Clinical trial NCT05451329
Conditions: Glaucoma, Open-Angle
Sponsor: VivaVision Biotech, Inc
Start date: 2022-07-12; Completion date: 2022-12-22
Enrollment: 68 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a prospective, parallel-comparison, multi-center, double-masked, randomized, vehicle-controlled dose-response study assessing the safety and ocular hypotensive efficacy of VVN539 in subjects with POAG or OHT. Three different dosing regimens (once a day \[q.d.\] in the morning, q.d. in the evening and twice a day \[b.i.d.\]) will be tested for 7-9 days, each.

Detailed description:

Arms: arm 1: VVN539 Ophthalmic Solution 0.02% arm 2: VVN539 Ophthalmic Solution 0.04% arm 3: VVN539 Ophthalmic Solution Vehicle
Interventions: intervention 1: VVN539 Ophthalmic Solution 0.02% intervention 2: VVN539 Ophthalmic Solution 0.04% intervention 3: VVN539 Ophthalmic Solution Vehicle
Intervention descriptions: intervention 1: Once a day \[q.d.\] in the morning, q.d. in the evening and twice a day \[b.i.d.\]) will be tested for 7-9 days intervention 2: Once a day \[q.d.\] in the morning, q.d. in the evening and twice a day \[b.i.d.\]) will be tested for 7-9 days intervention 3: Once a day \[q.d.\] in the morning, q.d. in the evening and twice a day \[b.i.d.\]) will be tested for 7-9 days

Protocol documents:
Prot_000.pdf: 
NCT05451329 
Study VVN559-CS201 
Clinical protocol 
 
A phase 2, double-masked, randomized, vehicle-controlled, dose-response study assessing the 
safety and ocular hypotensive efficacy of VVN539 in subjects with primary open angle 
glaucoma or ocular hypertension. 
 
Dated 7 June 2022 


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VVN539-CS-201 Protocol v2.0 
Title Page 
 
A phase 2, double-masked, randomized, vehicle-controlled, dose-
response study assessing the safety and ocular hypotensive efficacy of 
VVN539 in subjects with primary open angle glaucoma or ocular 
hypertension. 
 
Protocol No.: 
VVN539-CS-201 
Investigational New Drug (IND) No.: 
158012 
Study Phase: 
2 
Sponsor:  
VivaVision Biotech, Inc. 
304-866 Halei Rd, Pudong District, 
Shanghai, China 
Medical Monitor:  
 
 
 
 
 
Version No.: 
2.0 
Issue Date:  
07/Jun/2022 
 
Confidentiality Statement  
This document contains information from VivaVision and is of confidential, trade secret, and/or proprietary nature and may not be 
disclosed unless such disclosure is required by federal or state law or regulation. It is loaned to you for your confidential review on 
behalf of VivaVision only and is not to be photocopied, disclosed, or transmitted to any other person or party without the advance 
written approval of VivaVision.   


