 2019).  


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Kowa proposes to pursue development of K-321 (ripasudil ophthalmic solution) for the 
following indication: “the treatment of Fuchs endothelial corneal dystrophy after 
descemetorhexis.” This will be the first Kowa-sponsored study in patients for this indication.  


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2 Study Objectives  
2.1 Primary Objective 
The primary objective of this study is to investigate the effect of K-321 dosed for 12 weeks 
on central ECD in patients with FECD after descemetorhexis.  
2.2 Secondary Objectives 
The secondary objectives of this study are the following:  
• to investigate the effect of K-321 on central ECD, corneal thickness, and corneal 
clarity in patients with FECD at each visit after descemetorhexis out to 52 weeks 
• to assess the safety and tolerability of K-321 in patients with FECD at each visit after 
descemetorhexis out to 52 weeks 
2.3  Exploratory Objectives 
 
 
 
 
  


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3 Investigational Plan 
3.1 Study Design 
This is a multi-centre, double-masked, randomised, parallel-group, placebo-controlled, 
2-period study of patients with FECD after descemetorhexis. The study design is presented in 
Figure 3-1, and the schedule of study site activities (SOA) is presented in Table 6-1.  
The first period is the treatment period, consisting of a screening visit within a 1- to 4-week 
screening period, a descemetorhexis and randomisation visit, a 12-week full treatment period 
containing 7 interim visits (including 1 optional visit at Week 2) and an end-of-treatment 
(EOT) visit scheduled for Week 12. During the 2 weeks immediately following the EOT 
visit, each enrolled patient will taper dosing of study drug to zero (Section 5.2).  
The second period is a follow-up observation period of 40 weeks, including the tapering 
phase and containing 4 interim visits and an end-of-study (EOS) visit. Patients are not to 
self-administer study drug after approximately Week 14. The maximum total study period for 
each enrolled patient is 56 weeks, with a maximum of 15 visits (including the 1 optional 
visit). Patients will be considered to have completed the study with the completion of their 
EOS visit (scheduled for Week 52). The duration of the study is defined for each patient as 
the date signed written informed consent is provided through the last follow-up visit. 


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There are 3 treatment groups: K-321 ophthalmic solution 0.4% dosed QID, K-321 
ophthalmic solution 0.4% dosed BID, and placebo. The procedures for randomly assigning 
study drug are presented in Section 5.1. 
After obtaining informed consent at Visit 1, the investigator will select one eye as the study 
eye, and the patient will come back for the descemetorhexis operation and random 
assignment to treatment after 1 to 4 weeks (Visit 2). At Visit 2, each patient’s eligibility for 
study participation will be confirmed, including a successful descemetorhexis operation that 
meets the inclusion criteria (Section 4.1). Guidance for the descemetorhexis will be provided 
in the study manual. Recommended post-descemetorhexis concomitant therapy is described 
in Section 5.8.1. Documentation of the descemetorhexis is discussed in Section 6.1.  
After eligibility is confirmed, patients will be randomly assigned to the 3 treatment groups 
(in a 1:1:1 ratio). All patients will be instructed to apply the study drug only to the study eye.  
Except on days of study visits on Day 1 through Week 12 (not including Visit 9) during the 
treatment period, study drug will be dosed by the patient QID (Section 5.2): morning (9 AM 
±1 hour), mid-day (1 PM ±1 hour), evening (5 PM ±1 hour), and night (9 PM ±1 hour). Each 
patient will be provided with 2 sets of colour-coded 
, one set to be applied 


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morning and night (M/N) and the other to be applied mid-day and evening (MD/E). See 
Section 5.4.1 for a detailed description of packaging.  
On study visit days Visit 3 through Visit 9, patients will not apply any study drug until after 
all study measurements have been completed; thereafter on those days, patients will start 
dosing with the dose closest in timing to the dosing regimen and continue with the remaining 
doses for the day. Additionally, on Visit 3 they must first apply the study drug within 
 after the end of the descemetorhexis procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient applies 
any study drug on the day of Visit 9 before all study measurements have been completed, that 
patient’s Visit 9 should be rescheduled within the visit window allowance (eg, the next day). 
For visits other than Visit 9 during the treatment period, a patient who applies study drug 
before all study measurements have been completed will be assessed as planned, and a 
protocol deviation must be recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up period, and 
new study drug will be dispensed without M/N or MD/E colour coding (Section 5.2). All 
patients will self-administer their tapering-phase study drug BID (9 AM ±1 h and 9 PM ±1 h) 
for a week, then once daily (9 AM ±1 h) for a week, and then will discontinue study drug. At 
Visit 9, patients will apply the first dose of study drug 
 of completing study 
measurements and will apply the second dose at night, as scheduled.  
During the follow-up period, patients will attend 5 study visits (Figure 3-1), starting at 
Week 16 (Visit 10).  
Study activities and assessments will be performed at study visits as described in Table 6-1 
and in Section 6. 
 will determine 
central ECD by analysing digital images of the corneal endothelium (Table 11-1).  
Keratoplasty may be offered as a rescue surgical procedure to an enrolled patient if the 
investigator judges that endothelial healing is not complete at Visit 9 (Week 12), or earlier if 
the investigator considers that the patient needs urgent treatment (Section 5.8.2).  


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4 Patient Selection and Discontinuation/Withdrawal Criteria 
4.1 Selection of Study Population 
Approximately 60 patients will be enrolled at approximately 30 sites in the United States, 
Europe, and Australia. Patients will be assigned to study treatment only if they meet all of the 
inclusion criteria and none of the exclusion criteria. 
Deviations from the inclusion and exclusion criteria are not allowed because they can 
potentially jeopardise the scientific integrity of the study, regulatory acceptability, or patient 
safety. Therefore, adherence to the criteria as specified in the protocol is essential. 
Rescreening of patients that failed the initial screening may be done if the patient fulfils 
selection criteria upon a subsequent visit.  However, this may only be done for some of the 
criteria, as specified below. 
4.1.1 Inclusion Criteria 
Each patient must meet all of the following criteria to be enrolled in this study: 
1. Is at least 18 years old at the screening visit (Visit 1).  – Re-screening possible if initial 
screening was soon before 18th birthday. 
2. Has a diagnosis of FECD at Visit 1.  – Rescreening is not possible. 
3. Has confluent central guttae in the study eye that can be removed by descemetorhexis 
of a circular area of 
 diameter or less (at Visit 1).  – Rescreening is not possible.  
4. Has a study eye with best corrected visual acuity (BCVA) of 75 letters or fewer by 
Early Treatment Diabetic Retinopathy Study (ETDRS) testing (Snellen equivalent of 
20/32 or worse) at Visit 1.  – Rescreening is not possible. 
5. The study eye descemetorhexis at Visit 2 is confirmed to have excised a central area 
with confluent guttae and a diameter of 
. Guidance for performing the 
study eye descemetorhexis and measurement of the excised area will be provided in 
the study manual.  – Rescreening is not possible in that eye but the non-study eye can 
be used. 


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6. Can understand the written informed consent, provides signed and witnessed written 
informed consent, and agrees to comply with protocol requirements before any 
study-specific assessment is performed.  – Rescreening is not possible. 
4.1.2 Exclusion Criteria 
Patients meeting any of the following criteria will be excluded from the study: 
1. Is a female patient and any of the following is true:  
• 
is pregnant or lactating/breastfeeding, or  
• 
is not surgically sterile, not post-menopausal (no menses for the previous 
12 months), or not practising an effective method of birth control as 
determined by the investigator (eg, oral contraceptives, double barrier 
methods, hormonal injectable or implanted contraceptives, tubal ligation, 
or partner with vasectomy).  
If a patient is female and of childbearing potential, she must have a negative urine 
pregnancy test result at Visit 2 before the descemetorhexis. Women of 
childbearing potential must also agree to use effective contraception throughout 
the study. 
A female of childbearing potential is defined as a woman who has experienced 
menarche and has not undergone successful surgical sterilisation or is not 
post-menopausal, and a post-menopausal woman is defined as having had no 
menses for the previous 12 months (without a known cause). 
– Rescreening is possible if patient either stops nursing or initiates birth control. 
2. Has a study eye with confluent guttae in the periphery or confluent guttae outside the 
stripped area (individual guttae are allowed). 
Note that patients may have individual or small number of guttae remaining outside 
the stripped area, but there should be no areas of confluent guttae remaining after the 
descemetorhexis. For example, a patient who had 5 to 10 guttae remaining in a few 
spots around the circumference of the descemetorhexis would not be excluded by this 
criterion.  – Rescreening is not possible. 
3. Has a study eye with a history of cataract surgery within 90 days of Visit 1.  
– Rescreening is possible. 


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4. Has a study eye with a history of any previous ocular surgery other than for cataract.  
– Rescreening is not possible. 
5. Has a non-study eye with a history of any previous ocular surgery within 30 days of 
Visit 1.  – Rescreening is possible. 
6. Plans to receive any surgical treatment on the study eye during any study period, other 
than the study descemetorhexis.  – Re-screening possible if during initial screening 
subject had plans for surgical treatment on the study eye during the study period, but 
upon re-screening these plans for surgery were cancelled. 
7. Plans to receive any surgical treatment for FECD or cataract on the non-study eye in 
the screening or treatment period.  – Re-screening possible if during initial screening 
subject had plans for surgical treatment for FECD or cataract on the non-study eye 
during the screening or treatment period, but upon re-screening these plans for surgery 
were cancelled. 
8. Has advanced corneal stromal oedema defined as the presence of widespread haze or 
bullae on slit-lamp examination.  – Rescreening is not possible. 
9. Has a study eye with central corneal thickness ≥ 670 μm.  – Rescreening is not 
possible. 
10. Has known severe comorbidities that may interfere with descemetorhexis (e.g., a 
bacterial, viral, or fungal ophthalmic infection).  – Rescreening is not possible. 
11. Has any clinically significant ocular condition other than FECD or cataract that 
requires medication or ocular surgery (described in Section 5.8.3).  – Rescreening is 
not possible. 
12. Has diabetes with poor blood sugar control (eg, HbA1c value > 8.5%).  – Re-
screening possible if HbA1c has improved at the time of the re-screening. 
13. Has used contact lenses within 7 days of Visit 1.  – Rescreening is possible. 
14. Has hypersensitivity to any ophthalmic medication used for diagnosis or treatment, 
including eye drops containing antibiotic(s) or glucocorticoid(s).  – Rescreening is not 
possible. 
15. Has known hypersensitivity to any component of the study drugs.  – Rescreening is 
not possible. 


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16. Has previously used ripasudil, netarsudil, or other ROCK inhibitors.  – Rescreening is 
not possible. 
17. Has used any investigational medications within 30 days of Visit 1.  – Rescreening is 
possible. 
18. Has a positive urine test result for drugs of abuse (opiates, methadone, cocaine, 
amphetamines, barbiturates, or benzodiazepines) or alcohol at screening.  
– Rescreening is not possible. 
19. Has donated more than 450 mL of blood within 60 days of Visit 1.  – Rescreening is 
possible. 
20. Is a member or a family member of the professional or ancillary personnel working at 
the study site or the sponsor involved in the study.  – Rescreening is not possible. 
21. Has a concomitant medical or psychological condition that could interfere with study 
participation or is otherwise not suitable for entry into the study in the opinion of the 
investigator.  – Re-screening possible if investigator provides evidence that the 
concomitant medical or psychological condition resolved and the subject was suitable 
for entry into the study. 
 
4.2 Discontinuation of Treatment and Withdrawal of Patients From the 
Study 
The duration of the study is defined for each patient as the date signed written informed 
consent is provided through the date of the last study visit attended.  
There are 3 scenarios that result in interruption of the treatment regimen. Interruption of 
treatment is defined as a temporary stopping of study drug treatment that resumes during the 
treatment period, due to an AE or any other reason. Early discontinuation of treatment is 
defined as permanent stopping of study drug treatment before completing Visit 9, scheduled 
for Week 12. Investigators will strive to ensure that a patient who has interrupted treatment 
for a particular reason will not discontinue treatment unless discontinuation is medically 
imperative in the investigator’s judgement. Early withdrawal from the study is defined as 
failing to complete Visit 14, scheduled for Week 52.  


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Study drug administration may be discontinued early at the discretion of the investigator (or 
designee) if the patient does not tolerate the dosing regimen or if, in the investigator’s 
judgement, the patient’s health may be jeopardised by continuing study treatment. Patients 
who discontinue treatment during the study will be encouraged by investigators to continue 
to participate in all scheduled study site visits and assessments, and study data will be 
collected for these patients per protocol. Every effort must be made to keep patients in the 
study (see Section 5.8.2 for a discussion of treatment discontinuation after rescue surgical 
procedures).  
4.2.1 Reasons for Discontinuation or Withdrawal 
Patients may discontinue treatment or withdraw from the study at any time and for any 
reason without prejudice to their future medical care by the investigator or at the study site. 
Every effort must be made to keep patients in the study. The reasons for patients 
discontinuing treatment or withdrawing early will be recorded.  
A patient may discontinue treatment or be discontinued from treatment for any of the 
following reasons: 
1. The patient does not meet the protocol inclusion or exclusion criteria. 
2. The patient is non-compliant with the protocol. 
3. The patient has a serious or intolerable AE that, in the investigator’s opinion, requires 
withdrawal from the study.  
4. The patient has symptoms or an intercurrent illness not consistent with the protocol 
requirements or that justify withdrawal. 
5. The patient undergoes rescue keratoplasty (Section5.8.2). 
6. Other reasons (eg, pregnancy, development of contraindications of use of study drug). 
A patient may be withdrawn from the study for any of the following reasons: 
1. The patient withdraws consent.  
2. The patient is lost to follow-up. 


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The investigator will also withdraw all patients if Kowa Research Institute, Inc. terminates 
the study. The study may be terminated if it becomes apparent that the objectives of the trial 
cannot be met, or if the frequency or pattern of AEs reported in the trial indicate that the drug 
is no longer likely to have a positive benefit-to-risk ratio. 
4.2.2 Handling of Withdrawals  
 Patients who withdraw from the study or who are withdrawn from the study will no longer 
receive study drug. When a patient withdraws or is withdrawn from the study, the reason(s) 
for withdrawal shall be recorded by the investigator on the relevant page of the electronic 
case report form (eCRF).  
Whenever possible, all patients who withdraw from the study early will undergo all EOS 
assessments. Patients who fail to return for final assessments will be contacted by the site 
with 2 documented telephone calls followed by 1 registered letter, as needed, to have them 
comply with the protocol. 
It is vital to obtain follow-up data on any patient withdrawn because of an AE or serious AE 
(SAE). In every case, efforts must be made to undertake protocol-specified safety follow-up 
procedures. 
4.2.3 Replacements 
Patients who are screened for the study and receive a study descemetorhexis may be replaced 
if they never are randomly assigned study drug or are withdrawn from the study before they 
administer any study drug. The replacement patients will receive a new randomly assigned 
treatment and not the treatment assignment of the replaced patient. Patients who self-
administer any study drug and subsequently are withdrawn early from the study will not be 
replaced. Patients who fail to satisfy inclusion and exclusion criteria at screening may be 
rescreened 1 additional time at the discretion of the sponsor. A special case and conditions for 
replacement, in case the descemetorhexis fails on the first-selected study eye, is presented in 
Section 5.8.3.1.  


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5 Study Drug Treatments 
5.1 Method of Assigning Patients to Treatment Groups 
After confirmation that the study eye descemetorhexis satisfied inclusion criterion 5 
(Section 4.1.1) and any pregnancy test is negative (exclusion criterion 1, Section 4.1.2), 
patients will be randomly assigned at Visit 2 to either placebo, K-321 BID, or K-321 QID 
treatment (Section 5.2) using a 1:1:1 allocation ratio. An interactive web response system 
(IWRS) will be used to administer the randomisation schedule. An independent 
 
biostatistician will generate the randomisation schedules using SAS software Version 9.4 
(SAS Institute Inc, Cary, North Carolina) or later. The IWRS will link sequential patient 
randomisation numbers to treatment codes. The randomisation schedule will use an 
appropriate block size, which will not be revealed other than to the 
 and Kowa 
biostatisticians. 
5.2 Treatments Administered 
There are 3 treatment groups: K-321 ophthalmic solution 0.4% dosed QID (K-321 QID), 
K-321 ophthalmic solution 0.4% dosed BID (K-321 BID); and placebo. To preserve masking, 
all patients will self-administer assigned study drug QID and will dose from 
 
, one to be used M/N and the other to be used MD/E, as presented in 
Table 5-1. 
Table 5-1 
Vial Contents by Dosing Regimen and Dosing Time for Each 
Treatment Group From Day 1 to Visit 9 (Week 12) 
Treatment  
Morning 
Mid-day 
Evening 
Night 
K-321 QID 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 0.4% 
K-321 BID 
K-321 0.4% 
Placebo 
Placebo 
K-321 0.4% 
Placebo 
Placebo 
Placebo 
Placebo 
Placebo 
 
Except on days of study visits on Day 1 through Week 12 (not including Visit 9) during the 
treatment period, study drug will be dosed by the patient QID: morning (9 AM ±1 hour), 
mid-day (1 PM ±1 hour), evening (5 PM ±1 hour), and night (9 PM ±1 hour). Each patient 
will be provided with 2 sets of colour-coded 
, one set to be applied M/N and 
the other to be applied MD/E. See Section 5.4.1 for a detailed description of packaging.  


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On study visit days Visit 3 through Visit 9, patients will not apply any study drug until after 
all study measurements have been completed; thereafter on those days, patients will start 
dosing with the dose closest in timing to the dosing regimen and continue with the remaining 
doses for the day. Additionally, on Visit 3 they must first apply study drug within 
 after the end of the descemetorhexis procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient applies 
any study drug on the day of Visit 9 before all study measurements have been completed, that 
patient’s Visit 9 should be rescheduled within the visit window allowance (eg, the next day). 
For visits other than Visit 9 during the treatment period, a patient who applies study drug 
before all study measurements have been completed will be assessed as planned, and a 
protocol deviation must be recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up period, and 
new study drug will be dispensed without M/N or MD/E colour coding (Section 5.2). All 
patients will self-administer their tapering-phase study drug BID (9 AM ±1 hour and 
9 PM ± 1 hour) for a week, then once daily (9 AM ± 1 hour) for a week, and then will 
discontinue study drug. At Visit 9, patients will apply the first dose of study drug 
 
 of completing study measurements and will apply the second dose at night, as 
scheduled. However, if the first dose will be applied after 8 PM, the second dose must be 
skipped. 
For each dose, patients must instil 1 drop to the study eye only and then close the eyelids, 
pressing on the lacrimal punctum. Patients will be advised to keep the eye closed for 
1 to 5 minutes before opening the eye. Any other concurrent ocular medication(s) must be 
applied at least 5 minutes following instillation of study drug. Patients will be trained by site 
staff on the application of eye drops. 
5.3 Identity of Study Drug 
The K-321 ophthalmic solution contains ripasudil 0.4% (4.896 mg/mL of ripasudil 
hydrochloride hydrate, equivalent to 4.0 mg/mL as the free base) in a solution also containing 
the excipients sodium dihydrogen phosphate and sodium hydroxide at a pH in the range of 
5.0 to 7.0. The solution is isosmotic to normal saline, clear, colourless, and 
. The 
placebo ophthalmic solution is identical in appearance, osmolarity, and pH to the K-321 




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selected clinical supply depots where shipments to clinical sites are managed via the IWRS 
system.  
The materials schedule will be prepared by an independent 
 biostatistician and provided 
to the IWRS and the packaging vendor. At the time of randomisation, the IWRS will assign 
each patient a study drug kit corresponding to the patient’s randomly assigned treatment 
based on the drug supply inventory that is available at the site. The kit will be identified by a 
unique kit number that is separate from the patient or randomisation numbers. The IWRS 
will administer study drug dispensing at each visit indicated in Table 6-1. 
5.4.2 Study Drug Accountability 
The investigator will maintain accurate records of receipt of all study drug, including dates of 
receipt. In addition, accurate records will be kept regarding when and how much study drug 
is dispensed and used by each patient in the study. Reasons for departure from the expected 
dispensing regimen must also be recorded. At the completion of the study, to satisfy 
regulatory requirements regarding drug accountability, all study drug will be reconciled, 
shipped from study sites to Catalent depots in the United States and Germany, and destroyed 
according to applicable regulations. 
5.5 Medication Errors 
At Visit 3, patients will be given instructions on how to administer their assigned ophthalmic 
solution and how to avoid contaminating the 
 solution, and they will be observed 
instilling their own drops by the site staff. Re-education on the proper administration 
procedure will be given to patients by study site staff throughout the treatment period at each 
visit. To minimise medication errors during the 12-week treatment phase, 
 and 
 will be colour-coded differently for M/N and MD/E doses.  
5.5.1 Treatment of Medication Errors 
In the event of a medication error, the appropriate supportive clinical care must be provided 
as dictated by the patient’s clinical status.  