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Protocol Synopsis 
Title: 
A phase 2, double-masked, randomized, vehicle-controlled, dose-response 
study assessing the safety and ocular hypotensive efficacy of VVN539 in 
subjects with primary open angle glaucoma or ocular hypertension. 
Phase: 
2 
Design/Conduct: 
Prospective, parallel-comparison, multi-center, double-masked, randomized, 
vehicle-controlled dose-response study assessing the safety and ocular 
hypotensive efficacy of VVN539 in subjects with primary open angle 
glaucoma (POAG) or ocular hypertension (OHT) in the United States (US). 
Objective(s): 
Efficacy: To evaluate the ocular hypotensive efficacy of 2 concentrations of 
VVN539. 
Safety: To evaluate the ocular and systemic safety of 2 concentrations of 
VVN539 
Endpoint(s): 
Primary Efficacy: Mean intraocular pressure (IOP) at each diurnal time point 
(8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14) and Visit 6 (Day 
21). 
Secondary Efficacy: Mean change in IOP from Visit 2 (Baseline) to Visit 4 
(Day 7), Visit 5 (Day 14) and visit 6 (Day 21). 
Population studied: 
Approximately 60 (20 per cohort) evaluable adult males or females diagnosed 
with OHT or POAG. 
Inclusion criteria: 
1. 18 years of age or older. 
2. Diagnosis of POAG or OHT in both eyes that are untreated, or if 
treated, in the opinion of the investigator are well controlled on 2 or 
fewer ocular hypotensive medications prior to Visit 1.  
• 
Combination medications count as 2 medications 
3. Unmedicated IOP of ≥ 22 mm Hg and ≤ 36 mm Hg in the study eye, 
with no more than 5 mm Hg inter-eye difference at 08:00AM and 
10:00AM at Visit 2. 
4. Corrected visual acuity in each eye +1.0 logMAR (Snellen equivalent 
to 20/200) or better by ETDRS in each eye. 
5. Central corneal thickness of ≥ 400 and ≤ 620 µm in each eye. 
6. Able and willing to give signed informed consent and follow study 
instructions. 
7. In the opinion of the Investigator, the subject is able to safely 
discontinue current ocular hypotensive medication(s), when a 
washout is required. 
Exclusion criteria: 
Ophthalmic:  
1. Known hypersensitivity to any kinase inhibitors, any excipient or 
preservative of the formulation or to topical anesthetics or fluorescein. 
2. Unmedicated IOP of > 36 mm Hg in either eye at any time point at 
Visit 2. 


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3. History of intraocular implant for IOP treatment, glaucoma filtering 
surgery, placement, or removal of minimally invasive glaucoma 
implant (MIGS) in the study eye.   
4. History of laser IOP lowering surgery within 6 months of Visit 1 in 
the study eye.   
• 
Note: Laser peripheral iridotomy for narrow angle is allowed if 
performed 3 months or more prior to Visit 1. 
5. Ocular trauma within the past six months, or surgery involving the 
ocular surface (including cataract surgery) or non-IOP-lowering laser 
treatment within the past three months of Visit 1 in the study eye. 
6. Refractive surgery in the study eye (including but not limited to radial 
keratotomy, Photorefractive Keratectomy [PRK], Laser-Assisted in 
situ Keratomileusis [LASIK], etc.). 
• 
Refractive surgery planned during study participation in either 
eye. 
7. Have received intravitreal corticosteroid injection within 6 months or 
subtenon/subconjunctival steroid injection within 3 months of Visit 1 
in either eye. 
8. History or evidence of ocular infection, inflammation, clinically 
significant blepharitis, conjunctivitis, or of herpes simplex keratitis 
within 2 months of Visit 1 in either eye. 
9. Unable or unwilling to avoid wearing of contact lenses from Visit 1 
through the study.   
10. Currently using 3 or more ocular hypotensive medications prior to 
screening in either eye (combination medications count as 2 
medications). 
11. Ocular medication of any kind within 28 days of Visit 2 in either eye 
other than: 
• 
Ocular hypotensive medications which must be washed out 
according to the provided schedule 
• 
Ocular Medication as part of an eye exam 
• 
Lubricating drops for dry eye 
12. Clinically significant ocular disease in either eye (e.g. corneal edema, 
uveitis, severe keratoconjunctivitis sicca) which might interfere with 
the study. 
13. Pseudoexfoliative, 
pigmentary, 
congenital, 
developmental 
or 
secondary glaucoma (e.g. neovascular, uveitic, pigmentary, lens-
induced, steroid-induced, trauma induced or glaucoma associated 
with increased episcleral venous pressure) in either eye. 
14. Closed angle forms of glaucoma in either eye 
• 
Shaffer grade < 3 (in 1 or more quadrants) 
• 
Gonioscopy within 3 months prior to Visit 1 is acceptable. 
15. Cup-disc ratio of 0.7 or greater in either eye. 
16. Nerve fiber layer thickness assessment by Optical Coherence 
Tomography (OCT) documents thinning of the NFL into the abnormal 
range for either eye.  
• 
If, in the opinion of the investigator, the thinning of the NFL may 
be attributed to other ocular features (including but not limited to 


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optic nerve drusen, myopia, chorioretinal scars), then the subject 
is allowed. 
17. Subjects with visual fields with substantial glaucomatous loss (e.g., 
Bjerrum scotoma, blind spot enlargement) for whom washout and 
potential randomization would be unsafe. 
18. In addition to the above criteria, glaucomatous damage in either eye 
so severe that washout of ocular hypotensive medications for up to 5 
weeks and treatment for 3 weeks (for subjects randomized to vehicle 
control) is not judged safe in the opinion of the Investigator.  
• 
Note: Subjects may be changed to a another IOP lowering therapy 
with a shorter washout period as long as the appropriate washout 
is met prior to Visit 2. 
19. Any abnormality preventing reliable applanation tonometry of either 
eye. 
General/Systemic: 
20. Laboratory tests indicative of any clinically significant disease in the 
opinion of the Investigator. 
21. Clinically significant systemic disease (e.g., uncontrolled diabetes, 
myasthenia gravis, hepatic, renal, cardiovascular or endocrine 
disorders) which might interfere with the study in the opinion of the 
Investigator. 
22. Participation in any study involving an investigational product within 
the past 30 days of Visit 1. 
23. Changes of systemic beta blockers during the study that in the opinion 
of the Investigator could have a substantial effect on IOP within 30 
days prior to Visit 1 or anticipated during the study. 
24. Have received systemic corticosteroids (oral, injectable, inhaled) 
within 30 days of Visit 1 or planned use during the study.   
• 
Note: Topical dermatologic steroids are allowed if used for less 
than 3 consecutive days on no more than 3 separate areas of the 
body within 30 days of Visit 1 and the dosage is not expected to 
increase during the study.   
• 
Intranasal steroids are allowed if used less than or 4 times per 
week within 30 days of Visit 1 and the dosage is not expected to 
increase during the study. 
25. History of substance abuse within 1 year of Visit 1 
26. Subjects that are employees or relatives of employees at the clinical 
site. 
27. Women of childbearing potential who are pregnant, nursing, planning 
a pregnancy, or not using a medically acceptable form of birth control.  
• 
Females of childbearing potential with a positive urine pregnancy 
test (UPT) result at Visit 1 or Visit 2 are excluded. 
• 
An adult woman is considered to be of childbearing potential 
unless she is one year post-menopausal or three months post-
surgical sterilization.  
Investigational product(s): 
VVN539 ophthalmic solution 0.02% (3mL in 5 mL bottles) 
VVN539 ophthalmic solution 0.04% (3mL in 5 mL bottles) 
VVN539 ophthalmic solution vehicle (3mL in 5 mL bottles) 


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Dosing regimen: 
Both eyes (OU), q.d. (morning) for 7 days, then q.d. (evening) for 7 days, and 
then b.i.d. for 7 days. 
Assessments/Evaluations: 
Efficacy:  
• 
IOP for efficacy will be measured by Goldmann applanation 
tonometry at 8:00AM, 10:00AM and 4:00PM at Visits 2, 4, 5 and 6. 
Safety: 
• 
Adverse event monitoring 
• 
Best Corrected Visual Acuity (ETDRS) 
• 
IOP 
• 
Biomicroscopy of anterior segment  
• 
Central corneal thickness  
• 
Cup-disc ratio  
• 
Ophthalmoscopy (dilated) 
• 
Heart rate and blood pressure (BP) 
• 
Blood chemistry, hematology 
Duration of study: 
There are a total of 6 clinical visits over 8 weeks (up to 5 weeks of washout 
and 3 weeks of study treatment) 
Statistical methods: 
With a sample size in each group of 20, the study will have 80% power to detect 
a difference of 3.0 mm Hg between an VVN539 dose compared to vehicle at 
each diurnal time point assuming a common standard deviation of 3.3 mm Hg, 
α = 0.05 (two-tailed). There is no correction for multiplicity for multiple time 
points or comparisons for high or low VVN539 doses. 
 