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5.5.2 Overdose Management 
A symptomatic overdose from topical administration is unlikely, as no overdose was 
observed during the development of Glanatec (Kowa Company Ltd 2019). Patients will be 
instructed to contact the investigator or study coordinator immediately in the event of a 
suspected symptomatic overdose. Suspected symptomatic overdoses must be reported as AEs 
(or SAEs) in the eCRF. In the event of overdose, the appropriate supportive clinical care must 
be provided as dictated by the patient’s clinical status  
5.5.3 Missed Doses 
If a dose is missed, patients will be advised as follows: 
• If you notice you missed a dose 2 hours or less after a scheduled dose time, dose 
immediately 
• If you notice you missed a dose more than 2 hours after a scheduled dose time, skip 
the dose you missed and resume regular dosing at the next planned time according to 
the schedule. A missed night dose may be taken at any time until midnight of the 
same day.  
5.6 Masking 
Study drug will be double-masked. All study drug will be supplied in identical packaging, 
colour, smell, and appearance to enable double-masked conditions. K-321 and matching 
placebo will be provided in identical packaging so that all investigators, study site staff, 
patients, and clinical monitors will remain masked throughout the study. The IWRS will 
assign study drug to patients at the time of randomisation. Only personnel in IWRS, the 
independent randomisation team within 
 Biostatistics, and clinical supplies will be 
unmasked and will have access to treatment assignments; all other parties involved in the 
study will be fully masked. 
5.6.1 Unmasking Treatment 
A patient’s treatment assignment will not be unmasked for the investigator or study site staff 
until the EOS unless medical treatment of the patient depends on knowing the study 
treatment the patient received. In the rare event that unmasking is needed because of a 


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medical emergency, the investigator may unmask an individual patient through the IWRS. 
Reasons for treatment unmasking must be clearly explained and justified in the eCRF. The 
date on which the code was broken together with the identity of the person responsible must 
also be documented. 
Patients who are unmasked will be allowed to continue their participation in the study; 
however, their data may be excluded from per-protocol analyses. 
5.7 Treatment Compliance 
Patient compliance will be determined from 
 
according to the SOA (Table 6-1). The M/N and MD/E 
 will 
 separately. 
The calculations for determining percentage compliance is presented in Section 7.7.5.2. 
At each visit during the treatment period, study site staff will remind the patient of the need 
to administer the ophthalmic drops as instructed.  
Before each visit (Visit 4 through Visit 10), study site staff will call patients to remind them 
to bring all study drug 
 to the study site for their visit.  
5.8 Prior and Concomitant Therapy 
At screening, the following prior therapies will be recorded in the eCRF: 
• History of prior study eye surgical procedures 
• All medications used within the previous 30 days 
• All surgical procedures within the previous 30 days (any body part) 
At screening, all patients’ prior medications used and surgical procedures undergone within 
the previous 30 days will be recorded in the eCRF. A history of all prior study eye surgeries 
must be obtained and recorded (note exclusion criteria 3 and 4, Section 4.1.2). 
Use of all concomitant medications will be recorded in the patient’s eCRF. The minimum 
requirement is that drug name and the dates of administration are to be recorded. This will 
include all prescription drugs, herbal products, vitamins, minerals, and over-the-counter 


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medications. Any changes in concomitant medications also will be recorded in the patient’s 
eCRF.  
All patients’ surgical procedures undergone concomitantly during the study must be reported 
and recorded in the eCRF.  
Any concomitant medication or surgical procedure deemed necessary for the welfare of the 
patient during the study may be given at the discretion of the investigator. However, it is the 
responsibility of the investigator to ensure that details regarding the medication and surgical 
treatment are recorded in full in the eCRF.  
5.8.1 Descemetorhexis of the Study Eye 
As a condition of participating in the study, each patient will undergo descemetorhexis of the 
investigator-selected study eye at Visit 2 (Day –1). See Section 6.1.2 for details.  
In the absence of suitable local guidance or personal preference of the investigator, 
recommended post-descemetorhexis concomitant therapy consists 
 
 instilled 
 and 
 instilled 
 
. However, an investigator will be 
able to add 
 dosing after 
 if the 
investigator judges it necessary. When more than 1 different eye drop is administered 
(including study drug), the study drug must be administered first, followed by any non-study 
eye drop(s) after at least 5 minutes have passed (Section 5.2).  
A patient must not be randomly assigned to study drug unless the excised diameter satisfies 
inclusion criterion 5 (Section 4.1.1).  
5.8.2 Rescue Surgical Procedures 
Penetrating keratoplasty or endothelial keratoplasty may be offered as rescue surgical 
procedures to an enrolled patient if the investigator judges that endothelial healing is not 
complete at Week 12 (Visit 9), or earlier if the investigator considers that the patient needs 
urgent treatment. If the rescue surgery is performed, the dosing with study drug must be 


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discontinued immediately (without tapering) and the patient will attend all planned study 
visits including the follow-up period. 
5.8.3 Prohibited and Restricted Concomitant Therapy 
5.8.3.1 Prohibited and Restricted Surgical Intervention 
Throughout the study, study eyes must not undergo any surgical intervention other than the 
descemetorhexis at Visit 2. Non-study eyes must not undergo any surgical treatment for 
FECD or cataract in the screening or treatment periods, but they can be surgically treated 
during the follow-up period. The non-study eye will not be eligible for participation in the 
trial, except if the descemetorhexis fails on the first-selected study eye and that eye is not 
eligible to participate. In that case, if all other inclusion and exclusion criteria are met by the 
second eye and more than 30 days have passed since the first study descemetorhexis, then the 
patient can be enrolled using the second eye to qualify. 
Punctal occlusion plugs are considered to be surgical interventions.  
5.8.3.2 Prohibited and Restricted Medication 
The following medications and drugs identified as exclusion criteria are prohibited for use by 
patients throughout the study: 
• Netarsudil or other ROCK inhibitors 
• Eye drops and ointments containing 5% sodium chloride 
• Devices to improve any ocular symptoms (eg, TrueTear®, LipiFlow®, etc)  
• Drugs of abuse (opiates, methadone, cocaine, amphetamines, barbiturates, or 
benzodiazepines) or more than 2 standard drinks of alcohol per day.  Subjects in 
countries which have lower thresholds for alcohol intake must follow their country’s 
own recommendations and limits for alcohol consumption. 
• Contact lenses (except non-study eye) 


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• Any ophthalmic solution besides artificial tears, over-the-counter anti-allergic 
ophthalmic products, study drug, or drugs prescribed by the investigator for treatment 
of the underlying condition 
• Any investigational drug besides study drug 


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6 Study Assessments and Procedures 
Before performing any study procedures, all potential patients will sign an informed consent 
form (ICF). Patients will have the opportunity to have any questions answered before signing 
the ICF. The investigator must address all questions raised by the patient. The investigator or 
designee will also sign the ICF.  
The SOA is presented in Table 6-1. Detailed instructions for study site activities will be 
provided in the study manual. 
 


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Table 6-1 
Schedule of Study Site Activities 
 Activity 
Screening 
Treatment Period 
Follow-Up Period 
Time Point Day -28 
Day –1 
Day 1 
Week 1 Week2 Week 3 Week 5 Week 7 Week 9 Week 12 Week 16 Week 20 Week 24 Week 38 Week 52 
Visit Window (Day) -28 to -7 
- 
- 
5-9 
12-16 
18-24 
32-38 
46-52 
60-66 
81-87 
105-119 133-147 161-175 259-273 357-371 
Visit No. Visit 1 
Visit 2 
Visit 3 
Visit 4 Optional 
Visit 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and 
concomitant disease 
X 
X a 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination and 
vital sign measurements 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Urine drug screen 
X 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Urine pregnancy test (if 
applicable) 
 
X a 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Laboratory sampling  
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Eligibility evaluation 
X 
X b 
 
 
 
 
 
 
 
 
 
 
 
 
 
Descemetorhexis with photo 
or video documentation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense study drug 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 study drug c 
 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
 
 
 
 
Study drug administration 
 
 
X 
X 
(X) d 
X 
X 
X 
X 
X 
 
 
 
 
 
Slit-lamp evaluation 
X 
X a 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal ECD measurement e 
(specular microscopy, 
non-contact type) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal ECD measurement 
by HRT f 
(X) 
 
 
(X) 
(X) d 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 




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6.1 Eligibility Assessments and Procedures 
6.1.1 Medical History, Physical Examination, and Laboratory 
Assessments 
Medical history and assessments of concomitant disease, physical examination and vital sign 
measurements, urine screening for drugs of abuse, pregnancy screening for patients of 
childbearing potential, and clinical laboratory assessments will be conducted at screening 
primarily to assess eligibility to participate in the study. The limited physical examination 
includes a review of the patient’s medical and medication history and subsequent evaluation 
of relevant body system complaints. In case of relevant findings, full evaluation of the 
condition needs to be conducted. Demographics information will be collected at screening as 
follows: age, race, iris colour, and sex. 
6.1.2 Descemetorhexis of the Study Eye 
Each patient will undergo descemetorhexis of the investigator-selected study eye at Visit 2 
(Day –1). A digital photo of the study eye must be taken immediately before and immediately 
after descemetorhexis at Visit 2. The investigator will perform descemetorhexis to remove 
central confluent guttae, removing an area of Descemet membrane with a diameter of 
 
. The longest diameter and the shortest diameter must be recorded in the eCRF. The 
procedure must be recorded by video to record potential differences in technique among 
investigators. Also, immediately after the operation, the stripped area will be recorded by 
photo or video with an adjacent scale to measure the area removed. Detailed guidance for 
performing the study eye descemetorhexis will be provided in the study manual. 
Recommended post-descemetorhexis concomitant therapy is described in Section 5.8.1. 
6.2 Efficacy Assessments 
6.2.1 Corneal Endothelial Cell Density Measurement 
6.2.1.1 Non-Contact Specular Microscopy 
Corneal ECD will be measured by non-contact specular microscopy. The study eye must be 
assessed at all study visits as indicated in Table 6-1, whereas the non-study eye must be 
measured only at Visit 1 and Visit 14. The same instrument must be used for all study visits 


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for a given patient throughout the study. All specular microscope images taken will be sent to 
the 
 for analysis via a designated portal site. Please see the study manual for detailed 
instructions.  
6.2.1.2 Heidelberg Retina Tomography  
Heidelberg retina tomography (HRT) of the study eye will be performed according to the 
schedule in Table 6-1 if the necessary equipment is available at the study site before the start 
of the study at that site. All HRT images taken will be sent to the 
 for analysis via a 
designated portal site. Please see the study manual for detailed instructions.  
6.2.2 Corneal Thickness 
Corneal thickness of the study eye will be measured by contact ultrasound pachymetry on the 
central cornea. The same instrument must be used for all study visits for a given patient 
throughout the study. From a safety perspective, the corneal thickness of both eyes must be 
measured, and the results must be recorded. See the study manual for detailed instructions.  
6.2.3 Corneal Oedema  
Corneal oedema of the study eye will be categorised by epithelial, stromal, or endothelial, 
and assessed by the investigator as either present or absent.   
6.2.4 Corneal Morphology 
Corneal morphology of the study eye will be assessed by OCULUS Pentacam® or Bausch 
and Lomb Orbscan®. If the site has both instruments, the OCULUS Pentacam will be used. 
The same instrument must be used for all study visits for a given patient throughout the 
study. All data and images must be recorded.  
6.2.5 Best Corrected Visual Acuity  
The BCVA of the study eye will be assessed by ETDRS testing without pupil dilation. For all 
measurements, the BCVA letter score will be recorded. Please see the study manual for 
detailed instructions on the BCVA ETDRS assessment.  




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Ocular examination will be performed with pupil dilation using an indirect ophthalmoscope 
according to the current standard of practice to evaluate the condition of the vitreous, macula, 
retina, optic nerve, choroid, and retinal periphery.  
 
6.3.2 Adverse Events 
6.3.2.1 Definitions of Adverse Events 
An AE is defined as any untoward medical occurrence in a patient enrolled into this study 
regardless of its causal relationship to study drug. An AE can therefore be any unfavourable 
and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related 
to the medicinal product. Patients will be instructed to contact the investigator at any time 
after randomisation if any symptoms develop. 
An AE may include the following: 
• Exacerbation of a pre‑existing disease or symptom 
• Increase in frequency or intensity of a pre‑existing event or condition 
• A condition detected or diagnosed after study drug administration even though it may 
have been present prior to the start of the study 
An AE does not include the following:  
• Medical or surgical procedures (although the condition that leads to the procedure 
must be reported as an AE) 
• Pre‑planned medical procedures (eg, planned surgical interventions, investigations, 
social and/or convenience hospitalisations) 
• Pre‑existing disease or conditions present at the time of signing the ICF are 
considered concurrent medical conditions and must not be recorded as an AE 


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The investigator must attempt to establish a diagnosis of the event based on signs, symptoms, 
and other clinical information. In such cases, the diagnosis must be documented as the AE 
and not as the individual signs or symptoms.  
The investigator will evaluate any changes in laboratory values and other study investigations 
and determine whether the change is clinically important and whether it is related to the 
study drug. The investigator must record the AE or investigation abnormality in the eCRF 
regardless of the relationship to the study drug (ie, the AE will be recorded whether or not it 
is considered related to the study drug). 
Considerations for determining whether an abnormal objective test finding must be reported 
as an AE may include whether or not:: 
• The test result is associated with accompanying symptoms 
• The test result requires additional diagnostic testing or medical/surgical intervention 
• The test result leads to a change in study drug dosing or discontinuation from the 
study, significant additional concomitant drug treatment or other therapy and/or 
• The test result leads to any of the outcomes included in the definition of an SAE 
Merely repeating an abnormal test, in the absence of any of the above conditions, does not 
require reporting as an AE. An abnormal test result that is determined to be an error or 
artefact does not require reporting as an AE. 
A treatment-emergent AE (TEAE) is defined as any event not present before exposure to 
study drug or any event already present that worsens in either intensity or frequency after 
exposure to study drug. 
An SAE is defined as any event that 
• results in death.  
Any death resulting from an AE occurring during the study period or within 30 days after 
the last dose of the study drug, even if the death appears to be completely unrelated to the 
study or study drug. 


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• is immediately life threatening.  
The term ‘life‑threatening’ refers to an event in which the patient was at risk of death at 
the time of the event; it does not refer to an event that hypothetically might have caused 
death if it were more severe. 
• requires inpatient hospitalisation or prolongation of existing hospitalisation.  
The term ‘hospitalisation’ means a hospital admission or an overnight stay; prolongation 
means delay of a planned or anticipated discharge date by at least 1 overnight stay. 
Adverse events which require attendance at hospital but do not require admission may be 
considered medically important. 
• results in persistent or significant disability/incapacity. 
• results in impairment, damage, or disruption in the study patient’s body function or 
structure, or physical ability or quality of life.  
• is a congenital anomaly/birth defect.  
Additionally, events in which there is suspicion that exposure of either parent to the study 
drug resulted in an adverse outcome in the offspring is a medically important event. 
• Is medically important: an event that may not be immediately life‑threatening or fatal or 
result in hospitalisation, but may require intervention to prevent one of the serious 
outcomes listed above.  
Examples of such events are intensive treatment in an emergency room or at home for 
allergic bronchospasm; blood dyscrasias; convulsions that do not result in hospitalisation; 
or development of drug dependency or drug abuse.  
6.3.2.2 Eliciting and Documenting Adverse Events 
The investigator is responsible for reporting AEs that are observed or reported during the 
study, regardless of their relationship to study drug or their clinical significance. Serious AEs 
will be collected from the time that the patient gives consent to participate in the study until 
30 days after the date of last dosing or of completion or withdrawal from the study, 
whichever is latest. All AEs will be collected starting from the time that the patient gives 
consent until the date of completion or withdrawal from the study. 


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Serious AEs that occur more than 30 days after the last dose of study drug or after exit from 
the study, whichever is later, need not be reported unless the investigator considers them 
related to study drug. 
At each study visit, patients will be asked a standard non-leading question to elicit any 
medically related changes in their well-being. They will also be asked if they have been 
hospitalised, had any accidents, had any eye surgery, used any new medications, or changed 
concomitant medication regimens (both prescription and over-the-counter medications). 
In addition to patient observations, AEs identified from any study data (eg, laboratory values, 
physical examination findings) or identified from review of other documents  that are 
relevant to patient safety will be documented on the AE page in the eCRF. 
6.3.2.3 Reporting Adverse Events 
All AEs reported or observed  will be recorded on the AE page of the eCRF. Information to 
be collected includes event term, time of onset, date of resolution of the event, seriousness, 
severity, relatedness to study drug, any required treatment or evaluations, and outcome.  
The investigator will assess the severity of the event. The severity (intensity) of AEs must be 
classified using the following criteria:  
• Mild: An AE that is easily tolerated by the patient, causes minimal discomfort, and 
does not interfere with everyday activities 
• Moderate: An AE that causes enough discomfort to interfere with normal everyday 
activities and may require intervention 
• Severe: An AE that prevents normal everyday activities (treatment or intervention 
will usually be required). Severe events are usually incapacitating 
When changes in the severity of an AE occur more frequently than once a day, the maximum 
intensity for the event must be noted for that day. Any changes in intensity of signs and 
symptoms over multiple days will be captured by recording new AEs, with the amended 
intensity grades and the dates (and time, if known) of the change(s). Changes in the intensity 


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of an AE must be documented to allow an assessment of the duration of the event at each 
level of intensity.  
The investigator will assess the causal relationship between the study medication and the AE 
and decide whether, in his or her qualified medical judgement, there is a reasonable 
possibility that the event may have been caused by the study drug. There are 2 categories of 
causality:  
• Unrelated: If no valid reason exists for suggesting a relationship, or the event may 
plausibly be related to the patient's clinical condition, an underlying disease or other 
medication taken by the patient, then the AE must be considered unrelated.  
• Related: If there is any valid reason for supposing that there is a relationship between 
the study medicine and the AE, especially if the even improves on withdrawal of the 
drug (de-challenge) or recurs on recommencement of therapy (re-challenge), then the 
AE must be considered related. The AE must not be considered related simply 
because it is not possible to rule out an association between the AE and the study 
drug. 
The investigator’s assessment of an AE’s relationship to study drug is part of the 
documentation process, but it is not a factor in determining what is or is not reported in the 
study. If there is any doubt as to whether a clinical observation is an AE, the event must be 
reported. 
The action taken with the study medication must be recorded as treatment continued, 
treatment interrupted, or treatment discontinued. If the patient is no longer taking study 
medication at the time of the event, then action taken must be recorded as not applicable.  
All AEs will be followed to adequate resolution. The Medical Dictionary for Regulatory 
Activities (MedDRA) will be used to code all AEs.  
Any AE that meets SAE criteria (Section 6.3.2.1) must be reported to the 
 
Pharmacovigilance Department immediately (ie, within 24 hours) after the time site staff first 
learn about the event, using the electronic data capture (EDC) system. If for any reason the 
EDC is not available to the study site, the following contact information is to be used for 
SAE reporting: 


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 Pharmacovigilance Department 
 
 
 
 
If the initial contact to 
 Pharmacovigilance is made via the SAE Hotline or the SAE fax 
line, the SAE must still be reported, urgently, using the EDC system when it becomes 
available. Details on SAE reporting are presented in the study manual.  
6.3.2.4 Suspected Unexpected Serious Adverse Reactions  
The sponsor will promptly evaluate all suspected unexpected serious adverse reactions 
(SUSARs) against cumulative product experience to identify and expeditiously communicate 
possible new safety findings to investigators, IRBs/IECs, and applicable health authorities 
based on applicable legislation. 
To determine reporting requirements for single SUSAR cases, the sponsor will assess the 
expectedness of these events using the Reference Safety Information section of the study 
drug investigator's brochure.  
The sponsor will compare the severity of each SUSAR and the cumulative event frequency 
reported for the study with the severity and frequency reported in the applicable reference 
document. 
6.3.2.5 Follow-Up of Adverse Events 
All AEs must be reported in detail on the appropriate page in the eCRF and followed to 
resolution, until the investigator deems the event to be chronic or not clinically significant 
(NCS), or until the patient is considered stable. Adverse events that are ongoing at the time of 
the EOS visit are to be followed for 30 days or until resolution, until the investigator deems 
the no longer clinically significant (CS), or until the investigator considers the patient’s 
condition stable. 
6.3.3 Vital Sign Measurements 
The following vital signs will be measured at screening and at Visit 9: 


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• Blood pressure (measured supine, systolic and diastolic [mm Hg]) 
• Pulse (beats per minute) 
• Body temperature (°F ) 
• Height (cm) and body weight (kg ). Height is to be measured at screening only. 
Supine blood pressure, pulse, and body temperature (°F) will be recorded after the patient has 
been recumbent and at rest for at least 5 minutes. 
6.3.4 Eligibility 
Eligibility will be evaluated at screening (Visit 1) and post-operatively on Day –1 (Visit 2). 
Patients must meet all inclusion criterion and no exclusion criteria to be assigned study drug.  
6.4 Safety Monitoring Committee 
There will be no safety monitoring committee.  
6.5 Pregnancy 
Female patients of childbearing potential (defined in inclusion criterion 5 of Section 4.1.1) 
are required to use effective contraception throughout the study. 
Acceptable methods of contraception for female patients include oral contraceptives, other 
hormonal contraceptives (vaginal products, skin patches, or implanted/injectable products), 
or mechanical products such as an intrauterine device or barrier methods (diaphragm, 
condoms, spermicides). Note: female patients who are actively practising abstinence or who 
have a partner that is sterile (eg, vasectomy) will be permitted to participate in the study 
without contraception use. 
Pregnancy is not regarded as an AE unless there is a suspicion that an investigational product 
may have interfered with the effectiveness of a contraceptive medication. Any pregnancy that 
occurs during study participation must be reported using a clinical study pregnancy form. 
To ensure patient safety, each pregnancy must be reported to 
sponsor within 2 weeks 
of learning of its occurrence. The pregnancy must be followed up to determine outcome 
(including spontaneous miscarriage, elective termination, normal birth, or congenital 
abnormality) and status of mother and child even if the patient was discontinued from the 


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study. Pregnancy complications and elective terminations for medical reasons must be 
reported as an AE or SAE. Spontaneous miscarriages and congenital abnormalities of the 
child must be reported as an SAE. 
Any SAE occurring in association with a pregnancy, brought to the investigator’s attention 
after study completion, and considered by the investigator as possibly related to the study 
treatment, must be promptly reported to 
sponsor. 
6.6 Laboratory Analyses 
Routine laboratory analyses will be performed by a central laboratory and will include the 
following assessments: 
• Haematology: haemoglobin, haematocrit, red blood cell count, white blood cell 
count with differential, mean corpuscular volume, mean corpuscular haemoglobin, 
mean corpuscular haemoglobin concentration, and absolute platelet count. 
• Clinical chemistry: total protein, sodium, potassium, chloride, albumin, glucose, 
HbA1c, blood urea nitrogen, creatinine, bilirubin (total and direct), alkaline 
phosphatase, aspartate aminotransferase, alanine aminotransferase, 
γ-glutamyltransferase, lactate dehydrogenase. 
• Urinalysis: pH, glucose, ketones, specific gravity, nitrite, protein, bilirubin, 
urobilinogen, and blood. Microscopic urinalysis will be performed if urinalysis results 
are abnormal. 
Urine pregnancy testing will be performed for all patients of childbearing potential before 
descemetorhexis at Visit 2 and at Visit 10.  
Urine screening for drugs of abuse (opiates, methadone, cocaine, amphetamines, barbiturates 
benzodiazepines) and alcohol will be performed by dipstick.  
6.6.1 Future Genetic Testing 
A 6-mL blood sample will be obtained at Visit 1 for archiving and future genetic testing 
(Section 2.3). The blood sample will be collected only at sites in countries where allowed by 
local regulations and where approved by the IRB/IEC and relevant regulatory authorities.  