The primary endpoint for this study is to compare mean IOP at each diurnal 
time point (8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14) and 
Visit 6 (Day 21) for two doses of VVN539 compared to vehicle using data from 
the study eye. Time-matched longitudinal models adjusting for baseline and 
including a random effect for center will be used to test time-specific 
comparisons. Data from Days 7, 14, and 21 will be analyzed using mixed-
model repeated measures (MMRM) with an unstructured covariance assumed 
for each treatment with treatment, visit, and visit by treatment interaction as a 
fixed effects, baseline measurement as a covariate, and a random effect for site. 
Sensitivity analyses include a time-matched last observation carried forward 
(LOCF) of the last available IOP measurement as well as analysis of covariance 
(ANCOVA) analyses at each visit. Additional sensitivity analyses will be 
performed on the PP Analysis Set as necessary. 
 
Analyses of change from baseline and observed and change from baseline for 
diurnal IOP will be performed in a manner similar to the primary efficacy 
analysis. 
 
Safety analyses will be performed on all subjects in the Safety Analysis Set. 
The assessment of safety will be based on the summary of ocular and non-
ocular treatment emergent adverse events (TEAEs), Best Corrected Visual 
Acuity (BCVA), central corneal thickness, vital signs, clinical laboratory tests, 
and ophthalmic exams using slit-lamp biomicroscopy and dilated 
ophthalmoscopy. Summaries will be provided by treatment group, and for 
ocular assessments separately by study-eye and non-study-eye. 


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Table of Contents 
Title Page ....................................................................................................................................... 1 
Protocol Synopsis .......................................................................................................................... 2 
Table of Contents .......................................................................................................................... 6 
List of Tables ............................................................................................................................... 11 
List of Figures .............................................................................................................................. 11 
1 
Introduction ..................................................................................................................... 12 
1.1 
Background ........................................................................................................... 12 
1.2 
Study Rationale ..................................................................................................... 13 
1.3 
Risk/Benefit Assessment ...................................................................................... 13 
1.3.1 
Known Potential Risks ........................................................................... 13 
1.3.2 
Known Potential Benefits ...................................................................... 13 
1.3.3 
Assessment of Benefits and Risks ......................................................... 13 
2 
Study Objectives and Endpoints.................................................................................... 14 
2.1 
Study Objectives ................................................................................................... 14 
2.2 
Study Endpoints .................................................................................................... 14 
2.2.1 
Primary Endpoint(s) ............................................................................... 14 
2.2.2 
Secondary Endpoint(s) ........................................................................... 14 
2.2.3 
Safety Assessments ................................................................................ 14 
3 
Study Design .................................................................................................................... 16 
3.1 
Overall Design of the Study .................................................................................. 16 
3.2 
Rationale for the Study Design ............................................................................. 17 
3.3 
Dose Justification .................................................................................................. 17 
3.4 
End of Study Definition ........................................................................................ 17 
4 
Study Population ............................................................................................................. 18 
4.1 
Inclusion Criteria .................................................................................................. 18 
4.2 
Exclusion Criteria ................................................................................................. 18 
4.3 
Lifestyle Considerations ....................................................................................... 20 


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4.4 
Screen Failures ...................................................................................................... 21 
4.5 
Strategies for Recruitment and Retention ............................................................. 21 
5 
Study Treatment(s) or Intervention(s) .......................................................................... 22 
5.1 
Investigational Product(s) ..................................................................................... 22 
5.1.1 
Description ............................................................................................. 22 
5.1.2 
Dosage and Administration.................................................................... 22 
5.2 
Preparation/Storage/Handling/Accountability ...................................................... 23 
5.2.1 
Acquisition and Accountability ............................................................. 23 
5.2.2 
Product Packaging and Labeling ........................................................... 23 
5.2.3 
Product Storage and Stability................................................................. 24 
5.3 
Measures to Minimize Bias: Randomization and Blinding .................................. 24 
5.4 
Treatment Adherence ............................................................................................ 24 
5.5 
Concomitant Therapy............................................................................................ 24 
5.5.1 
Management of Elevated IOP During Treatment .................................. 26 
6 
Study Discontinuation/Subject Withdrawal ................................................................. 27 
6.1 
Discontinuation of Study Treatment or Intervention ............................................ 27 
6.2 
Subjects Discontinuation/Withdrawal from the Study ......................................... 27 
6.3 
Lost to Follow up .................................................................................................. 28 
7 
Study Procedures ............................................................................................................ 29 
7.1 
Visit Descriptions.................................................................................................. 29 
7.1.1 
Screening (Visit 1, Day –35 to -1) ......................................................... 29 
7.1.2 
Randomization (Visit 2, Day 0) ............................................................. 30 
7.1.3 
Visit 3 (Day 1) ....................................................................................... 31 
7.1.4 
Visit 4 (Day 7 + 2 days) ......................................................................... 31 
7.1.5 
Visit 5 (Day 14 + 2 days) ....................................................................... 32 
7.1.6 
End of Study (Visit 6, Day 21 + 2 days) ................................................ 33 
7.1.7 
Unscheduled Visits ................................................................................ 34 
7.1.8 
Early Termination .................................................................................. 34 