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Results of genetic testing will not be reported to the patient, relatives, or attending physician, 
and they will not be recorded in the participant's medical record. The participant may 
withdraw consent for genetic testing at any time up to analysis, even after the samples have 
been obtained. In the event of withdrawal of consent, the bio-repository will be notified to 
pull and destroy the sample. 
For all samples collected for genetic testing, precautions are to be taken to maintain 
confidentiality by de-identifying the sample to prevent the genetic data from being linked to 
the identity of the participant.  
6.7 Sample Collections 
Sample collection, processing, and shipping must be performed according to local laboratory 
requirements and consistent with detailed procedures described in the study manual. 
The volume of blood sampled is planned to be less than 20 mL in a single day and less than 
35 mL over the entire study.  


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7 Statistical and Analytical Plan 
A study eye will be identified for each patient. Unless otherwise specified, efficacy will be 
summarised for the study eye only. Ocular safety summaries by eye will be presented 
separately for the study eye and the non-study eye. Baseline measurements are the last 
measurements taken during the screening period (before Visit 2).  
7.1 Primary Efficacy Endpoint 
The primary efficacy endpoint is the central corneal ECD at Week 12 as assessed by the 
 
7.2 Secondary Efficacy Endpoints 
Secondary efficacy endpoints include: 
• Central corneal thickness change from baseline to each subsequent visit 
• Percent change of central corneal thickness from baseline to each subsequent visit 
• Central corneal ECD at each visit  
• Number and percentage of patients who achieve central corneal ECD of 
700 cells/mm2 or more at each visit 
• Time to achieve central corneal ECD 700 cells/mm2 or more  
• Number and percentage of patients who achieve cornea clearance at each visit where 
corneal clearance is defined as patients achieving a cornea thickness less than or 
equal to baseline cornea thickness and who have no corneal oedema 
o Number and percentage of patients who achieve cornea thickness less than or 
equal to baseline cornea thickness 
o Number and percentage of patients who achieve no corneal oedema 
• Time to cornea clearance  




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and K-321 was assumed to be distributed normally with 
. 
Proportion of unmeasurable value of Week 12 due to corneal oedema and proportion of 
 value of Week 12 due to the withdrawal were assumed to be about 10% and about 
5%, respectively. Here, unmeasurable value of Week 12 due to corneal oedema were imputed 
as zero, and 
 value of Week 12 due to the withdrawal were imputed by the last-
observation-carried-forward (LOCF) method. 
7.5 Analysis Sets 
The following analysis sets will be used in the statistical analyses. 
Full-analysis set (FAS): The FAS will consist of all participants who have a successful 
descemetorhexis procedure performed, were randomly assigned to receive double-mask 
study drug, received at least one dose of study drug in the study eye, and have at least one 
assessment of central corneal ECD performed after the descemetorhexis procedure on the 
study eye. (An assessment of “cannot perform due to corneal oedema” is considered a valid 
assessment for inclusion in the FAS.) All analyses using the FAS will group participants 
according to randomised treatment.  
Per-protocol set (PPS): The PPS will consist of all FAS participants who complete the 
12-week dosing treatment period for the study eye (for the primary analysis only), have at 
least 80% compliance with study treatment for the study eye, have not taken any prohibited 
medication, and have no major protocol deviations. Patients who receive rescue therapy will 
be considered to have completed study treatment for inclusion in the PPS. All analyses using 
the PPS will group participants according to treatment actually received.  
Safety set (SFS): The safety set will consist of all participants who received any study drug. 
All analyses using the safety set will group participants according to treatment actually 
received. 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy analyses 
will be repeated for the PPS. Safety will be summarised using the SFS.  
7.6 Description of Subgroups to be Analysed 
Due to the small sample size, no subgroup analyses are planned.  


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7.7 Statistical Analysis Methodology 
Statistical analysis will be performed using SAS software Version 9.4 or later. Continuous 
variables will be summarised using the mean, the SD, median, minimum value, and 
maximum value. Categorical variables will be summarised using frequency counts and 
percentages. Unless otherwise noted, the denominator to determine the percentage of 
participants in each category will be based on the number of participants with available data. 
Selected ordinal data may be summarised using both descriptive statistics and counts and 
percentages of participants in each category, as appropriate. Data will be listed in data 
listings. 
Details of the statistical analyses, methods, and data conventions are described in the 
statistical analysis plan (SAP). 
All statistical tests will be 2-sided and performed using a 0.05 significance level, leading to 
95% (2-sided) CIs. 
A closed 2-step ordered testing procedure will be used to control overall type I error for the 
primary efficacy endpoint, central corneal ECD at Week 12. In step 1, the K-321 0.4% QID 
group will be statistically compared with the placebo group. If the step 1 comparison is 
significant, step 2 will be performed to statistically compare K-321 0.4% BID group with the 
placebo group. If the statistical comparison made during step 1 is not significant, testing will 
still be performed to compare the BID group to placebo; however, all p values will be 
considered nominal/descriptive only. 
No other measures will be taken to control error for the multiple secondary and exploratory 
endpoints or multiple treatment comparisons. 
7.7.1 Analysis of Primary Efficacy Endpoint 
The primary analysis of central corneal ECD will be performed on the study eye within the 
FAS via a Wilcoxon rank sum test and will be based on assessments performed by specular 
microscopy. Testing will be performed in a pairwise manner with each dosing regimen (QID 
and BID) of K-321 0.4% compared to placebo. For each dosing regimen, the following 
2-sided hypothesis will be tested  


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Ho: Δ = 0 versus Ha: Δ ≠ 0 
where Δ is the shift in location in central corneal ECDs between the active and placebo 
groups being compared: ie,  
Xi = ei for i = 1 to n for the placebo patients 
Yj = ej + Δ for j = 1 to m for the K-321 patients 
The Hodges-Lehmann estimate (Hodges and Lehmann 1963) for the shift in location (eg, the 
median of all differences d=Yj – Xi) and Moses 95% CI (Moses 1965) will also be presented.  
Week 12 assessments that are unable to be performed due to corneal oedema will be 
 
 
 
. 
Week 12 assessments that are 
 due to patient withdrawal or missed visit will be 
. Patients who receive a 
rescue keratoplasty performed prior to Week 12 will be 
 
 
 
 
 
  
Sensitivity analyses will be performed by repeating the above analysis and 
 
 for the PPS. The primary analysis methods will also be repeated for the FAS using 
 
 Additionally, analyses will be performed using assessments performed by 
HRT for the subset of patients for which the assessments are available.  
7.7.2 Analysis of Secondary Efficacy Endpoints 
Secondary endpoints will be summarised for the FAS. Central ECD which cannot be 
performed due to corneal oedema will be 
 for analysis. Assessments of central 


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ECD, central corneal thickness, and epithelial oedema after rescue keratoplasty will be 
 
 Otherwise, secondary efficacy analyses will be 
 
Endpoints based on continuous measures (central ECD at each visit, central corneal thickness 
change from baseline to each visit, and percent change of central corneal thickness from 
baseline to each visit) will be analysed by pairwise Wilcoxon rank sum tests comparing each 
active K-321 0.4% treatment. The Hodges-Lehmann estimate and Moses 95% CI for the shift 
in location will also be presented regimen to placebo.  
Endpoints based on the number and percentage of patients achieving the endpoint (patients 
who achieve central ECD of 700 cells/mm2 or more, patients who achieve cornea clearance 
at each visit, patients who achieve a cornea thickness less than or equal to baseline cornea 
thickness at each visit, and patients who have no corneal oedema at each visit) will be 
analysed by pairwise Fisher’s Exact or Pearson chi-square tests comparing each active K-321 
0.4% treatment regimen to placebo. The 95% CI for the difference in percentage of patients 
achieving the endpoint will also be presented. 
Endpoints based on time-to-event endpoints (time to achieve central ECD 700 cells/mm2 or 
more, time to cornea clearance, time to return of cornea thickness to less than or equal to 
baseline cornea thickness, time to no corneal oedema) will be analysed by pairwise log-rank 
tests comparing each active K-321 0.4% treatment regimen to placebo. Kaplan-Meier 
estimates and plots will also be presented. Patients who complete follow-up or discontinue 
the study without achieving the specified endpoint will be 
 
 Patients who receive rescue keratoplasty will be 
 
  
7.7.3 Analyses of Exploratory Efficacy Endpoints 
Exploratory endpoints will be summarised for the FAS. Assessments after rescue keratoplasty 
will be 
. Endpoints will be analysed in a similar manner to the secondary 
efficacy endpoints.  
Endpoints based on continuous measures will be analysed by pairwise Wilcoxon rank sum 
tests comparing each active K-321 0.4% treatment regimen to placebo with the 
Hodges-Lehmann estimate for the different in medians and Moses 95% CI for the difference 
in medians also presented. 


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Endpoints based on the number and percentage of patients achieving the endpoint will be 
analysed by pairwise Pearson chi-square tests comparing each active K-321 0.4% treatment 
regimen to placebo with the 95% CI for the difference in percentage of patients achieving the 
endpoint also presented. 
Endpoints based on time-to-event endpoints will be analysed by pairwise log-rank tests 
comparing each active K-321 0.4% treatment regimen to placebo with Kaplan-Meier 
estimates and plots also presented. Patients who complete follow-up or discontinue the study 
without achieving the specified endpoint will be 
 
 Patients who receive rescue keratoplasty will be 
 
 
7.7.4 Safety Analyses 
Safety parameters include monitoring of AEs, TEAEs, vital sign measurements, laboratory 
examinations, slit-lamp biomicroscopy, IOP, and ophthalmoscopy. 
Safety data will be summarised and listed with no statistical hypothesis testing performed. 
Continuous variables will be summarised using the mean, the SD, median, minimum value, 
and maximum value. Categorical variables will be summarised using frequency counts and 
percentages. 
7.7.4.1 Adverse Events  
All AEs will be listed and all TEAEs will be summarised. Adverse events will be deemed 
treatment-emergent if the onset date is on or after the date of first treatment. Events which are 
not treatment-emergent will be listed separately and together with all other AEs. AEs will be 
summarised separately for the treatment period, the tapering phase, and the 9-month 
follow-up period. Ocular TEAEs will be summarised separately from non-ocular TEAEs. 
Ocular TEAEs will be summarised separately for the study eye and non-study eye.  
Treatment-emergent AEs will be summarised by treatment group, presenting the number and 
percentage of patients having a TEAE in each system organ class and having each individual 
TEAE based on the preferred term. Treatment-emergent AEs will also be tabulated according 
to intensity and causality. Patients who experienced multiple TEAEs for a preferred term will 
be counted once, similarly for patients with multiple TEAEs per system organ class. 


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Deaths, SAEs, and TEAEs leading to discontinuation of study treatment will be listed 
separately and, if appropriate, summarised by treatment group, system organ class and 
preferred term. 
7.7.4.2 Vital Signs 
Observed values at each visit and change from baseline values for vital sign measurements 
will be summarised descriptively by treatment group. All vital sign measurement data will be 
listed. 
7.7.4.3 Clinical Laboratory  
Observed values at each visit and change from baseline values for clinical laboratory 
evaluations will be summarised descriptively by treatment group. Additionally, shift tables 
will be presented for chemistry and hematology parameters from baseline to each post-
baseline assessment. All clinical laboratory data will be listed. 
7.7.4.4 Intra-Ocular Pressure 
All IOP data will be listed. Observed values at each visit and change from baseline values for 
IOP evaluations will be summarised descriptively by treatment group for the study eye and 
non-study eye.  
7.7.4.5 Slit-Lamp Biomicroscopy and Ophthalmoscopy 
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal (NCS) 
• Abnormal with clinical significance (CS) 
Data from all slit-lamp biomicroscopy and ophthalmoscopy assessments will be listed. The 
number and percentage of patients with at least 1 treatment-emergent CS abnormality by 
ocular assessment will be summarised for each treatment group for the study eye and 
non-study eye. An abnormality will be deemed treatment-emergent if the onset date is on or 


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after the date of first treatment. A patient will be counted once even if the patient experiences 
more than 1 treatment-emergent CS abnormality.  
7.7.5 Other Analyses 
7.7.5.1 Patient Demographics and Other Baseline Characteristics 
Descriptive statistics will be provided by treatment group for patient demographics and all 
ocular and baseline characteristics. Relevant medical history (ocular and non-ocular) and 
current medical conditions will be tabulated by treatment group. Ocular summaries will be 
presented for the study eye and the non-study eye. Other relevant baseline information will 
be listed and summarised as appropriate with descriptive statistics. Analyses will be based on 
the FAS. 
7.7.5.2 Treatment Exposure and Compliance 
Descriptive statistics will be provided to characterise study treatment exposure and 
compliance using the safety set. Summaries will also be provided for the FAS if it differs 
from the safety set. Duration of exposure will be calculated in days as the (date of last 
dose – date of first dose + 1). Percent compliance will be calculated as the total number of 
study drug instillations/planned number of instillations × 100. The planed number of 
instillations for Visit 3 (Day 1) will be adjusted based on the time the descemetorhexis was 
completed on Visit 2 (Day –1) and the time all study visit assessments are completed on Visit 
3. For all other study visits during the treatment period, the planed number of instillations 
will be adjusted based on the time all planned study visit assessments are completed on the 
respective study visit days, as activities on these days may reduce the number of doses that 
can be given. The details of dosing adjustment will be provided in the SAP. For all other 
study days, the number of planned instillations will be considered to be 4 doses. If the first 
and last doses are administered on the same day, the number of planned instillations will be 
based on the time of the first dose. Subjects will be considered compliant with study 
treatment if the calculated percent compliance is between 80% and 125%, inclusive. 
 


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7.7.6 Interim Analyses 
No interim analyses will be performed. The primary analysis will be conducted at the 
Week 12 database lock ie, after all patients have completed dose tapering at the Week 16 visit 
or have been discontinued from the study and/or treatment before this visit. Analysis of the 
complete study data (through Week 52) will be performed after all patients have completed 
the Week 52 visits or have been discontinued from the study before the Week 52 visit. As the 
primary efficacy endpoint is at the Week 12 visit, no alpha adjustments will be required. No 
changes to study conduct (eg, change to sample size or early stopping) are planned.  


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8 Data Quality Assurance 
The sites will maintain source documentation and enter patient data into the eCRF as 
accurately as possible and will rapidly respond to any reported discrepancies. Electronic 
CRFs are accessed through Medidata Rave® (Medidata Solutions Inc, New York, New York). 
This EDC system is validated and compliant with US Title 21 Code of Federal Regulations 
(CFR) Part 11. Each person involved with the study will have an individual user name and 
password that allows for record traceability. Thus, the system, and subsequently any 
investigative reviews, can identify coordinators, investigators, and individuals who have 
entered or modified records, as well as the time and date of any modifications. A quality 
review of the data will be performed by the site with additional reviews by the clinical 
monitor through source data verification.  
Each eCRF is presented as an electronic copy, allowing data entry by site staff, who can 
add and edit data, identify and resolve discrepancies, and view records. This system provides 
immediate direct data transfer to the database, as well as immediate detection of 
discrepancies, enabling site coordinators to resolve and manage discrepancies in a timely 
manner.  
Paper copies of the eCRFs and other database reports may be printed by the investigator. 
This system provides site staff, monitors, and reviewers with access to hard copy audits, 
discrepancy reviews, and investigator comment information.  
After all data reviews and query resolutions are complete, the SAP is approved and signed, 
and any summary/analysis populations are approved, the database will be locked. 
8.1 Data Management 
As part of the responsibilities assumed by participating in the study, the investigator agrees 
to maintain adequate case histories for patients treated as part of the research under this 
protocol. The investigator agrees to maintain accurate eCRFs and source documentation as 
part of the case histories. These source documents may include laboratory reports, etc. All 
eCRF information is to be completed. If an item is not available or is not applicable, this fact 
must be indicated. Blank spaces must not be present unless otherwise directed. 


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Investigative site personnel will enter patient data into an EDC system. The analysis data sets 
will be a combination of these data and data from other sources (eg, laboratory data). 
Clinical data management will be performed in accordance with applicable 
 standards 
and data cleaning procedures to ensure the integrity of the data (eg, removing errors and 
inconsistencies in the data). Adverse events and medical history will be coded using the 
MedDRA terminology. Concomitant medications will be coded using the most current 
available WHO Drug Dictionary. 
After database lock, each study site will receive an electronic copy of their site-specific 
eCRF data as entered into the EDC system for the study, including full discrepancy and audit 
history. Additionally, an electronic copy of all the study site’s data from the study will be 
created and sent to the sponsor for storage. 
 will maintain a duplicate electronic copy for 
their records. In all cases, patient initials will not be collected or transmitted to the sponsor. 


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9 Ethics 
9.1 Independent Ethics Committee or Institutional Review Board 
Country regulations, US federal regulations, and the International Council for Harmonisation 
(ICH) guidelines require that approval be obtained from an IRB or IEC before participation 
of human patients in research studies. Before study onset, the protocol, informed consent, 
advertisements to be used for the recruitment of study patients, and any other written 
information regarding this study to be provided to the patient or the patient’s legal guardian 
must be approved by the IRB/IEC. Documentation of all IRB/IEC approvals and of the 
IRB/IEC compliance with ICH harmonised tripartite guideline E6(R2): Good Clinical 
Practice (GCP) and with applicable regulations in the countries where the study will be 
conducted will be maintained by the site and will be available for review by the sponsor or its 
designee. 
All IRB/IEC approvals must be signed by the IRB/IEC chairman or designee and must 
identify the IRB/IEC name and address, the clinical protocol by title or protocol number or 
both, and the date approval or a favourable opinion was granted. 
The investigator is responsible for providing written summaries of the progress and status of 
the study at intervals not exceeding 1 year or otherwise specified by the IRB/IEC. The 
investigator must promptly supply the sponsor or its designee, the IRB/IEC, and, where 
applicable, the institution, with written reports on any changes significantly affecting the 
conduct of the study or increasing the risk to patients. 
9.2 Ethical Conduct of the Study 
The study will be performed in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki, ICH GCP, the protocol, and all applicable country and local 
regulations. 
9.3 Patient Information and Consent 
A written informed consent in compliance with regulatory authority regulations or 
US Title 21 CFR Part 50 shall be obtained from each patient before entering the study or 
performing any unusual or non-routine procedure that involves risk to the patient. An 
informed consent template may be provided by the sponsor to investigative sites. If any 


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institution-specific modifications to study-related procedures are proposed or made by the 
site, the consent must be reviewed by the sponsor or its designee or both before IRB/IEC 
submission. Once reviewed, the consent will be submitted by the investigator to his or her 
IRB/IEC for review and approval before the start of the study. If the ICF is revised during the 
course of the study, all active participating patients must sign the revised form. 
Before recruitment and enrollment, each prospective patient or his or her legal guardian will 
be given a full explanation of the study and be allowed to read the approved ICF. Once the 
investigator is assured that the patient/legal guardian understands the implications of 
participating in the study, the patient/legal guardian will be asked to give consent to 
participate in the study by signing the ICF. The investigator will then sign the consent signed 
by the patient/legal guardian.  In Germany, only patients capable of giving informed consent 
personally will be included in the study. 
The investigator shall retain the signed original ICF(s) and give a copy of the signed original 
form to the patient or legal guardian. 