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8 
Study Assessments .......................................................................................................... 35 
8.1 
Efficacy Evaluations ............................................................................................. 35 
8.1.1 
IOP ......................................................................................................... 35 
8.2 
Safety Evaluations ................................................................................................ 35 
8.2.1 
Adverse Events (AEs) and Serious Adverse Events (SAEs) ................. 35 
8.2.1.1 Definitions.............................................................................................. 36 
8.2.1.2 Classification of Adverse Events ........................................................... 36 
8.2.1.3 Adverse Event Reporting Requirements ................................................ 37 
8.2.1.4 Pregnancy ............................................................................................... 38 
9 
Statistical Considerations ............................................................................................... 39 
9.1 
Statistical Hypothesis ............................................................................................ 39 
9.2 
Sample Size Determination ................................................................................... 39 
9.3 
Analysis Populations ............................................................................................. 39 
9.4 
Statistical Analyses ............................................................................................... 40 
9.4.1 
Baseline Descriptive Analyses ............................................................... 40 
9.4.2 
Efficacy Analyses .................................................................................. 40 
9.4.2.1 Primary Efficacy Analyses .................................................................... 40 
9.4.2.2 Secondary Efficacy Analyses ................................................................ 41 
9.4.3 
Safety Analyses ...................................................................................... 41 
9.4.3.1 Adverse Events ...................................................................................... 42 
9.4.3.2 Clinical Laboratory Tests ....................................................................... 42 
9.4.3.3 Other Safety Evaluations ....................................................................... 42 
9.5 
Interim Analysis .................................................................................................... 42 
9.6 
Subgroup Analyses ............................................................................................... 42 
9.7 
Exploratory Analyses ............................................................................................ 42 
9.8 
Missing or Unused Data........................................................................................ 42 
9.9 
Tabulation of Individual Subject Data .................................................................. 42 
10 
Supporting Documentation and Operational Considerations .................................... 43 
10.1 
Regulatory Issues, Ethical Concerns, and Study Oversight .................................. 43 
10.1.1 
Informed Consent Process ..................................................................... 43 


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10.1.1.1 Consent/Assent Documents ................................................................... 43 
10.1.1.2 Consent Procedures and Documentation ............................................... 43 
10.1.2 
Study Discontinuation and Closure ....................................................... 43 
10.1.3 
Confidentiality and Privacy ................................................................... 44 
10.1.4 
Key Roles and Study Governance ......................................................... 45 
10.1.5 
Clinical Monitoring ................................................................................ 45 
10.1.6 
Quality Assurance and Quality Control ................................................. 45 
10.1.7 
Data Handling and Record Keeping ...................................................... 45 
10.1.7.1 Data Collection and Management Responsibilities ............................... 45 
10.1.7.2 Study Records Retention ........................................................................ 46 
10.1.8 
Protocol Deviations ................................................................................ 46 
10.1.9 
Publication and Data Sharing Policy ..................................................... 46 
11 
References ........................................................................................................................ 47 
12 
Appendices ....................................................................................................................... 49 
12.1 
Appendix 1: Schedule of Procedures and Assessments ........................................ 49 
12.2 
Appendix 2: Abbreviations and Definition of Terms ........................................... 51 
12.3 
Appendix 3: Visual Acuity ................................................................................... 54 
12.3.1 
Manifest Refraction ............................................................................... 54 
12.3.2 
Best Corrected Visual Acuity ................................................................ 54 
12.4 
Appendix 4: Biomicroscopy ................................................................................. 55 
12.5 
Appendix 5: Intraocular Pressure .......................................................................... 56 
12.6 
Appendix 6: Dilated Ophthalmoscopy .................................................................. 56 
12.7 
Appendix 7: Pachymetry ....................................................................................... 57 
12.8 
Appendix 8: Gonioscopy ...................................................................................... 57 
12.9 
Appendix 9: Visual Field ...................................................................................... 57 
12.10 Appendix 10: Optical Coherence Tomography (OCT) ........................................ 58 
12.11 Appendix 11: Blood Chemistry, Hematology ...................................................... 58 
12.12 Appendix 12: Heart Rate & Blood Pressure ......................................................... 59 
12.12.1 Heart Rate .............................................................................................. 59 
12.12.2 Blood Pressure ....................................................................................... 59 