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10Investigator’s Obligations 
The following administrative items are meant to guide the investigator in the conduct of the 
study but may be patient to change based on industry and government standard operating 
procedures, working practice documents, or guidelines. Changes will be reported to the 
IRB/IEC but will not result in protocol amendments. 
10.1 Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain patient confidentiality. All records will be kept in a secure 
storage area with limited access. Clinical information will not be released without the written 
permission of the patient (or the patient’s legal guardian), except as necessary for monitoring 
and auditing by the sponsor, its designee, the US Food and Drug Administration (FDA), the 
Medicines and Healthcare Products Regulatory Agency (MHRA), other agencies, or the 
IRB/IEC. 
The investigator and all employees and coworkers involved with this study may not disclose 
or use for any purpose other than performance of the study any data, record, or other 
unpublished, confidential information disclosed to those individuals for the purpose of the 
study. Prior written agreement from the sponsor or its designee must be obtained for the 
disclosure of any said confidential information to other parties. 
10.2 Financial Disclosure and Obligations 
Investigators are required to provide financial disclosure information to allow the sponsor to 
submit the complete and accurate certification or disclosure statements required under 
21 CFR 54. In addition, the investigator must provide to the sponsor a commitment to 
promptly update this information if any relevant changes occur during the course of the 
investigation and for 1 year following the completion of the study. 
Neither the sponsor nor 
 is financially responsible for further testing or treatment of any 
medical condition that may be detected during the screening process. In addition, in the 
absence of specific arrangements, neither the sponsor nor 
 is financially responsible for 
further treatment of the patient’s disease. 


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10.3 Investigator Documentation 
Prior to beginning the study, the investigator will be asked to comply with ICH E6(R2) 8.2 
and Title 21 of the CFR by providing the following essential documents, including but not 
limited to: 
• IRB/IEC approval. 
• Original investigator-signed investigator agreement page of the protocol. 
• Form FDA 1572, fully executed, and all updates on a new fully executed Form 
FDA 1572. 
• Curriculum vitae (CV) for the investigator and each sub-investigator listed on Form 
FDA 1572. 
• Financial disclosure information to allow the sponsor to submit complete and accurate 
certification or disclosure statements required under 21 CFR 54. In addition, the 
investigators must provide to the sponsor a commitment to promptly update this 
information if any relevant changes occur during the course of the investigation and for 
1 year after the completion of the study. 
• IRB/IEC-approved informed consent, samples of site advertisements for recruitment for 
this study, and any other written information regarding this study that is to be provided to 
the patient or legal guardian, and 
• Laboratory certifications and normal ranges for any local laboratories used by the site, in 
accordance with 42 CFR 493. 
10.4 Study Conduct 
The investigator agrees that the study will be conducted according to the principles of ICH 
E6(R2). The investigator will conduct all aspects of this study in accordance with all 
applicable national, state, and local laws or regulations. Study information from this protocol 
will be posted on publicly available clinical trial registers before enrollment of patients 
begins. 




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Data are the property of the sponsor and cannot be published without prior authorisation from 
the sponsor, but data and publication thereof will not be unduly withheld. 




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11.1.2 Monitoring of the Study 
The clinical monitor, as a representative of the sponsor, has the obligation to follow the study 
closely. In doing so, the monitor will visit the investigator and study site at periodic intervals, 
in addition to maintaining necessary telephone and letter contact. The monitor will maintain 
current personal knowledge of the study through observation, review of study records and 
source documentation, and discussion of the conduct of the study with the investigator and 
personnel. 
All aspects of the study will be carefully monitored, by the sponsor or its designee, for 
compliance with applicable government regulation with respect to current GCP and current 
standard operating procedures. 
11.1.3 Inspection of Records 
Investigators and institutions involved in the study will permit study-related monitoring, 
audits, IRB/IEC review, and regulatory inspections by providing direct access to all study 
records. In the event of an audit, the investigator agrees to allow the sponsor, representatives 
of the sponsor, or a regulatory agency (eg, FDA or other regulatory agency) access to all 
study records. 
The investigator must promptly notify the sponsor and 
 of any audits scheduled by any 
regulatory authorities and promptly forward copies of any audit reports received to the 
sponsor. 
11.2 Management of Protocol Amendments and Deviations 
11.2.1 Modification of the Protocol 
Any changes in this research activity, except those necessary to remove an apparent, 
immediate hazard to the patient, must be reviewed and approved by the sponsor or its 
designee before implementation. Amendments to the protocol must be submitted in writing to 
the investigator’s IRB/IEC for approval before patients can be enrolled into an amended 
protocol or before the amendment can be implemented for patients already participating in 
the study. 


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11.2.2 Protocol Deviations 
The investigator or designee must document and explain in the patient’s source 
documentation any deviation from the approved protocol. The investigator may implement a 
deviation from, or a change of, the protocol to eliminate an immediate hazard to study 
patients without prior IRB/IEC approval. As soon as possible after such an occurrence, the 
implemented deviation or change, the reasons for it, and any proposed protocol amendments 
must be submitted to the IRB/IEC for review and approval, to the sponsor for agreement, and 
to the regulatory authorities, if required. 
A deviation from the protocol is an unintended or unanticipated departure from the 
procedures or processes approved by the sponsor and the IRB/IEC and agreed to by the 
investigator. A significant deviation occurs when there is non-adherence to the protocol by 
the patient or investigator that results in a significant, additional risk to the patient or to the 
integrity of the study. Significant deviations can include non-adherence to inclusion or 
exclusion criteria, or non-adherence to FDA regulations or ICH GCP guidelines, and will 
lead either to the patient being discontinued from study drug treatment or withdrawn from the 
study (Section 4.2). 
Protocol deviations will be documented by the clinical monitor throughout the course of 
monitoring visits. Principal investigators will be notified in writing by the monitor of 
deviations. The IRB/IEC must be notified of all protocol deviations in a timely manner. 
11.3 Study Termination 
Although Kowa Research Institute, Inc. has every intention of completing the study, Kowa 
Research Institute, Inc. reserves the right to discontinue the study at any time for clinical or 
administrative reasons. 
The end of the study is defined as the date on which the last patient completes his or her last 
visit.  
11.4 Final Report 
Whether the study is completed or prematurely terminated, the sponsor will ensure that the 
clinical study reports are prepared and provided to the regulatory agency(ies) as required by 
the applicable regulatory requirement(s). The sponsor will also ensure that the clinical study 


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reports in marketing applications meet the standards of the ICH harmonised tripartite 
guideline E3: Structure and content of clinical study reports and the standards of regulatory 
agencies to which the clinical study reports will be submitted.  
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the clinical study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results. 
Upon completion of the clinical study report, the sponsor will provide the investigator with 
the full summary of the study results. The investigator is encouraged to share the summary 
results with the study patients, as appropriate. The study results will be posted on publicly 
available clinical trial registers. 


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12 Reference List 
Borkar DS, Veldman P, Colby KA. Treatment of Fuchs Endothelial Dystrophy by Descemet 
Stripping Without Endothelial Keratoplasty. Cornea. 2016;35(10):1267-73. 
Elhalis H, Azizi B, Jurkunas. UV. Fuchs endothelial corneal dystrophy. Ocul Surf. 
2010;8(4):173-84.  
Garcerant D, Hirnschall N, Toalster N, et al. Descemet's stripping without endothelial 
keratoplasty. Curr Opin Ophthalmol. 2019;30(4):275-85. 
Hodges JL Jr, Lehmann EL. Estimates of location based on rank test. Ann. Math Statist. 
1963;34(2):598-611. 
Ianchulev T, Lane S, Masis M, et al. Corneal endothelial cell density and morphology after 
phacoemulsification in patients with primary open-angle glaucoma and cataracts: 2-year 
results of a randomised multicenter trial. Cornea. 2019;38(3):325-31. 
Iovieno A, Neri A, Soldani AM, et al. Descemetorhexis Without Graft Placement for the 
Treatment of Fuchs Endothelial Dystrophy: Preliminary Results and Review of the 
Literature. Cornea. 2017;36(6):637-641. 
Jullienne R, Manoli P, Tiffet T, et al. Corneal endothelium self-healing mathematical model 
after inadvertent descemetorhexis. J Cataract Refract Surg. 2015;41(10):2313–8. 
Kowa Company Ltd. Ripasudil hydrochloride hydrate ophthalmic solution. Investigator’s 
Brochure, version 00.01. Tokyo (Japan); 2019. 115 p.  
Mangione CM, Lee PP, Gutierrez PR, et al. Development of the 25-item National Eye 
Institute Visual Function Questionnaire. Arch Ophthalmol. 2001;119(7):1050-8.  
Macsai MS, Shiloach M. Use of topical rho kinase inhibitors in the treatment of Fuchs 
dystrophy after Descemet stripping only. Cornea. 2019;38(5):529-34.  
Moloney G, Petsoglou C, Ball M, et al. Descemetorhexis without grafting for Fuchs 
endothelial dystrophy-supplementation with topical ripasudil. Cornea. 2017;36(6):642-8. 


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Moses LE. Query 10: Confidence limits from rank tests. Technometrics. 1965;7(2):257-60. 
Van den Bogerd B, Dhubhghaill SN, Koppen C, et al. A review of the evidence for in vivo 
corneal endothelial regeneration. Surv Ophthalmol. 2018;63(2):149–65. 
Watanabe S, Oie Y, Fujimoto H, et al. Relationship between corneal guttae and quality of 
vision in patients with mild Fuchs’ endothelial corneal dystrophy. Ophthalmology. 
2015;122(10):2103–9. 

Prot_000.pdf: 
 
 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-
Group, 12 Week, Phase 2 Study to Investigate the Safety and 
Efficacy of Ripasudil (K-321) Eye Drops After Descemetorhexis in 
Patients with Fuchs Endothelial Corneal Dystrophy 
 
NCT04250207 
Clinical Study Protocol Ver.: 1.0 
Document Date: 16 August 2019 






Kowa Research Institute, Inc. 
K-321 
Protocol: K-321-201, version 1.0 (Original) 
16 August 2019 
 
Declaration of Investigator 
I have read and understood all sections of the protocol entitled “A double-masked, 
randomised, placebo-controlled, parallel-group, 12-week, Phase 2 study to investigate the 
safety and efficacy of ripasudil (K-321) eye drops after descemetorhexis in patients with 
Fuchs endothelial corneal dystrophy” and the accompanying investigator’s brochure. 
I agree to supervise all aspects of the protocol and to conduct the clinical investigation in 
accordance with the Protocol Version 1.0, dated 16 August 2019, the International Council 
for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), 
Integrated Addendum to E6 (R1): Guideline For Good Clinical Practice E6 (R2), the 
principles of the Declaration of Helsinki, and applicable regulations in the countries where 
the study will be conducted. I will not make changes to the protocol before consulting with 
Kowa Research Institute, Inc. or implement protocol changes without independent ethics 
committee approval except to eliminate an immediate risk to patients. I agree to administer 
study treatment only to patients under my personal supervision or the supervision of a 
sub-investigator. 
I will not supply the investigational drug to any person not authorised to receive it. 
Confidentiality will be protected. Patient identity will not be disclosed to third parties or 
appear in any study reports or publications. 
I will not disclose information regarding this clinical investigation or publish results of the 
investigation without authorisation from Kowa Research Institute, Inc.  
 
 
 
Signature of Principal Investigator 
 
Date 
 
 
 
Printed Name of Principal Investigator 
 
 
 
Page 3


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Page 4 
Table of Contents 
Table of Contents ....................................................................................................................... 4 
List of Tables .............................................................................................................................. 8 
List of Figures ............................................................................................................................ 8 
Protocol Synopsis....................................................................................................................... 9 
List of Abbreviations................................................................................................................ 17 
List of Terms ............................................................................................................................ 18 
1 
Introduction ...................................................................................................................... 19 
2 
Study Objectives ............................................................................................................... 22 
2.1 
Primary Objective ................................................................................................... 22 
2.2 
Secondary Objectives.............................................................................................. 22 
2.3 
Exploratory Objectives ........................................................................................... 22 
3 
Investigational Plan .......................................................................................................... 23 
3.1 
Study Design ........................................................................................................... 23 
4 
Patient Selection and Discontinuation/Withdrawal Criteria ............................................. 26 
4.1 
Selection of Study Population ................................................................................. 26 
4.1.1 
Inclusion Criteria ..................................................................................... 26 
4.1.2 
Exclusion Criteria .................................................................................... 26 
4.2 
Discontinuation of Treatment and Withdrawal of Patients From the Study ........... 28 
4.2.1 
Reasons for Discontinuation or Withdrawal ............................................ 29 
4.2.2 
Handling of Withdrawals ......................................................................... 30 
4.2.3 
Replacements ........................................................................................... 30 
5 
Study Drug Treatments ..................................................................................................... 31 
5.1 
Method of Assigning Patients to Treatment Groups ............................................... 31 
5.2 
Treatments Administered ........................................................................................ 31 
5.3 
Identity of Study Drug ............................................................................................ 32 
5.4 
Management of Clinical Supplies ........................................................................... 33 
5.4.1 
Study Drug Packaging and Storage ......................................................... 33 
5.4.2 
Study Drug Accountability ...................................................................... 34 


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5.5 
Medication Errors ................................................................................................... 34 
5.5.1 
Treatment of Medication Errors ............................................................... 34 
5.5.2 
Overdose Management ............................................................................ 34 
5.5.3 
Missed Doses ........................................................................................... 35 
5.6 
Masking .................................................................................................................. 35 
5.6.1 
Unmasking Treatment .............................................................................. 35 
5.7 
Treatment Compliance ............................................................................................ 36 
5.8 
Prior and Concomitant Therapy .............................................................................. 36 
5.8.1 
Descemetorhexis of the Study Eye .......................................................... 37 
5.8.2 
Rescue Surgical Procedures ..................................................................... 37 
5.8.3 
Prohibited and Restricted Concomitant Therapy ..................................... 37 
5.8.3.1 
Prohibited and Restricted Surgical Intervention ...................................... 37 
5.8.3.2 
Prohibited and Restricted Medication ..................................................... 38 
6 
Study Assessments and Procedures .................................................................................. 39 
6.1 
Eligibility Assessments and Procedures ................................................................. 42 
6.1.1 
Medical History, Physical Examination, and Laboratory Assessments ... 42 
6.1.2 
Descemetorhexis of the Study Eye .......................................................... 42 
6.2 
Efficacy Assessments .............................................................................................. 42 
6.2.1 
Corneal Endothelial Cell Density Measurement ..................................... 42 
6.2.1.1 
Non-Contact Specular Microscopy .......................................................... 42 
6.2.1.2 
Heidelberg Retina Tomography ............................................................... 43 
6.2.2 
Corneal Thickness .................................................................................... 43 
6.2.3 
Corneal Oedema ...................................................................................... 43 
6.2.4 
Corneal Morphology ................................................................................ 43 
6.2.5 
Best Corrected Visual Acuity ................................................................... 43 
6.2.6 
Contrast Sensitivity .................................................................................. 43 
6.2.7 
 ........................................................... 44 
6.3 
Safety Assessments ................................................................................................. 44 
6.3.1 
Ocular Safety Assessments ...................................................................... 44 
6.3.2 
Adverse Events ........................................................................................ 45 
6.3.2.1 
Definitions of Adverse Events ................................................................. 45 
6.3.2.2 
Eliciting and Documenting Adverse Events ............................................ 47 
6.3.2.3 
Reporting Adverse Events ....................................................................... 48 
6.3.2.4 
Suspected Unexpected Serious Adverse Reactions ................................. 50 


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6.3.2.5 
Follow-Up of Adverse Events ................................................................. 50 
6.3.3 
Vital Sign Measurements ......................................................................... 50 
6.3.4 
Eligibility ................................................................................................. 51 
6.4 
Safety Monitoring Committee ................................................................................ 51 
6.5 
Pregnancy ................................................................................................................ 51 
6.6 
Laboratory Analyses ............................................................................................... 52 
6.6.1 
Future Genetic Testing ............................................................................. 52 
6.7 
Sample Collections ................................................................................................. 53 
7 
Statistical and Analytical Plan .......................................................................................... 54 
7.1 
Primary Efficacy Endpoint ..................................................................................... 54 
7.2 
Secondary Efficacy Endpoints ................................................................................ 54 
7.3 
Exploratory Endpoints ............................................................................................ 55 
7.4 
Sample Size Calculations ........................................................................................ 55 
7.5 
Analysis Sets ........................................................................................................... 56 
7.6 
Description of Subgroups to be Analysed ............................................................... 56 
7.7 
Statistical Analysis Methodology ........................................................................... 56 
7.7.1 
Analysis of Primary Efficacy Endpoint ................................................... 57 
7.7.2 
Analysis of Secondary Efficacy Endpoints ............................................. 58 
7.7.3 
Analyses of Exploratory Efficacy Endpoints ........................................... 59 
7.7.4 
Safety Analyses ........................................................................................ 60 
7.7.4.1 
Adverse Events ........................................................................................ 60 
7.7.4.2 
Vital Signs ................................................................................................ 61 
7.7.4.3 
Clinical Laboratory .................................................................................. 61 
7.7.4.4 
Intra-Ocular Pressure ............................................................................... 61 
7.7.4.5 
Slit-Lamp Biomicroscopy and Ophthalmoscopy ..................................... 61 
7.7.5 
Other Analyses ......................................................................................... 62 
7.7.5.1 
Patient Demographics and Other Baseline Characteristics ...................... 62 
7.7.5.2 
Treatment Exposure and Compliance ...................................................... 62 
7.7.6 
Interim Analyses ...................................................................................... 62 
8 
Data Quality Assurance .................................................................................................... 64 
8.1 
Data Management ................................................................................................... 64 
9 
Ethics ................................................................................................................................ 66 


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9.1 
Independent Ethics Committee or Institutional Review Board .............................. 66 
9.2 
Ethical Conduct of the Study .................................................................................. 66 
9.3 
Patient Information and Consent ............................................................................ 66 
10 Investigator’s Obligations ................................................................................................ 68 
10.1 Confidentiality ........................................................................................................ 68 
10.2 Financial Disclosure and Obligations ..................................................................... 68 
10.3 Investigator Documentation .................................................................................... 69 
10.4 Study Conduct ......................................................................................................... 69 
10.5 Adherence to Protocol ............................................................................................ 70 
10.6 Adverse Events and Study Report Requirements ................................................... 70 
10.7 Investigator’s Final Report ..................................................................................... 70 
10.8 Records Retention ................................................................................................... 70 
10.9 Publications ............................................................................................................. 70 
11 Study Management ........................................................................................................... 72 
11.1 Monitoring .............................................................................................................. 72 
11.1.1 
External Data Monitoring Committee ..................................................... 72 
11.1.2 
Monitoring of the Study ........................................................................... 73 
11.1.3 
Inspection of Records .............................................................................. 73 
11.2 Management of Protocol Amendments and Deviations.......................................... 73 
11.2.1 
Modification of the Protocol .................................................................... 73 
11.2.2 
Protocol Deviations ................................................................................. 74 
11.3 Study Termination ................................................................................................... 74 
11.4 Final Report ............................................................................................................ 74 
12 Reference List................................................................................................................... 76 


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List of Tables 
Table 5-1 
Vial Contents by Dosing Regimen and Dosing Time for Each Treatment 
Group From Day 1 to Visit 9 (Week 12) ......................................................... 31 
Table 6-1 
Schedule of Study Site Activities .................................................................... 40 
Table 11-1 
Study Administration ...................................................................................... 72 
 
List of Figures 
Figure 3-1 
Study Design ................................................................................................... 24 
 