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12.13 Appendix 13: Pregnancy Test ............................................................................... 59 
12.14 Appendix 14: Investigator Agreement .................................................................. 60 
12.15 Appendix 15: Compliance Statement ................................................................... 61 
12.16 Appendix 16: Protocol Amendments .................................................................... 62 
Last page ........................................................................................................................... 63 
 


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List of Tables 
Table 1: VVN539 Ophthalmic Solution General Information ..................................................... 22 
Table 2: Medications and Procedures Not Permitted ................................................................... 25 
 
 
List of Figures 
Figure 1: Study Treatment ............................................................................................................ 17 
 
 


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1 Introduction 
1.1 
Background 
A relatively new class of topical agents being evaluated for the treatment of glaucoma are Rho 
kinase (ROCK) inhibitors (Kopczynski 2014). The most commonly prescribed ocular hypotensive 
medications reduce intraocular pressure (IOP) by increasing uveoscleral aqueous outflow 
(prostaglandin analogues) or decreasing aqueous humor production (ß-adrenoceptor antagonists, 
α-adrenoceptor agonists, and carbonic anhydrase inhibitors). ROCK inhibitors lower IOP through 
a different mechanism of action, increasing aqueous outflow through the trabecular outflow 
pathway by decreasing actomyosin-driven cellular contraction and reducing production of fibrotic 
extracellular matrix proteins (Tian 2005; Tokushige 2007; Honjo 2001; Wang 2015; Rao 2017).  
In this class of ROCK inhibitors are netarsudil (approved in the U.S. as Rhopressa® (NDA 
2085254) in 2017, and as a component of the fixed dose product, Rocklatan® (NDA 201259) in 
2019 (Serle 2018), and ripasudil (approved in Japan in 2014 as Glanatec®, Tanihara 2015). 
VivaVision is developing VVN539, a ROCK inhibitor with nanomolar potency. Upon contact 
with tissue, it releases nitric oxide (NO) from the nitrate (ONO2) functional group, and is 
metabolized to VIP-5156, a ROCK inhibitor with subnanomolar potency. The release of NO 
from VVN539 is a characteristic like latanoprostene bunod (approved in the U.S. as Vyzulta® in 
2017, Medeiros 2016). It has been demonstrated “NO” alone can lower the IOP by 10-20% (2-
4 mmHg) by increasing outflow facility of aqueous humor (Cavet 2018).  
Nitric oxide released by organic nitrates such as VVN539 stimulates soluble guanylate cyclase 
(GC), leading to an increase of cGMP in TM cells (Torfard 1994). This leads to the relaxation of 
trabecular meshwork, a smooth muscle like tissue. In addition, NO can also alter calcium-
dependent potassium channel conductance, which leads to channel membrane activation and 
hyperpolarization with lower calcium ions resulting in vascular smooth muscle relaxation 
(Garcia-Calvo 1994). 
The IOP lowering mechanisms of action by NO is different from the IOP lowering MOA by 
ROCK kinase inhibition. The sponsor believes that there may be synergy or additivity of NO 
releasing capacity and ROCK inhibition. Thus, the Sponsor suggests this molecule may provide 
a favorable benefit/risk profile in the lowering of elevated IOP in patients with open angle 
glaucoma (OAG) and ocular hypertension (OHT). 
VVN539 ophthalmic solution is a preserved multi-dose product, intended for topical ocular 
instillation. As a new chemical entity (NCE), the intended regulatory route is 505(b)(1).  
 