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Protocol Synopsis 
Protocol Number: 
K-321-201 
Title: 
A Double-Masked, Randomised, Placebo-Controlled, Parallel-Group, 12-Week, Phase 2 
Study to Investigate the Safety and Efficacy of Ripasudil (K-321) Eye Drops After 
Descemetorhexis in Patients with Fuchs Endothelial Corneal Dystrophy 
Sponsor:  
Kowa Research Institute, Inc. 
430 Davis Drive, Suite 200 
Morrisville, NC 27560 
Study Phase: 
Phase 2 
Study Sites: 
Approximately 30 sites in the United States, Europe, and Australia 
Indication: 
The treatment of Fuchs endothelial corneal dystrophy after descemetorhexis 
Rationale: 
In Fuchs endothelial corneal dystrophy (FECD), there is an increased rate of loss of 
endothelial cells, starting in the centre of Descemet’s membrane and spreading to the 
periphery. There is an increased deposition of extracellular matrix on Descemet’s 
membrane, resulting in excrescences known as guttae, which are a marker of the 
condition. Guttae may coalesce and inhibit the migration of endothelial cells. Eventually 
the corneal endothelium ceases to function effectively, and the cornea begins to cloud, 
leading eventually to blindness.  
Currently, most patients undergoing keratoplasty for FECD have Stage 2 disease and the 
procedure starts with descemetorhexis, removing an area of the roughened and irregular 
Descemet’s membrane (including the central guttae) from the diseased cornea. The 
excised area is replaced with a graft of endothelial cells: either donor Descemet’s 
membrane with a thin layer of corneal stroma (Descemet stripping endothelial 
keratoplasty [DSEK]), or, increasingly commonly, donor Descemet’s membrane alone 
(Descemet’s membrane endothelial keratoplasty [DMEK]). 
Experience with failed grafts has indicated that even though there is an underlying 
abnormality of endothelial cells in FECD, areas of stroma exposed by descemetorhexis 
can be re-endothelialised and the migrated endothelial cells can maintain corneal 
transparency. Mathematical modelling of the healing of an inadvertent 
 diameter 
descemetorhexis in an elderly man without FECD suggested that normal healing is 
achieved by a redistribution of peripheral endothelial cells into the central defect without 
any increase in the number of endothelial cells. This led to the proposal that some cases 






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During the follow-up period, patients will attend 5 study visits, starting at Week 16 
(Visit 10).  
 
 will determine central ECD by 
analysing digital images of the corneal endothelium.  
Keratoplasty may be offered as a rescue surgical procedure to an enrolled patient if the 
investigator judges that endothelial healing is not complete at Visit 9 (Week 12), or earlier 
if the investigator considers that the patient needs urgent treatment.  
Study Population: 
Inclusion Criteria: 
Each patient must meet all of the following criteria to be enrolled in this study: 
1. Is at least 18 years old at the screening visit (Visit 1). 
2. Has a diagnosis of FECD at Visit 1. 
3. Has confluent central guttae in the study eye that can be removed by descemetorhexis 
of a circular area of 
 diameter or less (at Visit 1).  
4. Has a study eye with best corrected visual acuity (BCVA) of 75 letters or fewer by 
Early Treatment Diabetic Retinopathy Study (ETDRS) testing (Snellen equivalent of 
20/32 or worse) at Visit 1.  
5. The study eye descemetorhexis at Visit 2 is confirmed to have excised a central area 
with confluent guttae and a diameter of 
 Guidance for performing the 
study eye descemetorhexis and measurement of the excised area will be provided in 
the study manual.  
6. Can understand the written informed consent, provides signed and witnessed written 
informed consent, and agrees to comply with protocol requirements before any 
study-specific assessment is performed. 
Exclusion Criteria:  
Patients meeting any of the following criteria will be excluded from the study:  
1. Is a female patient and any of the following is true:  
• 
is pregnant or lactating/breastfeeding, or  
• 
is not surgically sterile, not post-menopausal (no menses for the previous 12 
months), or not practising an effective method of birth control as 
determined by the investigator (eg, oral contraceptives, double barrier 
methods, hormonal injectable or implanted contraceptives, tubal ligation, 
or partner with vasectomy).  
If a patient is female and of childbearing potential, she must have a negative urine 
pregnancy test result at Visit 2 before the descemetorhexis. Women of childbearing 
potential must also agree to use effective contraception throughout the study. 
A female of childbearing potential is defined as a woman who has experienced menarche 
and has not undergone successful surgical sterilisation or is not post-menopausal, and a 


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post-menopausal woman is defined as having had no menses for the previous 12 months 
(without a known cause). 
2. Has a study eye with confluent guttae in the periphery or confluent guttae outside the 
stripped area (individual guttae are allowed). 
Note that patients may have individual or small number of guttae remaining outside 
the stripped area, but there should be no areas of confluent guttae remaining after the 
descemetorhexis. For example, a patient who had 5 to 10 guttae remaining in a few 
spots around the circumference of the descemetorhexis would not be excluded by this 
criterion.  
3. Has a study eye with a history of cataract surgery within 90 days of Visit 1. 
4. Has a study eye with a history of any previous ocular surgery other than for cataract.  
5. Has a non-study eye with a history of any previous ocular surgery within 30 days of 
Visit 1. 
6. Plans to receive any surgical treatment on the study eye during any study period, other 
than the study descemetorhexis.  
7. Plans to receive any surgical treatment for FECD or cataract on the non-study eye in 
the screening or treatment period. 
8. Has advanced corneal stromal oedema defined as the presence of widespread haze or 
bullae on slit-lamp examination. 
9. Has a study eye with central corneal thickness ≥ 670 μm. 
10. Has known severe comorbidities that may interfere with descemetorhexis (eg, a 
bacterial, viral, or fungal ophthalmic infection). 
11. Has any clinically significant ocular condition other than FECD or cataract that 
requires medication or ocular surgery. 
12. Has diabetes with poor blood sugar control (eg, HbA1c value > 8.5%). 
13. Has used contact lenses within 7 days of Visit 1. 
14. Has hypersensitivity to any ophthalmic medication used for diagnosis or treatment, 
including eye drops containing antibiotic(s) or glucocorticoid(s).  
15. Has known hypersensitivity to any component of the study drugs. 
16. Has previously used ripasudil, netarsudil, or other ROCK inhibitors. 
17. Has used any investigational medications within 30 days of Visit 1. 
18. Has a positive urine test result for drugs of abuse (opiates, methadone, cocaine, 
amphetamines, barbiturates, or benzodiazepines) or alcohol at screening. 
19. Has donated more than 450 mL of blood within 60 days of Visit 1. 
20. Is a member or a family member of the professional or ancillary personnel working at 
the study site or the sponsor involved in the study. 
21. Has a concomitant medical or psychological condition that could interfere with study 
participation or is otherwise not suitable for entry into the study in the opinion of the 
investigator.  






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will be summarised and listed with no statistical hypothesis testing performed. 
Continuous variables will be summarised using the mean, the SD, median, minimum 
value, and maximum value. Categorical variables will be summarised using frequency 
counts and percentages. 
Version and Date of Protocol: 
Version 1.0 (Original), 16 August 2019 


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List of Abbreviations 
Abbreviation 
Definition 
AE 
adverse event 
BCVA 
best controlled visual acuity 
BID 
twice daily 
CFR 
Code of Federal Regulations 
CI 
confidence interval 
 
 
CRO 
contract research organisation 
CS 
clinically significant 
CV 
curriculum vitae 
DMEK 
Descemet membrane endothelial keratoplasty 
DSEK 
Descemet stripping endothelial keratoplasty 
DSO 
Descemet stripping only 
ECD 
endothelial cell density 
eCRF 
electronic case report form 
EDC 
electronic data capture 
EOS 
end-of-study 
EOT 
end-of-treatment 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FAS 
full-analysis set 
FDA 
US Food and Drug Administration 
FECD 
Fuchs endothelial corneal dystrophy 
GCP 
Good Clinical Practice 
HbA1c 
haemoglobin A1c (glycated haemoglobin) 
HRT 
Heidelberg retina tomography 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
IEC 
independent ethics committee 
IOP 
intra-ocular pressure 
IRB 
institutional review board 
IWRS 
interactive web response system 
LOCF 
last-observation-carried-forward 


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Abbreviation 
Definition 
M/N 
morning and night 
MD/E 
mid-day and evening 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHRA 
Medicines and Healthcare Products Regulatory Agency 
NCS 
not clinically significant 
PPS 
per-protocol set 
QID 
4 times daily 
ROCK 
Rho-associated protein kinase 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SFS 
safety set 
SOA 
schedule of study site activities 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE 
treatment-emergent adverse event 
UK 
United Kingdom 
US 
United States 
 
 
 
List of Terms 
Term 
Definition 
Descemetorhexis 
Removal of an area of roughened and irregular Descemet membrane 
along with central guttae (also referred to herein as Descemet 
membrane stripping) 
 


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1 
Introduction 
In the early 20th century, Ernst Fuchs described a bilateral corneal dystrophy now referred to 
as Fuchs endothelial corneal dystrophy (FECD). The primary defect in FECD seems to be 
related to the endothelial cells that normally sit on a modified basement membrane 
(Descemet’s membrane) that lines the inner surface of the cornea.  
In the normal adult human cornea, the endothelial cell density (ECD) on Descemet’s 
membrane is in the range of 2,000 to 3,500 cells/mm2 (Ianchulev et al 2019) and the 
endothelial cells maintain the cornea in a state of relative dehydration, which preserves 
corneal transparency. During adulthood there is continuous loss of endothelial cells at an 
approximate rate of 0.6% per year. Since the number of endothelial cells cannot increase 
because of a state of mitotic arrest, gaps caused by apoptosis are filled by the migration of 
adjacent cells that increase in size and change shape. The function of the corneal endothelium 
can be maintained while the ECD is maintained above approximately 400 cells/mm2. Below 
that threshold (due to disease, trauma or old age), there is decompensation of the endothelial 
pumping mechanism and corneal oedema produces a visual haze (Van den Bogerd et al 
2018).  
In FECD, there is an increased rate of loss of endothelial cells, starting in the centre of 
Descemet’s membrane and spreading to the periphery. There is an increased deposition of 
extracellular matrix on Descemet’s membrane, resulting in excrescences known as guttae, 
which are a marker of the condition. Guttae may coalesce and inhibit the migration of 
endothelial cells. Eventually the corneal endothelium ceases to function effectively, and the 
cornea begins to cloud, eventually leading to blindness (Elhalis et al. 2010).  
There are clinical staging systems for FECD that describe the progression of the disease 
(Elhalis et al. 2010). Stage 1 is characterised by the presence of corneal guttae that are 
centrally located, non-confluent, and asymptomatic. Guttae may also form in response to 
trauma, toxins, or infection and may not be indicative of FECD. Guttae appearing with age 
are considered a normal finding in the elderly if they appear in the periphery. In Stage 2, 
central guttae start to coalesce and spread to the periphery and there is loss of endothelial 
cells, with ECD being inversely proportional to the number of guttae. The cells become much 
more variable in size and lose their regular shape (pleomorphism), and painless visual 
symptoms start to appear. The irregularity of the Descemet’s membrane, and particularly the 
presence of central confluent guttae, may lead to forward scatter of light in the eye, loss of 


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visual contrast sensitivity, and impairment of visual acuity, even in the absence of oedema 
(Watanabe et al 2015). In the early stages of the disease, oedema of the stroma causes 
blurriness of vision in the morning that clears during the day, but this progresses to 
permanent loss of visual acuity with glare. In Stage 3, there is more marked corneal oedema 
and painful sub-epithelial and epithelial bullae may form. Rupture of the bullae disrupts the 
outer surface of the cornea and exposes the patient to risk of infection. Finally, in Stage 4, the 
cornea becomes opaque and new vessel growth may occur. In addition, there may be 
sub-epithelial fibrous tissue formed due to long-standing oedema. In this stage, the eye 
becomes blind, but the pain may subside (Elhalis et al. 2010).  
Fuchs endothelial corneal dystrophy is currently treated by a variety of keratoplasty 
techniques, including full-thickness cornea transplantation (for Stages 3 and 4) and 
2 techniques involving transplantation of a graft of donor endothelial cells on donor 
Descemet’s membrane. Currently, most patients undergoing keratoplasty for FECD have 
Stage 2 disease and the procedure starts with descemetorhexis, removing an area of the 
roughened and irregular Descemet’s membrane (including the central guttae) from the 
diseased cornea. The excised area is replaced with a graft of endothelial cells: either donor 
Descemet’s membrane with a thin layer of corneal stroma (Descemet stripping endothelial 
keratoplasty [DSEK]), or, increasingly commonly, donor Descemet’s membrane alone 
(Descemet’s membrane endothelial keratoplasty [DMEK]). 
Experience with failed grafts has indicated that even though there is an underlying 
abnormality of endothelial cells in FECD, areas of stroma exposed by descemetorhexis can 
be re-endothelialised and the migrated endothelial cells can maintain corneal transparency. 
Mathematical modelling of the healing of an inadvertent 5-mm diameter descemetorhexis in 
an elderly man without FECD suggested that normal healing is achieved by redistribution of 
peripheral endothelial cells into the central defect without any increase in the number of 
endothelial cells (Jullienne et al 2015). This led to the proposal that some cases of FECD, 
particularly those with relatively preserved peripheral endothelial cells, can be treated by 
descemetorhexis alone (Garcerant et al 2019). 
There are limits to the ability of the endothelium to heal. In a recent analysis of cases of 
patients who underwent descemetorhexis without grafting as a possible treatment for FECD 
(compiled from multiple papers), 31 (65%) out of 47 cases eventually achieved a clear 
cornea, with healing taking place as early as 1 month after the procedure (Van den Bogerd et 


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al 2018). If there was persistent oedema at 6 months, then it was unlikely to resolve. If 
corneal oedema did not clear, some patients’ corneas cleared without residual stromal 
damage after DMEK performed as rescue therapy between 4 and 8.5 months after 
Descemet’s membrane stripping (Van den Bogerd et al 2018). All keratoplasty procedures, 
including DMEK, are associated with risks of graft detachment, graft failure or immune 
rejection. There is a medical need for medication that can speed healing after 
descemetorhexis and that can help establish a high, functional ECD to maintain corneal 
transparency. 
Ripasudil ophthalmic solution 0.4% (K-321) is a Rho-associated protein kinase (ROCK) 
inhibitor that has been marketed in Japan since December 2014 as Glanatec®, with twice 
daily (BID) dosing for glaucoma and ocular hypertension. Studies in endothelial cells have 
indicated that K-321 can lead to reduction of apoptosis, enhancement of cell proliferation, 
and increased rates of migration, all of which may support endothelial healing (Kowa 
Company Ltd 2019). Moloney et al (2017) first reported use of a ROCK inhibitor to promote 
healing in patients with FECD treated with Descemet stripping only (DSO). More recently, 
Macsai et al (2019) reported results from a small study of 18 patients with FECD and central 
confluent guttae of up to 
 diameter who underwent DSO and were subsequently treated 
with either ripasudil ophthalmic solution 0.4% four times daily (QID) or no ripasudil for 
2 months. Overall, patients who underwent DSO with ripasudil recovered vision more 
quickly 
 
 
 The patients in the no-ripasudil 
group had 
 
 while in the ripasudil group there was 
 
. In the short-term 
studies (8 weeks duration and less) for the development of Glanatec, there were no 
 
 
 
 (Kowa Company Ltd 2019).  
Kowa proposes to pursue development of K-321 (ripasudil ophthalmic solution) for the 
following indication: “the treatment of Fuchs endothelial corneal dystrophy after 
descemetorhexis.” This will be the first Kowa-sponsored study in patients for this indication.  


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2 
Study Objectives  
2.1 
Primary Objective 
The primary objective of this study is to investigate the effect of K-321 dosed for 12 weeks 
on central ECD in patients with FECD after descemetorhexis.  
2.2 
Secondary Objectives 
The secondary objectives of this study are the following:  
• to investigate the effect of K-321 on central ECD, corneal thickness, and corneal 
clarity in patients with FECD at each visit after descemetorhexis out to 52 weeks 
• to assess the safety and tolerability of K-321 in patients with FECD at each visit after 
descemetorhexis out to 52 weeks 
2.3 
 Exploratory Objectives 
 
 
 
 
  


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3 
Investigational Plan 
3.1 
Study Design 
This is a multi-centre, double-masked, randomised, parallel-group, placebo-controlled, 
2-period study of patients with FECD after descemetorhexis. The study design is presented in 
Figure 3-1, and the schedule of study site activities (SOA) is presented in Table 6-1.  
The first period is the treatment period, consisting of a screening visit within a 1- to 4-week 
screening period, a descemetorhexis and randomisation visit, a 12-week full treatment period 
containing 7 interim visits (including 1 optional visit at Week 2) and an end-of-treatment 
(EOT) visit scheduled for Week 12. During the 2 weeks immediately following the EOT 
visit, each enrolled patient will taper dosing of study drug to zero (Section 5.2).  
The second period is a follow-up observation period of 40 weeks, including the tapering 
phase and containing 4 interim visits and an end-of-study (EOS) visit. Patients are not to 
self-administer study drug after approximately Week 14. The maximum total study period for 
each enrolled patient is 56 weeks, with a maximum of 15 visits (including the 1 optional 
visit). Patients will be considered to have completed the study with the completion of their 
EOS visit (scheduled for Week 52). The duration of the study is defined for each patient as 
the date signed written informed consent is provided through the last follow-up visit. 


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There are 3 treatment groups: K-321 ophthalmic solution 0.4% dosed QID, K-321 
ophthalmic solution 0.4% dosed BID, and placebo. The procedures for randomly assigning 
study drug are presented in Section 5.1. 
After obtaining informed consent at Visit 1, the investigator will select one eye as the study 
eye, and the patient will come back for the descemetorhexis operation and random 
assignment to treatment after 1 to 4 weeks (Visit 2). At Visit 2, each patient’s eligibility for 
study participation will be confirmed, including a successful descemetorhexis operation that 
meets the inclusion criteria (Section 4.1). Guidance for the descemetorhexis will be provided 
in the study manual. Recommended post-descemetorhexis concomitant therapy is described 
in Section 5.8.1. Documentation of the descemetorhexis is discussed in Section 6.1.  
After eligibility is confirmed, patients will be randomly assigned to the 3 treatment groups 
(in a 1:1:1 ratio). All patients will be instructed to apply the study drug only to the study eye.  
Except on days of study visits on Day 1 through Week 12 (not including Visit 9) during the 
treatment period, study drug will be dosed by the patient QID (Section 5.2): morning (9 AM 
±1 hour), mid-day (1 PM ±1 hour), evening (5 PM ±1 hour), and night (9 PM ±1 hour). Each 
patient will be provided with 2 sets of colour-coded 
 one set to be applied 


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morning and night (M/N) and the other to be applied mid-day and evening (MD/E). See 
Section 5.4.1 for a detailed description of packaging.  
On study visit days Visit 3 through Visit 9, patients will not apply any study drug until after 
all study measurements have been completed; thereafter on those days, patients will start 
dosing with the dose closest in timing to the dosing regimen and continue with the remaining 
doses for the day. Additionally, on Visit 3 they must first apply the study drug within 
 after the end of the descemetorhexis procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient applies 
any study drug on the day of Visit 9 before all study measurements have been completed, that 
patient’s Visit 9 should be rescheduled within the visit window allowance (eg, the next day). 
For visits other than Visit 9 during the treatment period, a patient who applies study drug 
before all study measurements have been completed will be assessed as planned, and a 
protocol deviation must be recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up period, and 
new study drug will be dispensed without M/N or MD/E colour coding (Section 5.2). All 
patients will self-administer their tapering-phase study drug BID (9 AM ±1 h and 9 PM ±1 h) 
for a week, then once daily (9 AM ±1 h) for a week, and then will discontinue study drug. At 
Visit 9, patients will apply the first dose of study drug 
 of completing study 
measurements and will apply the second dose at night, as scheduled.  
During the follow-up period, patients will attend 5 study visits (Figure 3-1), starting at 
Week 16 (Visit 10).  
Study activities and assessments will be performed at study visits as described in Table 6-1 
and in Section 6. 
 will determine 
central ECD by analysing digital images of the corneal endothelium (Table 11-1).  
Keratoplasty may be offered as a rescue surgical procedure to an enrolled patient if the 
investigator judges that endothelial healing is not complete at Visit 9 (Week 12), or earlier if 
the investigator considers that the patient needs urgent treatment (Section 5.8.2).  


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4 
Patient Selection and Discontinuation/Withdrawal Criteria 
4.1 
Selection of Study Population 
Approximately 60 patients will be enrolled at approximately 30 sites in the United States, 
Europe, and Australia. Patients will be assigned to study treatment only if they meet all of the 
inclusion criteria and none of the exclusion criteria. 
Deviations from the inclusion and exclusion criteria are not allowed because they can 
potentially jeopardise the scientific integrity of the study, regulatory acceptability, or patient 
safety. Therefore, adherence to the criteria as specified in the protocol is essential. 
4.1.1 
Inclusion Criteria 
Each patient must meet all of the following criteria to be enrolled in this study: 
1. Is at least 18 years old at the screening visit (Visit 1). 
2. Has a diagnosis of FECD at Visit 1. 
3. Has confluent central guttae in the study eye that can be removed by descemetorhexis 
of a circular area of 
 diameter or less (at Visit 1).  
4. Has a study eye with best corrected visual acuity (BCVA) of 75 letters or fewer by 
Early Treatment Diabetic Retinopathy Study (ETDRS) testing (Snellen equivalent of 
20/32 or worse) at Visit 1.  
5. The study eye descemetorhexis at Visit 2 is confirmed to have excised a central area 
with confluent guttae and a diameter of 
 Guidance for performing the 
study eye descemetorhexis and measurement of the excised area will be provided in 
the study manual.  
6. Can understand the written informed consent, provides signed and witnessed written 
informed consent, and agrees to comply with protocol requirements before any 
study-specific assessment is performed. 
4.1.2 
Exclusion Criteria 
Patients meeting any of the following criteria will be excluded from the study: 
1. Is a female patient and any of the following is true:  


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• 
is pregnant or lactating/breastfeeding, or  
• 
is not surgically sterile, not post-menopausal (no menses for the previous 
12 months), or not practising an effective method of birth control as 
determined by the investigator (eg, oral contraceptives, double barrier 
methods, hormonal injectable or implanted contraceptives, tubal ligation, 
or partner with vasectomy).  
If a patient is female and of childbearing potential, she must have a negative urine 
pregnancy test result at Visit 2 before the descemetorhexis. Women of 
childbearing potential must also agree to use effective contraception throughout 
the study. 
A female of childbearing potential is defined as a woman who has experienced 
menarche and has not undergone successful surgical sterilisation or is not 
post-menopausal, and a post-menopausal woman is defined as having had no 
menses for the previous 12 months (without a known cause). 
2. Has a study eye with confluent guttae in the periphery or confluent guttae outside the 
stripped area (individual guttae are allowed). 
Note that patients may have individual or small number of guttae remaining outside 
the stripped area, but there should be no areas of confluent guttae remaining after the 
descemetorhexis. For example, a patient who had 5 to 10 guttae remaining in a few 
spots around the circumference of the descemetorhexis would not be excluded by this 
criterion.  
3. Has a study eye with a history of cataract surgery within 90 days of Visit 1. 
4. Has a study eye with a history of any previous ocular surgery other than for cataract.  
5. Has a non-study eye with a history of any previous ocular surgery within 30 days of 
Visit 1. 
6. Plans to receive any surgical treatment on the study eye during any study period, other 
than the study descemetorhexis.  
7. Plans to receive any surgical treatment for FECD or cataract on the non-study eye in 
the screening or treatment period. 
8. Has advanced corneal stromal oedema defined as the presence of widespread haze or 
bullae on slit-lamp examination. 


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9. Has a study eye with central corneal thickness ≥ 670 μm. 
10. Has known severe comorbidities that may interfere with descemetorhexis (eg, a 
bacterial, viral, or fungal ophthalmic infection). 
11. Has any clinically significant ocular condition other than FECD or cataract that 
requires medication or ocular surgery (described in Section 5.8.3). 
12. Has diabetes with poor blood sugar control (eg, HbA1c value > 8.5%). 
13. Has used contact lenses within 7 days of Visit 1. 
14. Has hypersensitivity to any ophthalmic medication used for diagnosis or treatment, 
including eye drops containing antibiotic(s) or glucocorticoid(s).  
15. Has known hypersensitivity to any component of the study drugs. 
16. Has previously used ripasudil, netarsudil, or other ROCK inhibitors. 
17. Has used any investigational medications within 30 days of Visit 1. 
18. Has a positive urine test result for drugs of abuse (opiates, methadone, cocaine, 
amphetamines, barbiturates, or benzodiazepines) or alcohol at screening. 
19. Has donated more than 450 mL of blood within 60 days of Visit 1. 
20. Is a member or a family member of the professional or ancillary personnel working at 
the study site or the sponsor involved in the study. 
21. Has a concomitant medical or psychological condition that could interfere with study 
participation or is otherwise not suitable for entry into the study in the opinion of the 
investigator.  
4.2 
Discontinuation of Treatment and Withdrawal of Patients From the 
Study 
The duration of the study is defined for each patient as the date signed written informed 
consent is provided through the date of the last study visit attended.  
There are 3 scenarios that result in interruption of the treatment regimen. Interruption of 
treatment is defined as a temporary stopping of study drug treatment that resumes during the 
treatment period, due to an AE or any other reason. Early discontinuation of treatment is 
defined as permanent stopping of study drug treatment before completing Visit 9, scheduled 


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for Week 12. Investigators will strive to ensure that a patient who has interrupted treatment 
for a particular reason will not discontinue treatment unless discontinuation is medically 
imperative in the investigator’s judgement. Early withdrawal from the study is defined as 
failing to complete Visit 14, scheduled for Week 52.  
Study drug administration may be discontinued early at the discretion of the investigator (or 
designee) if the patient does not tolerate the dosing regimen or if, in the investigator’s 
judgement, the patient’s health may be jeopardised by continuing study treatment. Patients 
who discontinue treatment during the study will be encouraged by investigators to continue 
to participate in all scheduled study site visits and assessments, and study data will be 
collected for these patients per protocol. Every effort must be made to keep patients in the 
study (see Section 5.8.2 for a discussion of treatment discontinuation after rescue surgical 
procedures).  
4.2.1 
Reasons for Discontinuation or Withdrawal 
Patients may discontinue treatment or withdraw from the study at any time and for any 
reason without prejudice to their future medical care by the investigator or at the study site. 
Every effort must be made to keep patients in the study. The reasons for patients 
discontinuing treatment or withdrawing early will be recorded.  
A patient may discontinue treatment or be discontinued from treatment for any of the 
following reasons: 
1. The patient does not meet the protocol inclusion or exclusion criteria. 
2. The patient is non-compliant with the protocol. 
3. The patient has a serious or intolerable AE that, in the investigator’s opinion, requires 
withdrawal from the study.  
4. The patient has symptoms or an intercurrent illness not consistent with the protocol 
requirements or that justify withdrawal. 
5. The patient undergoes rescue keratoplasty (Section5.8.2). 
6. Other reasons (eg, pregnancy, development of contraindications of use of study drug). 
A patient may be withdrawn from the study for any of the following reasons: 


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1. The patient withdraws consent.  
2. The patient is lost to follow-up. 
The investigator will also withdraw all patients if Kowa Research Institute, Inc. terminates 
the study. The study may be terminated if it becomes apparent that the objectives of the trial 
cannot be met, or if the frequency or pattern of AEs reported in the trial indicate that the drug 
is no longer likely to have a positive benefit-to-risk ratio. 
4.2.2 
Handling of Withdrawals  
 Patients who withdraw from the study or who are withdrawn from the study will no longer 
receive study drug. When a patient withdraws or is withdrawn from the study, the reason(s) 
for withdrawal shall be recorded by the investigator on the relevant page of the electronic 
case report form (eCRF). Whenever possible, all patients who withdraw from the study early 
will undergo all EOS assessments. Patients who fail to return for final assessments will be 
contacted by the site with 2 documented telephone calls followed by 1 registered letter, as 
needed, to have them comply with the protocol. 
It is vital to obtain follow-up data on any patient withdrawn because of an AE or serious AE 
(SAE). In every case, efforts must be made to undertake protocol-specified safety follow-up 
procedures. 
4.2.3 
Replacements 
Patients who are screened for the study and receive a study descemetorhexis may be replaced 
if they never are randomly assigned study drug or are withdrawn from the study before they 
administer any study drug. The replacement patients will receive a new randomly assigned 
treatment and not the treatment assignment of the replaced patient. Patients who self-
administer any study drug and subsequently are withdrawn early from the study will not be 
replaced. Patients who fail to satisfy inclusion and exclusion criteria at screening may be 
rescreened 1 additional time at the discretion of the sponsor. A special case and conditions for 
replacement, in case the descemetorhexis fails on the first-selected study eye, is presented in 
Section 5.8.3.1.  




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On study visit days Visit 3 through Visit 9, patients will not apply any study drug until after 
all study measurements have been completed; thereafter on those days, patients will start 
dosing with the dose closest in timing to the dosing regimen and continue with the remaining 
doses for the day. Additionally, on Visit 3 they must first apply study drug within 
 after the end of the descemetorhexis procedure on Day –1 (Visit 2). 
With the primary efficacy endpoint being assessed at Visit 9 (Week 12), if a patient applies 
any study drug on the day of Visit 9 before all study measurements have been completed, that 
patient’s Visit 9 should be rescheduled within the visit window allowance (eg, the next day). 
For visits other than Visit 9 during the treatment period, a patient who applies study drug 
before all study measurements have been completed will be assessed as planned, and a 
protocol deviation must be recorded.  
At Visit 9 (Week 12), patients will enter the drug-tapering phase of the follow-up period, and 
new study drug will be dispensed without M/N or MD/E colour coding (Section 5.2). All 
patients will self-administer their tapering-phase study drug BID (9 AM ±1 hour and 
9 PM ± 1 hour) for a week, then once daily (9 AM ± 1 hour) for a week, and then will 
discontinue study drug. At Visit 9, patients will apply the first dose of study drug 
 
 of completing study measurements and will apply the second dose at night, as 
scheduled. However, if the first dose will be applied after 8 PM, the second dose must be 
skipped. 
For each dose, patients must instil 1 drop to the study eye only and then close the eyelids, 
pressing on the lacrimal punctum. Patients will be advised to keep the eye closed for 
1 to 5 minutes before opening the eye. Any other concurrent ocular medication(s) must be 
applied at least 5 minutes following instillation of study drug. Patients will be trained by site 
staff on the application of eye drops. 
5.3 
Identity of Study Drug 
The K-321 ophthalmic solution contains ripasudil 0.4% (4.896 mg/mL of ripasudil 
hydrochloride hydrate, equivalent to 4.0 mg/mL as the free base) in a solution also containing 
the excipients sodium dihydrogen phosphate and sodium hydroxide at a pH in the range of 
5.0 to 7.0. The solution is isosmotic to normal saline, clear, colourless, and 
 The 
placebo ophthalmic solution is identical in appearance, osmolarity, and pH to the K-321 




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selected clinical supply depots where shipments to clinical sites are managed via the IWRS 
system.  
The materials schedule will be prepared by an independent 
 biostatistician and provided 
to the IWRS and the packaging vendor. At the time of randomisation, the IWRS will assign 
each patient a study drug kit corresponding to the patient’s randomly assigned treatment 
based on the drug supply inventory that is available at the site. The kit will be identified by a 
unique kit number that is separate from the patient or randomisation numbers. The IWRS 
will administer study drug dispensing at each visit indicated in Table 6-1. 
5.4.2 
Study Drug Accountability 
The investigator will maintain accurate records of receipt of all study drug, including dates of 
receipt. In addition, accurate records will be kept regarding when and how much study drug 
is dispensed and used by each patient in the study. Reasons for departure from the expected 
dispensing regimen must also be recorded. At the completion of the study, to satisfy 
regulatory requirements regarding drug accountability, all study drug will be reconciled, 
shipped from study sites to Catalent depots in the United States and Germany, and destroyed 
according to applicable regulations. 
5.5 
Medication Errors 
At Visit 3, patients will be given instructions on how to administer their assigned ophthalmic 
solution and how to avoid contaminating the 
solution, and they will be observed 
instilling their own drops by the site staff. Re-education on the proper administration 
procedure will be given to patients by study site staff throughout the treatment period at each 
visit. To minimise medication errors during the 12-week treatment phase, 
 and 
 will be colour-coded differently for M/N and MD/E doses.  
5.5.1 
Treatment of Medication Errors 
In the event of a medication error, the appropriate supportive clinical care must be provided 
as dictated by the patient’s clinical status.  
5.5.2 
Overdose Management 
A symptomatic overdose from topical administration is unlikely, as no overdose was 
observed during the development of Glanatec (Kowa Company Ltd 2019). Patients will be 


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instructed to contact the investigator or study coordinator immediately in the event of a 
suspected symptomatic overdose. Suspected symptomatic overdoses must be reported as AEs 
(or SAEs) in the eCRF. In the event of overdose, the appropriate supportive clinical care must 
be provided as dictated by the patient’s clinical status  
5.5.3 
Missed Doses 
If a dose is missed, patients will be advised as follows: 
• If you notice you missed a dose 2 hours or less after a scheduled dose time, dose 
immediately 
• If you notice you missed a dose more than 2 hours after a scheduled dose time, skip 
the dose you missed and resume regular dosing at the next planned time according to 
the schedule. A missed night dose may be taken at any time until midnight of the 
same day.  
5.6 
Masking 
Study drug will be double-masked. All study drug will be supplied in identical packaging, 
colour, smell, and appearance to enable double-masked conditions. K-321 and matching 
placebo will be provided in identical packaging so that all investigators, study site staff, 
patients, and clinical monitors will remain masked throughout the study. The IWRS will 
assign study drug to patients at the time of randomisation. Only personnel in IWRS, the 
independent randomisation team within 
 Biostatistics, and clinical supplies will be 
unmasked and will have access to treatment assignments; all other parties involved in the 
study will be fully masked. 
5.6.1 
Unmasking Treatment 
A patient’s treatment assignment will not be unmasked for the investigator or study site staff 
until the EOS unless medical treatment of the patient depends on knowing the study 
treatment the patient received. In the rare event that unmasking is needed because of a 
medical emergency, the investigator may unmask an individual patient through the IWRS. 
Reasons for treatment unmasking must be clearly explained and justified in the eCRF. The 
date on which the code was broken together with the identity of the person responsible must 
also be documented. 




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Any concomitant medication or surgical procedure deemed necessary for the welfare of the 
patient during the study may be given at the discretion of the investigator. However, it is the 
responsibility of the investigator to ensure that details regarding the medication and surgical 
treatment are recorded in full in the eCRF.  
5.8.1 
Descemetorhexis of the Study Eye 
As a condition of participating in the study, each patient will undergo descemetorhexis of the 
investigator-selected study eye at Visit 2 (Day –1). See Section 6.1.2 for details.  
Recommended post-descemetorhexis concomitant therapy consists 
 
 instilled 
 and 
 instilled 
 
. However, an investigator will be 
able to add 
 dosing after 
 if the 
investigator judges it necessary. When more than 1 different eye drop is administered 
(including study drug), the study drug must be administered first, followed by any non-study 
eye drop(s) after at least 5 minutes have passed (Section 5.2).  
A patient must not be randomly assigned to study drug unless the excised diameter satisfies 
inclusion criterion 5 (Section 4.1.1).  
5.8.2 
Rescue Surgical Procedures 
Penetrating keratoplasty or endothelial keratoplasty may be offered as rescue surgical 
procedures to an enrolled patient if the investigator judges that endothelial healing is not 
complete at Week 12 (Visit 9), or earlier if the investigator considers that the patient needs 
urgent treatment. If the rescue surgery is performed, the dosing with study drug must be 
discontinued immediately (without tapering) and the patient will attend all planned study 
visits including the follow-up period. 
5.8.3 
Prohibited and Restricted Concomitant Therapy 
5.8.3.1 
Prohibited and Restricted Surgical Intervention 
Throughout the study, study eyes must not undergo any surgical intervention other than the 
descemetorhexis at Visit 2. Non-study eyes must not undergo any surgical treatment for 
FECD or cataract in the screening or treatment periods, but they can be surgically treated 


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during the follow-up period. The non-study eye will not be eligible for participation in the 
trial, except if the descemetorhexis fails on the first-selected study eye and that eye is not 
eligible to participate. In that case, if all other inclusion and exclusion criteria are met by the 
second eye and more than 30 days have passed since the first study descemetorhexis, then the 
patient can be enrolled using the second eye to qualify. 
Punctal occlusion plugs are considered to be surgical interventions.  
5.8.3.2 
Prohibited and Restricted Medication 
The following medications and drugs identified as exclusion criteria are prohibited for use by 
patients throughout the study: 
• Netarsudil or other ROCK inhibitors 
• Eye drops and ointments containing 5% sodium chloride 
• Devices to improve any ocular symptoms (eg, TrueTear®, LipiFlow®, etc)  
• Drugs of abuse (opiates, methadone, cocaine, amphetamines, barbiturates, or 
benzodiazepines) or more than 2 standard drinks of alcohol per day 
• Contact lenses (except non-study eye) 
• Any ophthalmic solution besides artificial tears, over-the-counter anti-allergic 
ophthalmic products, study drug, or drugs prescribed by the investigator for treatment 
of the underlying condition 
• Any investigational drug besides study drug 


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6 
Study Assessments and Procedures 
Before performing any study procedures, all potential patients will sign an informed consent 
form (ICF). Patients will have the opportunity to have any questions answered before signing 
the ICF. The investigator must address all questions raised by the patient. The investigator or 
designee will also sign the ICF.  
The SOA is presented in Table 6-1. Detailed instructions for study site activities will be 
provided in the study manual. 
 


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Table 6-1 
Schedule of Study Site Activities 
 Activity 
Screening 
Treatment Period 
Follow-Up Period 
Time Point Day -28 
Day –1 
Day 1 
Week 1 Week2 Week 3 Week 5 Week 7 Week 9 Week 12 Week 16 Week 20 Week 24 Week 38 Week 52 
Visit Window (Day) -28 to -7 
- 
- 
5-9 
12-16 
18-24 
32-38 
46-52 
60-66 
81-87 
105-119 133-147 161-175 259-273 357-371 
Visit No. Visit 1 
Visit 2 
Visit 3 
Visit 4 Optional 
Visit 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and 
concomitant disease 
X 
X a 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination and 
vital sign measurements 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine drug screen 
X 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Urine pregnancy test (if 
applicable) 
 
X a 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Laboratory sampling  
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
Eligibility evaluation 
X 
X b 
 
 
 
 
 
 
 
 
 
 
 
 
 
Descemetorhexis with photo 
or video documentation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense study drug 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
study drug c 
 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
 
 
 
 
Study drug administration 
 
 
X 
X 
(X) d 
X 
X 
X 
X 
X 
 
 
 
 
 
Slit-lamp evaluation 
X 
X a 
X 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal ECD measurement e 
(specular microscopy, 
non-contact type) 
X 
 
 
X 
(X) d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Corneal ECD measurement 
by HRT f 
(X) 
 
 
(X) 
(X) d 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 




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6.1 
Eligibility Assessments and Procedures 
6.1.1 
Medical History, Physical Examination, and Laboratory 
Assessments 
Medical history and assessments of concomitant disease, physical examination and vital sign 
measurements, urine screening for drugs of abuse, pregnancy screening for patients of 
childbearing potential, and clinical laboratory assessments will be conducted at screening 
primarily to assess eligibility to participate in the study. The limited physical examination 
includes a review of the patient’s medical and medication history and subsequent evaluation 
of relevant body system complaints. In case of relevant findings, full evaluation of the 
condition needs to be conducted. Demographics information will be collected at screening as 
follows: age, race, iris colour, and sex. 
6.1.2 
Descemetorhexis of the Study Eye 
Each patient will undergo descemetorhexis of the investigator-selected study eye at Visit 2 
(Day –1). A digital photo of the study eye must be taken immediately before and immediately 
after descemetorhexis at Visit 2. The investigator will perform descemetorhexis to remove 
central confluent guttae, removing an area of Descemet membrane with a diameter of 
 
 The longest diameter and the shortest diameter must be recorded in the eCRF. The 
procedure must be recorded by video to record potential differences in technique among 
investigators. Also, immediately after the operation, the stripped area will be recorded by 
photo or video with an adjacent scale to measure the area removed. Detailed guidance for 
performing the study eye descemetorhexis will be provided in the study manual. 
Recommended post-descemetorhexis concomitant therapy is described in Section 5.8.1. 
6.2 
Efficacy Assessments 
6.2.1 
Corneal Endothelial Cell Density Measurement 
6.2.1.1 
Non-Contact Specular Microscopy 
Corneal ECD will be measured by non-contact specular microscopy. The study eye must be 
assessed at all study visits as indicated in Table 6-1, whereas the non-study eye must be 
measured only at Visit 1 and Visit 14. The same instrument must be used for all study visits 
for a given patient throughout the study. All specular microscope images taken will be sent to 


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the 
 for analysis via a designated portal site. Please see the study manual for detailed 
instructions.  
6.2.1.2 
Heidelberg Retina Tomography  
Heidelberg retina tomography (HRT) of the study eye will be performed according to the 
schedule in Table 6-1 if the necessary equipment is available at the study site before the start 
of the study at that site. All HRT images taken will be sent to the 
 for analysis via a 
designated portal site. Please see the study manual for detailed instructions.  
6.2.2 
Corneal Thickness 
Corneal thickness of the study eye will be measured by contact ultrasound pachymetry on the 
central cornea. The same instrument must be used for all study visits for a given patient 
throughout the study. From a safety perspective, the corneal thickness of both eyes must be 
measured, and the results must be recorded. See the study manual for detailed instructions.  
6.2.3 
Corneal Oedema  
Corneal oedema of the study eye will be categorised by epithelial, stromal, or endothelial, 
and assessed by the investigator as either present or absent.   
6.2.4 
Corneal Morphology 
Corneal morphology of the study eye will be assessed by OCULUS Pentacam® or Bausch 
and Lomb Orbscan®. If the site has both instruments, the OCULUS Pentacam will be used. 
The same instrument must be used for all study visits for a given patient throughout the 
study. All data and images must be recorded.  
6.2.5 
Best Corrected Visual Acuity  
The BCVA of the study eye will be assessed by ETDRS testing without pupil dilation. For all 
measurements, the BCVA letter score will be recorded. Please see the study manual for 
detailed instructions on the BCVA ETDRS assessment.  
6.2.6 
Contrast Sensitivity 
Visual contrast sensitivity of the study eye will be assessed using the VectorVision CSV-1000 
instrument if the necessary equipment is available at the study site before the start of the 




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6.3.2 
Adverse Events 
6.3.2.1 
Definitions of Adverse Events 
An AE is defined as any untoward medical occurrence in a patient enrolled into this study 
regardless of its causal relationship to study drug. An AE can therefore be any unfavourable 
and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related 
to the medicinal product. Patients will be instructed to contact the investigator at any time 
after randomisation if any symptoms develop. 
An AE may include the following: 
• Exacerbation of a pre‑existing disease or symptom 
• Increase in frequency or intensity of a pre‑existing event or condition 
• A condition detected or diagnosed after study drug administration even though it may 
have been present prior to the start of the study 
An AE does not include the following:  
• Medical or surgical procedures (although the condition that leads to the procedure 
must be reported as an AE) 
• Pre‑planned medical procedures (eg, planned surgical interventions, investigations, 
social and/or convenience hospitalisations) 
• Pre‑existing disease or conditions present at the time of signing the ICF are 
considered concurrent medical conditions and must not be recorded as an AE 
The investigator must attempt to establish a diagnosis of the event based on signs, symptoms, 
and other clinical information. In such cases, the diagnosis must be documented as the AE 
and not as the individual signs or symptoms.  