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1.2 
Study Rationale 
Please refer to the Investigator’s Brochure (IB) for a summary of findings from the VVN539 non-
clinical studies.  
This first-in-human clinical trial with VVN539 is a phase 2 study intended to assess the efficacy 
as well as the ocular and systemic safety of two doses of VVN539 and two dosing regimens as 
compared to vehicle control in subjects with OHT or primary open angle glaucoma (POAG).  
1.3 
Risk/Benefit Assessment 
1.3.1 
Known Potential Risks 
The risks associated with VVN539 are not well understood at this time as this is a first-in-human 
clinical trial. Clinical trials of other ROCK inhibitors found the most common side effect to be 
transient, mild ocular hyperemia (Kopczynski, 2014). Other frequent adverse events reported 
include conjunctival hemorrhage (Serle et al 2018, Kahook et al 2019). 
1.3.2 
Known Potential Benefits 
There are no known direct benefits as this is the first-in-human study for VVN539. Currently 
approved treatments for OHT and POAG are often not well tolerated and / or are not sufficient for 
disease progression. As VVN539 has a different mechanism of action, it may provide an 
alternative for subjects replacing other therapeutics or acting synergistically. Subjects in this study 
contribute to the development of this new drug that may help others with OHT and POAG. 
1.3.3 
Assessment of Benefits and Risks 
Topical ocular VVN539 has demonstrated an excellent safety and tolerability profile during pre-
clinical testing. Based on clinical experience with other ROCK inhibitors and based on preclinical 
data, it is expected that transient conjunctival hyperemia will be observed. No other potential risks 
have been identified in association with VVN539 during its development and clinical trials with 
netarsudil have shown a similar pattern of systemic adverse events (AEs) as timolol, minimal 
treatment related serious AEs overall, and manageable ocular AEs (Singh, 2020). Even though 
blood levels of VVN539 after ocular instillation in animals have been minimal in the nonclinical 
studies to date, the Sponsor intends to conduct clinical laboratory and hematology testing at Visit 
1 (screening) and Visit 6 (end of treatment). 
Given the experience with this product in pre-clinical studies as well as with similar approved 
products used in a dry eye population, there is a favorable benefit-risk ratio. Further the risks of 
this trial will be mitigated via routine monitoring and reporting of potential new adverse events. 


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2 
Study Objectives and Endpoints 
2.1 
Study Objectives 
The primary objective of this study is to evaluate the ocular hypotensive efficacy of 2 
concentrations of VVN539 in subjects with OHT or POAG. 
The secondary objective is to evaluate the ocular and systemic safety of the 2 concentrations in 
this subject population.  
2.2 
Study Endpoints 
2.2.1 
Primary Endpoint(s) 
The primary endpoint for this study is to compare mean IOP at each diurnal time point (8AM, 
10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14) and Visit 6 (Day 21) for two doses of 
VVN539 compared to vehicle. The primary statistical analysis will be performed using the data 
from the study eye. 
The study eye is defined as meeting all inclusion criteria and no exclusion criteria, and with the 
higher IOP at 8:00AM at Visit 2. Should both eyes have the same IOP and meet all inclusion 
criteria and no exclusion criteria, then the right eye will be the study eye. 
2.2.2 
Secondary Endpoint(s) 
Secondary efficacy endpoints include: 
• Mean change from baseline (Visit 2) in IOP at each diurnal time point to Visit 4 (Day 7), 
Visit 5 (Day 14) and Visit 6 (Day 21); 
• Mean change from baseline (Visit 2) in diurnal IOP to Visit 4 (Day 7), Visit 5 (Day 14) 
and Visit 6 (Day 21). Diurnal IOP is the average of 8AM, 10AM, and 4PM measurements 
at a given visit. 
2.2.3 
Safety Assessments 
The safety assessments include: 
• Frequency of treatment emergent ocular and systemic adverse events and serious adverse 
events (SAEs). 
• Clinically relevant changes in: 
o Best Corrected Visual Acuity (by Early Treatment Diabetic Retinopathy Study 
[ETDRS]) 
o Slit-lamp Biomicroscopy  


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o Central corneal thickness  
o Ophthalmoscopy (dilated) 
o Vital signs (heart rate (HR) and blood pressure (BP)) 
o Clinical laboratory tests (blood chemistry, hematology) 
Data from both eyes will be used for the safety analysis. 
 