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The investigator will evaluate any changes in laboratory values and other study investigations 
and determine whether the change is clinically important and whether it is related to the 
study drug. The investigator must record the AE or investigation abnormality in the eCRF 
regardless of the relationship to the study drug (ie, the AE will be recorded whether or not it 
is considered related to the study drug). 
The criteria for determining whether an abnormal objective test finding must be reported as 
an AE are as follows: 
• The test result is associated with accompanying symptoms 
• The test result requires additional diagnostic testing or medical/surgical intervention 
• The test result leads to a change in study drug dosing or discontinuation from the 
study, significant additional concomitant drug treatment or other therapy and/or 
• The test result leads to any of the outcomes included in the definition of an SAE 
Merely repeating an abnormal test, in the absence of any of the above conditions, does not 
require reporting as an AE. An abnormal test result that is determined to be an error or 
artefact does not require reporting as an AE. 
A treatment-emergent AE (TEAE) is defined as any event not present before exposure to 
study drug or any event already present that worsens in either intensity or frequency after 
exposure to study drug. 
An SAE is defined as any event that 
• results in death.  
Any death resulting from an AE occurring during the study period or within 30 days after 
the last dose of the study drug, even if the death appears to be completely unrelated to the 
study or study drug. 
• is immediately life threatening.  
The term ‘life‑threatening’ refers to an event in which the patient was at risk of death at 
the time of the event; it does not refer to an event that hypothetically might have caused 
death if it were more severe. 


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• requires inpatient hospitalisation or prolongation of existing hospitalisation.  
The term ‘hospitalisation’ means a hospital admission or an overnight stay; prolongation 
means delay of a planned or anticipated discharge date by at least 1 overnight stay. 
Adverse events which require attendance at hospital but do not require admission may be 
considered medically important. 
• results in persistent or significant disability/incapacity. 
• results in impairment, damage, or disruption in the study patient’s body function or 
structure, or physical ability or quality of life.  
• is a congenital anomaly/birth defect.  
Additionally, events in which there is suspicion that exposure of either parent to the study 
drug resulted in an adverse outcome in the offspring is a medically important event. 
• Is medically important: an event that may not be immediately life‑threatening or fatal or 
result in hospitalisation, but may require intervention to prevent one of the serious 
outcomes listed above.  
Examples of such events are intensive treatment in an emergency room or at home for 
allergic bronchospasm; blood dyscrasias; convulsions that do not result in hospitalisation; 
or development of drug dependency or drug abuse.  
6.3.2.2 
Eliciting and Documenting Adverse Events 
The investigator is responsible for reporting AEs that are observed or reported during the 
study, regardless of their relationship to study drug or their clinical significance. Serious AEs 
will be collected from the time that the patient gives consent to participate in the study until 
30 days after the date of last dosing or of completion or withdrawal from the study, 
whichever is latest. All AEs will be collected starting from the time that the patient gives 
consent until the date of completion or withdrawal from the study. 
Serious AEs that occur more than 30 days after the last dose of study drug or after exit from 
the study, whichever is later, need not be reported unless the investigator considers them 
related to study drug. 
At each study visit, patients will be asked a standard non-leading question to elicit any 
medically related changes in their well-being. They will also be asked if they have been 


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hospitalised, had any accidents, had any eye surgery, used any new medications, or changed 
concomitant medication regimens (both prescription and over-the-counter medications). 
In addition to patient observations, AEs identified from any study data (eg, laboratory values, 
physical examination findings) or identified from review of other documents  that are 
relevant to patient safety will be documented on the AE page in the eCRF. 
6.3.2.3 
Reporting Adverse Events 
All AEs reported or observed  will be recorded on the AE page of the eCRF. Information to 
be collected includes event term, time of onset, date of resolution of the event, seriousness, 
severity, relatedness to study drug, any required treatment or evaluations, and outcome.  
The investigator will assess the severity of the event. The severity (intensity) of AEs must be 
classified using the following criteria:  
• Mild: An AE that is easily tolerated by the patient, causes minimal discomfort, and 
does not interfere with everyday activities 
• Moderate: An AE that causes enough discomfort to interfere with normal everyday 
activities and may require intervention 
• Severe: An AE that prevents normal everyday activities (treatment or intervention 
will usually be required). Severe events are usually incapacitating 
When changes in the severity of an AE occur more frequently than once a day, the maximum 
intensity for the event must be noted for that day. Any changes in intensity of signs and 
symptoms over multiple days will be captured by recording new AEs, with the amended 
intensity grades and the dates (and time, if known) of the change(s). Changes in the intensity 
of an AE must be documented to allow an assessment of the duration of the event at each 
level of intensity.  
The investigator will assess the causal relationship between the study medication and the AE 
and decide whether, in his or her qualified medical judgement, there is a reasonable 
possibility that the event may have been caused by the study drug. There are 2 categories of 
causality:  


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• Unrelated: If no valid reason exists for suggesting a relationship, or the event may 
plausibly be related to the patient's clinical condition, an underlying disease or other 
medication taken by the patient, then the AE must be considered unrelated.  
• Related: If there is any valid reason for supposing that there is a relationship between 
the study medicine and the AE, especially if the even improves on withdrawal of the 
drug (de-challenge) or recurs on recommencement of therapy (re-challenge), then the 
AE must be considered related. The AE must not be considered related simply 
because it is not possible to rule out an association between the AE and the study 
drug. 
The investigator’s assessment of an AE’s relationship to study drug is part of the 
documentation process, but it is not a factor in determining what is or is not reported in the 
study. If there is any doubt as to whether a clinical observation is an AE, the event must be 
reported. 
The action taken with the study medication must be recorded as treatment continued, 
treatment interrupted, or treatment discontinued. If the patient is no longer taking study 
medication at the time of the event, then action taken must be recorded as not applicable.  
All AEs will be followed to adequate resolution. The Medical Dictionary for Regulatory 
Activities (MedDRA) will be used to code all AEs.  
Any AE that meets SAE criteria (Section 6.3.2.1) must be reported to the 
 
Pharmacovigilance Department immediately (ie, within 24 hours) after the time site staff first 
learn about the event, using the electronic data capture (EDC) system. If for any reason the 
EDC is not available to the study site, the following contact information is to be used for 
SAE reporting: 
 
 Pharmacovigilance Department 
 
 
 
 
If the initial contact to 
 Pharmacovigilance is made via the SAE Hotline or the SAE fax 
line, the SAE must still be reported, urgently, using the EDC system when it becomes 
available. Details on SAE reporting are presented in the study manual.  


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6.3.2.4 
Suspected Unexpected Serious Adverse Reactions  
The sponsor will promptly evaluate all suspected unexpected serious adverse reactions 
(SUSARs) against cumulative product experience to identify and expeditiously communicate 
possible new safety findings to investigators, IRBs/IECs, and applicable health authorities 
based on applicable legislation. 
To determine reporting requirements for single SUSAR cases, the sponsor will assess the 
expectedness of these events using the Reference Safety Information section of the study 
drug investigator's brochure.  
The sponsor will compare the severity of each SUSAR and the cumulative event frequency 
reported for the study with the severity and frequency reported in the applicable reference 
document. 
6.3.2.5 
Follow-Up of Adverse Events 
All AEs must be reported in detail on the appropriate page in the eCRF and followed to 
resolution, until the investigator deems the event to be chronic or not clinically significant 
(NCS), or until the patient is considered stable. Adverse events that are ongoing at the time of 
the EOS visit are to be followed for 30 days or until resolution, until the investigator deems 
the no longer clinically significant (CS), or until the investigator considers the patient’s 
condition stable. 
6.3.3 
Vital Sign Measurements 
The following vital signs will be measured: 
• Blood pressure (measured supine, systolic and diastolic [mm Hg]) 
• Pulse (beats per minute) 
• Body temperature (°F) 
• Height (cm) and body weight (kg). Height is to be measured at screening only. 
Supine blood pressure, pulse, and body temperature (°F) will be recorded after the patient has 
been recumbent and at rest for at least 5 minutes. 


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6.3.4 
Eligibility 
Eligibility will be evaluated at screening (Visit 1) and post-operatively on Day –1 (Visit 2). 
Patients must meet all inclusion criterion and no exclusion criteria to be assigned study drug.  
6.4 
Safety Monitoring Committee 
There will be no safety monitoring committee.  
6.5 
Pregnancy 
Female patients of childbearing potential (defined in inclusion criterion 5 of Section 4.1.1) 
are required to use effective contraception throughout the study. 
Acceptable methods of contraception for female patients include oral contraceptives, other 
hormonal contraceptives (vaginal products, skin patches, or implanted/injectable products), 
or mechanical products such as an intrauterine device or barrier methods (diaphragm, 
condoms, spermicides). Note: female patients who are actively practising abstinence or who 
have a partner that is sterile (eg, vasectomy) will be permitted to participate in the study 
without contraception use. 
Pregnancy is not regarded as an AE unless there is a suspicion that an investigational product 
may have interfered with the effectiveness of a contraceptive medication. Any pregnancy that 
occurs during study participation must be reported using a clinical study pregnancy form. 
To ensure patient safety, each pregnancy must be reported to 
sponsor within 2 weeks 
of learning of its occurrence. The pregnancy must be followed up to determine outcome 
(including spontaneous miscarriage, elective termination, normal birth, or congenital 
abnormality) and status of mother and child even if the patient was discontinued from the 
study. Pregnancy complications and elective terminations for medical reasons must be 
reported as an AE or SAE. Spontaneous miscarriages and congenital abnormalities of the 
child must be reported as an SAE. 
Any SAE occurring in association with a pregnancy, brought to the investigator’s attention 
after study completion, and considered by the investigator as possibly related to the study 
treatment, must be promptly reported to 
sponsor. 


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6.6 
Laboratory Analyses 
Routine laboratory analyses will be performed by a central laboratory and will include the 
following assessments: 
• Haematology: haemoglobin, haematocrit, red blood cell count, white blood cell 
count with differential, mean corpuscular volume, mean corpuscular haemoglobin, 
mean corpuscular haemoglobin concentration, and absolute platelet count. 
• Clinical chemistry: total protein, sodium, potassium, chloride, albumin, glucose, 
HbA1c, blood urea nitrogen, creatinine, bilirubin (total and direct), alkaline 
phosphatase, aspartate aminotransferase, alanine aminotransferase, 
γ-glutamyltransferase, lactate dehydrogenase. 
• Urinalysis: pH, glucose, ketones, specific gravity, nitrite, protein, bilirubin, 
urobilinogen, and blood. Microscopic urinalysis will be performed if urinalysis results 
are abnormal. 
Urine pregnancy testing will be performed for all patients of childbearing potential before 
descemetorhexis at Visit 2 and at Visit 10.  
Urine screening for drugs of abuse (opiates, methadone, cocaine, amphetamines, barbiturates 
benzodiazepines) and alcohol will be performed by dipstick.  
6.6.1 
Future Genetic Testing 
A 6-mL blood sample will be obtained at Visit 1 for archiving and future genetic testing 
(Section 2.3). The blood sample will be collected only at sites in countries where allowed by 
local regulations and where approved by the IRB/IEC and relevant regulatory authorities.  
Results of genetic testing will not be reported to the patient, relatives, or attending physician, 
and they will not be recorded in the participant's medical record. The participant may 
withdraw consent for genetic testing at any time up to analysis, even after the samples have 
been obtained. In the event of withdrawal of consent, the bio-repository will be notified to 
pull and destroy the sample. 


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For all samples collected for genetic testing, precautions are to be taken to maintain 
confidentiality by de-identifying the sample to prevent the genetic data from being linked to 
the identity of the participant.  
6.7 
Sample Collections 
Sample collection, processing, and shipping must be performed according to local laboratory 
requirements and consistent with detailed procedures described in the study manual. 
The volume of blood sampled is planned to be less than 20 mL in a single day and less than 
35 mL over the entire study.  


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7 
Statistical and Analytical Plan 
A study eye will be identified for each patient. Unless otherwise specified, efficacy will be 
summarised for the study eye only. Ocular safety summaries by eye will be presented 
separately for the study eye and the non-study eye. Baseline measurements are the last 
measurements taken during the screening period (before Visit 2).  
7.1 
Primary Efficacy Endpoint 
The primary efficacy endpoint is the central corneal ECD at Week 12 as assessed by the 
 
7.2 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include: 
• Central corneal thickness change from baseline to each subsequent visit 
• Percent change of central corneal thickness from baseline to each subsequent visit 
• Central corneal ECD at each visit  
• Number and percentage of patients who achieve central corneal ECD of 
700 cells/mm2 or more at each visit 
• Time to achieve central corneal ECD 700 cells/mm2 or more  
• Number and percentage of patients who achieve cornea clearance at each visit where 
corneal clearance is defined as patients achieving a cornea thickness less than or 
equal to baseline cornea thickness and who have no corneal oedema 
o Number and percentage of patients who achieve cornea thickness less than or 
equal to baseline cornea thickness 
o Number and percentage of patients who achieve no corneal oedema 
• Time to cornea clearance  


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o Time to return of cornea thickness to less than or equal to baseline cornea 
thickness 
o Time to no corneal oedema 
7.3 
Exploratory Endpoints 
Exploratory endpoints include the following: 
• BCVA (letters) by ETDRS change from
 to 
  
• Time to achieve BCVA by ETDRS letter score 
 
  
• Number and percentage of patients who achieve 
 or more 
improvement (BCVA by ETDRS) 
• 
corneal ECD at 
  
• 
 corneal ECD change from 
 at 
  
• Percent change of 
 corneal ECD from 
 at 
 
• Changes in 
 parameters from
 
  
• Contrast sensitivity change (log values) from 
 
   
• 
 domain and total scores change from 
 
  
7.4 
Sample Size Calculations 
A sample size of 20 patients in each group will be needed to detect a difference of 
 in the 
measurement in the corneal ECD at Week 12 between K-321 0.4% QID/BID and placebo 
groups with a power of 
 for Step 1 and a power of 
 for Step 2 using a Wilcoxon 
rank sum test with a 0.050 two-sided significance level with closed testing procedures. 
Sample size was calculated by simulation. For the simulation, based on the existing literature, 
placebo was assumed to be distributed normally with 
 
 and K-321 was assumed to be distributed normally with 
 


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 Proportion of unmeasurable value of Week 12 due to corneal oedema and 
proportion of 
 value of Week 12 due to the withdrawal were assumed to be about 
10% and about 5%, respectively. 
7.5 
Analysis Sets 
The following analysis sets will be used in the statistical analyses. 
Full-analysis set (FAS): The FAS will consist of all participants who have a successful 
descemetorhexis procedure performed, were randomly assigned to receive double-mask 
study drug, received at least one dose of study drug in the study eye, and have at least one 
assessment of central corneal ECD performed after the descemetorhexis procedure on the 
study eye. (An assessment of “cannot perform due to corneal oedema” is considered a valid 
assessment for inclusion in the FAS.) All analyses using the FAS will group participants 
according to randomised treatment.  
Per-protocol set (PPS): The PPS will consist of all FAS participants who complete the 
12-week dosing treatment period for the study eye (for the primary analysis only), have at 
least 80% compliance with study treatment for the study eye, have not taken any prohibited 
medication, and have no major protocol deviations. Patients who receive rescue therapy will 
be considered to have completed study treatment for inclusion in the PPS. All analyses using 
the PPS will group participants according to treatment actually received.  
Safety set (SFS): The safety set will consist of all participants who received any study drug. 
All analyses using the safety set will group participants according to treatment actually 
received. 
The FAS will be the primary analysis set for efficacy analyses. Selected efficacy analyses 
will be repeated for the PPS. Safety will be summarised using the SFS.  
7.6 
Description of Subgroups to be Analysed 
Due to the small sample size, no subgroup analyses are planned.  
7.7 
Statistical Analysis Methodology 
Statistical analysis will be performed using SAS software Version 9.4 or later. Continuous 
variables will be summarised using the mean, the SD, median, minimum value, and 


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maximum value. Categorical variables will be summarised using frequency counts and 
percentages. Unless otherwise noted, the denominator to determine the percentage of 
participants in each category will be based on the number of participants with available data. 
Selected ordinal data may be summarised using both descriptive statistics and counts and 
percentages of participants in each category, as appropriate. Data will be listed in data 
listings. 
Details of the statistical analyses, methods, and data conventions are described in the 
statistical analysis plan (SAP). 
All statistical tests will be 2-sided and performed using a 0.05 significance level, leading to 
95% (2-sided) CIs. 
A closed 2-step ordered testing procedure will be used to control overall type I error for the 
primary efficacy endpoint, central corneal ECD at Week 12. In step 1, the K-321 0.4% QID 
group will be statistically compared with the placebo group. If the step 1 comparison is 
significant, step 2 will be performed to statistically compare K-321 0.4% BID group with the 
placebo group. If the statistical comparison made during step 1 is not significant, testing will 
still be performed to compare the BID group to placebo; however, all p values will be 
considered nominal/descriptive only. 
No other measures will be taken to control error for the multiple secondary and exploratory 
endpoints or multiple treatment comparisons. 
7.7.1 
Analysis of Primary Efficacy Endpoint 
The primary analysis of central corneal ECD will be performed on the study eye within the 
FAS via a Wilcoxon rank sum test and will be based on assessments performed by specular 
microscopy. Testing will be performed in a pairwise manner with each dosing regimen (QID 
and BID) of K-321 0.4% compared to placebo. For each dosing regimen, the following 
2-sided hypothesis will be tested  
Ho: Δ = 0 versus Ha: Δ ≠ 0 
where Δ is the shift in location in central corneal ECDs between the active and placebo 
groups being compared: ie,  


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Xi = ei for i = 1 to n for the placebo patients 
Yj = ej + Δ for j = 1 to m for the K-321 patients 
The Hodges-Lehmann estimate (Hodges and Lehmann 1963) for the shift in location (eg, the 
median of all differences d=Yj – Xi) and Moses 95% CI (Moses 1965) will also be presented.  
Week 12 assessments that are unable to be performed due to corneal oedema will be 
 
 
 
 
Week 12 assessments that are 
 due to patient withdrawal or missed visit will be 
 Patients who receive a 
rescue keratoplasty performed prior to Week 12 will be 
 
 
 
 
 
  
Sensitivity analyses will be performed by repeating the above analysis and 
 
 for the PPS. The primary analysis methods will also be repeated for the FAS using 
 
 Additionally, analyses will be performed using assessments performed by 
HRT for the subset of patients for which the assessments are available.  
7.7.2 
Analysis of Secondary Efficacy Endpoints 
Secondary endpoints will be summarised for the FAS. Central ECD which cannot be 
performed due to corneal oedema will be 
 for analysis. Assessments of central 
ECD, central corneal thickness, and epithelial oedema after rescue keratoplasty will be 
 
 Otherwise, secondary efficacy analyses will be 
 
Endpoints based on continuous measures (central ECD at each visit, central corneal thickness 
change from baseline to each visit, and percent change of central corneal thickness from 
baseline to each visit) will be analysed by pairwise Wilcoxon rank sum tests comparing each 


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active K-321 0.4% treatment. The Hodges-Lehmann estimate and Moses 95% CI for the shift 
in location will also be presented regimen to placebo.  
Endpoints based on the number and percentage of patients achieving the endpoint (patients 
who achieve central ECD of 700 cells/mm2 or more, patients who achieve cornea clearance 
at each visit, patients who achieve a cornea thickness less than or equal to baseline cornea 
thickness at each visit, and patients who have no corneal oedema at each visit) will be 
analysed by pairwise Fisher’s Exact or Pearson chi-square tests comparing each active K-321 
0.4% treatment regimen to placebo. The 95% CI for the difference in percentage of patients 
achieving the endpoint will also be presented. 
Endpoints based on time-to-event endpoints (time to achieve central ECD 700 cells/mm2 or 
more, time to cornea clearance, time to return of cornea thickness to less than or equal to 
baseline cornea thickness, time to no corneal oedema) will be analysed by pairwise log-rank 
tests comparing each active K-321 0.4% treatment regimen to placebo. Kaplan-Meier 
estimates and plots will also be presented. Patients who complete follow-up or discontinue 
the study without achieving the specified endpoint will be 
 
 Patients who receive rescue keratoplasty will be 
 
  
7.7.3 
Analyses of Exploratory Efficacy Endpoints 
Exploratory endpoints will be summarised for the FAS. Assessments after rescue keratoplasty 
will be 
 Endpoints will be analysed in a similar manner to the secondary 
efficacy endpoints.  
Endpoints based on continuous measures will be analysed by pairwise Wilcoxon rank sum 
tests comparing each active K-321 0.4% treatment regimen to placebo with the 
Hodges-Lehmann estimate for the different in medians and Moses 95% CI for the difference 
in medians also presented. 
Endpoints based on the number and percentage of patients achieving the endpoint will be 
analysed by pairwise Pearson chi-square tests comparing each active K-321 0.4% treatment 
regimen to placebo with the 95% CI for the difference in percentage of patients achieving the 
endpoint also presented. 


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Endpoints based on time-to-event endpoints will be analysed by pairwise log-rank tests 
comparing each active K-321 0.4% treatment regimen to placebo with Kaplan-Meier 
estimates and plots also presented. Patients who complete follow-up or discontinue the study 
without achieving the specified endpoint will be 
 
 Patients who receive rescue keratoplasty will be 
 
 
7.7.4 
Safety Analyses 
Safety parameters include monitoring of AEs, TEAEs, vital sign measurements, laboratory 
examinations, slit-lamp biomicroscopy, IOP, and ophthalmoscopy. 
Safety data will be summarised and listed with no statistical hypothesis testing performed. 
Continuous variables will be summarised using the mean, the SD, median, minimum value, 
and maximum value. Categorical variables will be summarised using frequency counts and 
percentages. 
7.7.4.1 
Adverse Events  
All AEs will be listed and all TEAEs will be summarised. Adverse events will be deemed 
treatment-emergent if the onset date is on or after the date of first treatment. Events which are 
not treatment-emergent will be listed separately and together with all other AEs. AEs will be 
summarised separately for the treatment period, the tapering phase, and the 9-month 
follow-up period. Ocular TEAEs will be summarised separately from non-ocular TEAEs. 
Ocular TEAEs will be summarised separately for the study eye and non-study eye.  
Treatment-emergent AEs will be summarised by treatment group, presenting the number and 
percentage of patients having a TEAE in each system organ class and having each individual 
TEAE based on the preferred term. Treatment-emergent AEs will also be tabulated according 
to intensity and causality. Patients who experienced multiple TEAEs for a preferred term will 
be counted once, similarly for patients with multiple TEAEs per system organ class. 
Deaths, SAEs, and TEAEs leading to discontinuation of study treatment will be listed 
separately and, if appropriate, summarised by treatment group, system organ class and 
preferred term. 


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7.7.4.2 
Vital Signs 
Observed values at each visit and change from baseline values for vital sign measurements 
will be summarised descriptively by treatment group. All vital sign measurement data will be 
listed. 
7.7.4.3 
Clinical Laboratory  
Observed values at each visit and change from baseline values for clinical laboratory 
evaluations will be summarised descriptively by treatment group. Additionally, shift tables 
will be presented for chemistry and hematology parameters from baseline to each post-
baseline assessment. All clinical laboratory data will be listed. 
7.7.4.4 
Intra-Ocular Pressure 
All IOP data will be listed. Observed values at each visit and change from baseline values for 
IOP evaluations will be summarised descriptively by treatment group for the study eye and 
non-study eye.  
7.7.4.5 
Slit-Lamp Biomicroscopy and Ophthalmoscopy 
Ocular findings will be evaluated by the following criteria: 
• Normal 
• Abnormal (NCS) 
• Abnormal with clinical significance (CS) 
Data from all slit-lamp biomicroscopy and ophthalmoscopy assessments will be listed. The 
number and percentage of patients with at least 1 treatment-emergent CS abnormality by 
ocular assessment will be summarised for each treatment group for the study eye and 
non-study eye. An abnormality will be deemed treatment-emergent if the onset date is on or 
after the date of first treatment. A patient will be counted once even if the patient experiences 
more than 1 treatment-emergent CS abnormality.  


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7.7.5 
Other Analyses 
7.7.5.1 
Patient Demographics and Other Baseline Characteristics 
Descriptive statistics will be provided by treatment group for patient demographics and all 
ocular and baseline characteristics. Relevant medical history (ocular and non-ocular) and 
current medical conditions will be tabulated by treatment group. Ocular summaries will be 
presented for the study eye and the non-study eye. Other relevant baseline information will 
be listed and summarised as appropriate with descriptive statistics. Analyses will be based on 
the FAS. 
7.7.5.2 
Treatment Exposure and Compliance 
Descriptive statistics will be provided to characterise study treatment exposure and 
compliance using the safety set. Summaries will also be provided for the FAS if it differs 
from the safety set. Duration of exposure will be calculated in days as the (date of last dose –
 date of first dose + 1). Percent compliance will be calculated as the total number of study 
drug instillations/planned number of instillations × 100. The planed number of instillations 
for Visit 3 (Day 1) will be adjusted based on the time the descemetorhexis was completed on 
Visit 2 (Day –1) and the time all study visit assessments are completed on Visit 3. For all 
other study visits during the treatment period, the planed number of instillations will be 
adjusted based on the time all planned study visit assessments are completed on the 
respective study visit days, as activities on these days may reduce the number of doses that 
can be given. The details of dosing adjustment will be provided in the SAP. For all other 
study days, the number of planned instillations will be considered to be 4 doses. If the first 
and last doses are administered on the same day, the number of planned instillations will be 
based on the time of the first dose. Subjects will be considered compliant with study 
treatment if the calculated percent compliance is between 80% and 125%, inclusive. 
 
7.7.6 
Interim Analyses 
No interim analyses will be performed. The primary analysis will be conducted at the 
Week 12 database lock ie, after all patients have completed dose tapering at the Week 16 visit 
or have been discontinued from the study and/or treatment before this visit. Analysis of the 
complete study data (through Week 52) will be performed after all patients have completed 


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the Week 52 visits or have been discontinued from the study before the Week 52 visit. As the 
primary efficacy endpoint is at the Week 12 visit, no alpha adjustments will be required. No 
changes to study conduct (eg, change to sample size or early stopping) are planned.  


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8 
Data Quality Assurance 
The sites will maintain source documentation and enter patient data into the eCRF as 
accurately as possible and will rapidly respond to any reported discrepancies. Electronic 
CRFs are accessed through Medidata Rave® (Medidata Solutions Inc, New York, New York). 
This EDC system is validated and compliant with US Title 21 Code of Federal Regulations 
(CFR) Part 11. Each person involved with the study will have an individual user name and 
password that allows for record traceability. Thus, the system, and subsequently any 
investigative reviews, can identify coordinators, investigators, and individuals who have 
entered or modified records, as well as the time and date of any modifications. A quality 
review of the data will be performed by the site with additional reviews by the clinical 
monitor through source data verification.  
Each eCRF is presented as an electronic copy, allowing data entry by site staff, who can 
add and edit data, identify and resolve discrepancies, and view records. This system provides 
immediate direct data transfer to the database, as well as immediate detection of 
discrepancies, enabling site coordinators to resolve and manage discrepancies in a timely 
manner.  
Paper copies of the eCRFs and other database reports may be printed by the investigator. 
This system provides site staff, monitors, and reviewers with access to hard copy audits, 
discrepancy reviews, and investigator comment information.  
After all data reviews and query resolutions are complete, the SAP is approved and signed, 
and any summary/analysis populations are approved, the database will be locked. 
8.1 
Data Management 
As part of the responsibilities assumed by participating in the study, the investigator agrees 
to maintain adequate case histories for patients treated as part of the research under this 
protocol. The investigator agrees to maintain accurate eCRFs and source documentation as 
part of the case histories. These source documents may include laboratory reports, etc. All 
eCRF information is to be completed. If an item is not available or is not applicable, this fact 
must be indicated. Blank spaces must not be present unless otherwise directed. 
Investigative site personnel will enter patient data into an EDC system. The analysis data sets 
will be a combination of these data and data from other sources (eg, laboratory data). 


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Clinical data management will be performed in accordance with applicable 
 standards 
and data cleaning procedures to ensure the integrity of the data (eg, removing errors and 
inconsistencies in the data). Adverse events and medical history will be coded using the 
MedDRA terminology. Concomitant medications will be coded using the most current 
available WHO Drug Dictionary. 
After database lock, each study site will receive an electronic copy of their site-specific 
eCRF data as entered into the EDC system for the study, including full discrepancy and audit 
history. Additionally, an electronic copy of all the study site’s data from the study will be 
created and sent to the sponsor for storage. 
 will maintain a duplicate electronic copy for 
their records. In all cases, patient initials will not be collected or transmitted to the sponsor. 


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9 
Ethics 
9.1 
Independent Ethics Committee or Institutional Review Board 
Country regulations, US federal regulations, and the International Council for Harmonisation 
(ICH) guidelines require that approval be obtained from an IRB or IEC before participation 
of human patients in research studies. Before study onset, the protocol, informed consent, 
advertisements to be used for the recruitment of study patients, and any other written 
information regarding this study to be provided to the patient or the patient’s legal guardian 
must be approved by the IRB/IEC. Documentation of all IRB/IEC approvals and of the 
IRB/IEC compliance with ICH harmonised tripartite guideline E6(R2): Good Clinical 
Practice (GCP) and with applicable regulations in the countries where the study will be 
conducted will be maintained by the site and will be available for review by the sponsor or its 
designee. 
All IRB/IEC approvals must be signed by the IRB/IEC chairman or designee and must 
identify the IRB/IEC name and address, the clinical protocol by title or protocol number or 
both, and the date approval or a favourable opinion was granted. 
The investigator is responsible for providing written summaries of the progress and status of 
the study at intervals not exceeding 1 year or otherwise specified by the IRB/IEC. The 
investigator must promptly supply the sponsor or its designee, the IRB/IEC, and, where 
applicable, the institution, with written reports on any changes significantly affecting the 
conduct of the study or increasing the risk to patients. 
9.2 
Ethical Conduct of the Study 
The study will be performed in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki, ICH GCP, the protocol, and all applicable country and local 
regulations. 
9.3 
Patient Information and Consent 
A written informed consent in compliance with regulatory authority regulations or 
US Title 21 CFR Part 50 shall be obtained from each patient before entering the study or 
performing any unusual or non-routine procedure that involves risk to the patient. An 
informed consent template may be provided by the sponsor to investigative sites. If any 


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institution-specific modifications to study-related procedures are proposed or made by the 
site, the consent must be reviewed by the sponsor or its designee or both before IRB/IEC 
submission. Once reviewed, the consent will be submitted by the investigator to his or her 
IRB/IEC for review and approval before the start of the study. If the ICF is revised during the 
course of the study, all active participating patients must sign the revised form. 
Before recruitment and enrollment, each prospective patient or his or her legal guardian will 
be given a full explanation of the study and be allowed to read the approved ICF. Once the 
investigator is assured that the patient/legal guardian understands the implications of 
participating in the study, the patient/legal guardian will be asked to give consent to 
participate in the study by signing the ICF. The investigator will then sign the consent signed 
by the patient/legal guardian. 
The investigator shall retain the signed original ICF(s) and give a copy of the signed original 
form to the patient or legal guardian. 


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10 Investigator’s Obligations 
The following administrative items are meant to guide the investigator in the conduct of the 
study but may be patient to change based on industry and government standard operating 
procedures, working practice documents, or guidelines. Changes will be reported to the 
IRB/IEC but will not result in protocol amendments. 
10.1 Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain patient confidentiality. All records will be kept in a secure 
storage area with limited access. Clinical information will not be released without the written 
permission of the patient (or the patient’s legal guardian), except as necessary for monitoring 
and auditing by the sponsor, its designee, the US Food and Drug Administration (FDA), the 
Medicines and Healthcare Products Regulatory Agency (MHRA), other agencies, or the 
IRB/IEC. 
The investigator and all employees and coworkers involved with this study may not disclose 
or use for any purpose other than performance of the study any data, record, or other 
unpublished, confidential information disclosed to those individuals for the purpose of the 
study. Prior written agreement from the sponsor or its designee must be obtained for the 
disclosure of any said confidential information to other parties. 
10.2 Financial Disclosure and Obligations 
Investigators are required to provide financial disclosure information to allow the sponsor to 
submit the complete and accurate certification or disclosure statements required under 
21 CFR 54. In addition, the investigator must provide to the sponsor a commitment to 
promptly update this information if any relevant changes occur during the course of the 
investigation and for 1 year following the completion of the study. 
Neither the sponsor nor 
 is financially responsible for further testing or treatment of any 
medical condition that may be detected during the screening process. In addition, in the 
absence of specific arrangements, neither the sponsor nor 
 is financially responsible for 
further treatment of the patient’s disease. 


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10.3 Investigator Documentation 
Prior to beginning the study, the investigator will be asked to comply with ICH E6(R2) 8.2 
and Title 21 of the CFR by providing the following essential documents, including but not 
limited to: 
• IRB/IEC approval. 
• Original investigator-signed investigator agreement page of the protocol. 
• Form FDA 1572, fully executed, and all updates on a new fully executed Form 
FDA 1572. 
• Curriculum vitae (CV) for the investigator and each sub-investigator listed on Form 
FDA 1572. 
• Financial disclosure information to allow the sponsor to submit complete and accurate 
certification or disclosure statements required under 21 CFR 54. In addition, the 
investigators must provide to the sponsor a commitment to promptly update this 
information if any relevant changes occur during the course of the investigation and for 
1 year after the completion of the study. 
• IRB/IEC-approved informed consent, samples of site advertisements for recruitment for 
this study, and any other written information regarding this study that is to be provided to 
the patient or legal guardian, and 
• Laboratory certifications and normal ranges for any local laboratories used by the site, in 
accordance with 42 CFR 493. 
10.4 Study Conduct 
The investigator agrees that the study will be conducted according to the principles of ICH 
E6(R2). The investigator will conduct all aspects of this study in accordance with all 
applicable national, state, and local laws or regulations. Study information from this protocol 
will be posted on publicly available clinical trial registers before enrollment of patients 
begins. 


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10.5 Adherence to Protocol 
The investigator agrees to conduct the study as outlined in this protocol in accordance with 
ICH E6(R2) and all applicable guidelines, regulations, and laws, and consistent with the 
principles of the Declaration of Helsinki. 
10.6 Adverse Events and Study Report Requirements 
By participating in this study, the investigator agrees to submit reports of SAEs to the 
contract research organization 
 and/or IRB/IEC according to the time line and method 
outlined in the protocol. In addition, the investigator agrees to submit annual reports to the 
study site IRB/IEC as appropriate. 
10.7 Investigator’s Final Report 
Upon completion of the study, the investigator, when applicable, must inform the institution. 
Also, the investigator/institution must provide the IRB/IEC with a summary of the study’s 
outcome and provide the sponsor and regulatory authority(ies) with any reports required. 
10.8 Records Retention 
Essential documents must be retained for at least 25 years. These documents must be retained 
for a longer period, however, if required by the applicable regulatory requirements or by an 
agreement with the sponsor. It is the responsibility of the sponsor to inform the 
investigator/institution as to when these documents no longer need to be retained. The 
patients’ medical records, however, need only be retained as long as required by the 
institution, in accordance with national law.  
10.9 Publications 
After completion of the study, the data may be considered for reporting at a scientific 
meeting or for publication in a scientific journal. In these cases, the sponsor will be 
responsible for these activities and will work with the investigators to determine how the 
manuscript is written and edited, the number and order of authors, the publication to which it 
will be submitted, and other related issues. The sponsor has final approval authority over all 
such issues. 


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Data are the property of the sponsor and cannot be published without prior authorisation from 
the sponsor, but data and publication thereof will not be unduly withheld. 




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11.1.2 
Monitoring of the Study 
The clinical monitor, as a representative of the sponsor, has the obligation to follow the study 
closely. In doing so, the monitor will visit the investigator and study site at periodic intervals, 
in addition to maintaining necessary telephone and letter contact. The monitor will maintain 
current personal knowledge of the study through observation, review of study records and 
source documentation, and discussion of the conduct of the study with the investigator and 
personnel. 
All aspects of the study will be carefully monitored, by the sponsor or its designee, for 
compliance with applicable government regulation with respect to current GCP and current 
standard operating procedures. 
11.1.3 
Inspection of Records 
Investigators and institutions involved in the study will permit study-related monitoring, 
audits, IRB/IEC review, and regulatory inspections by providing direct access to all study 
records. In the event of an audit, the investigator agrees to allow the sponsor, representatives 
of the sponsor, or a regulatory agency (eg, FDA or other regulatory agency) access to all 
study records. 
The investigator must promptly notify the sponsor and 
 of any audits scheduled by any 
regulatory authorities and promptly forward copies of any audit reports received to the 
sponsor. 
11.2 Management of Protocol Amendments and Deviations 
11.2.1 
Modification of the Protocol 
Any changes in this research activity, except those necessary to remove an apparent, 
immediate hazard to the patient, must be reviewed and approved by the sponsor or its 
designee before implementation. Amendments to the protocol must be submitted in writing to 
the investigator’s IRB/IEC for approval before patients can be enrolled into an amended 
protocol or before the amendment can be implemented for patients already participating in 
the study. 


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11.2.2 
Protocol Deviations 
The investigator or designee must document and explain in the patient’s source 
documentation any deviation from the approved protocol. The investigator may implement a 
deviation from, or a change of, the protocol to eliminate an immediate hazard to study 
patients without prior IRB/IEC approval. As soon as possible after such an occurrence, the 
implemented deviation or change, the reasons for it, and any proposed protocol amendments 
must be submitted to the IRB/IEC for review and approval, to the sponsor for agreement, and 
to the regulatory authorities, if required. 
A deviation from the protocol is an unintended or unanticipated departure from the 
procedures or processes approved by the sponsor and the IRB/IEC and agreed to by the 
investigator. A significant deviation occurs when there is non-adherence to the protocol by 
the patient or investigator that results in a significant, additional risk to the patient or to the 
integrity of the study. Significant deviations can include non-adherence to inclusion or 
exclusion criteria, or non-adherence to FDA regulations or ICH GCP guidelines, and will 
lead either to the patient being discontinued from study drug treatment or withdrawn from the 
study (Section 4.2). 
Protocol deviations will be documented by the clinical monitor throughout the course of 
monitoring visits. Principal investigators will be notified in writing by the monitor of 
deviations. The IRB/IEC must be notified of all protocol deviations in a timely manner. 
11.3 Study Termination 
Although Kowa Research Institute, Inc. has every intention of completing the study, Kowa 
Research Institute, Inc. reserves the right to discontinue the study at any time for clinical or 
administrative reasons. 
The end of the study is defined as the date on which the last patient completes his or her last 
visit.  
11.4 Final Report 
Whether the study is completed or prematurely terminated, the sponsor will ensure that the 
clinical study reports are prepared and provided to the regulatory agency(ies) as required by 
the applicable regulatory requirement(s). The sponsor will also ensure that the clinical study 


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reports in marketing applications meet the standards of the ICH harmonised tripartite 
guideline E3: Structure and content of clinical study reports and the standards of regulatory 
agencies to which the clinical study reports will be submitted.  
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the clinical study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results. 
Upon completion of the clinical study report, the sponsor will provide the investigator with 
the full summary of the study results. The investigator is encouraged to share the summary 
results with the study patients, as appropriate. The study results will be posted on publicly 
available clinical trial registers. 


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