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3 
Study Design 
3.1 
Overall Design of the Study 
This is a prospective, parallel-comparison, multi-center, double-masked, randomized, vehicle-
controlled dose-response study assessing the safety and ocular hypotensive efficacy of VVN539 
in subjects with POAG or OHT. Approximately 60 subjects will be randomized into one of two 
active treatment arms or a control arm (20 subjects per cohort): 
• Arm 1: 0.02% VVN539 ophthalmic solution 
• Arm 2: 0.04% VVN539 ophthalmic solution 
• Arm 3: VVN539 ophthalmic solution vehicle 
Treatment assignments will be masked to VivaVision, study subjects, Investigators, and site staff.  
The study involves 6 clinic visits, including screening, baseline, and treatment visits. The study is 
open to subjects with OHT and POAG who are either treatment-naïve or are already on ocular 
hypotensive medication. Subjects on ocular hypotensive who meet screening inclusion and do not 
meet screening exclusion criteria at Visit 1 will discontinue their prior ocular hypotensive 
medication for up to 35 days before returning to the site for Visit 2 (baseline). Treatment naïve 
subjects can return for the baseline visit as early as the day after Visit 1. Subjects meeting all 
baseline inclusion criteria and none of the baseline exclusion criteria will be randomized into one 
of the study arms and will return to the study site the next day for the first treatment. Three different 
dosing regimens (once a day [q.d.] in the morning, q.d. in the evening and twice a day [b.i.d.]) will 
be tested for 7-9 days, each. 


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Figure 1: Study Treatment 
3.2 
Rationale for the Study Design 
The design of this study, including choice of control, is similar to that of the first-in-human study 
of netarsudil, a ROCK inhibitor currently approved in the US. 
3.3 
Dose Justification 
The concentrations of VVN539 ophthalmic solution and dosing schedule were chosen based on 
data from non-clinical ocular hypotensive effect, pharmacokinetic (PK) and toxicology studies. 
For details, please refer to the Investigator’s Brochure. 
Dosing will be q.d. in the morning for 7 days, followed by q.d. dosing in the evening for 7 days 
and b.i.d. dosing for 7 days. The route of administration for this study is ocular topical as this is 
the intended route for VVN539. 
3.4 
End of Study Definition 
A subject is considered to have completed the study if he or she has completed all phases of the 
study including the last visit or the last scheduled procedure shown in the Schedule of Procedures 
and Assessments. The end of the study is defined as completion of the last visit or procedure shown 
in the schedule in the study 


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4 
Study Population 
The study population will consist of adult subjects who have/are diagnosed with OHT or POAG. 
Subjects can be treatment naïve subjects or on ocular hypotensive medication. Ocular eligibility 
criteria in general apply only to the study eye, unless specified otherwise.  
Treatment during the study will be both eyes (OU), only data from the study eye will be used for 
efficacy analysis. Data from both eyes will be used for safety analysis. The study eye will be 
defined as meeting all inclusion criteria and no exclusion criteria, and with the higher IOP at 
8:00AM at Visit 2. Should both eyes have the same IOP and meet all inclusion criteria and no 
exclusion criteria, then the right eye will be the study eye. 
4.1 
Inclusion Criteria 
Subjects must meet all of the following in order to be enrolled into the study: 
1. 18 years of age or older. 
2. Diagnosis of POAG or OHT in both eyes that are untreated, or if treated, well controlled 
with a stable regimen of 2 or fewer ocular hypotensive medications 30 days prior to Visit 
1.  
• Combination medications count as 2 medications 
3. Unmedicated IOP of ≥ 22 mm Hg and ≤ 36 mm Hg in the study eye, with no more than 5 
mm Hg inter-eye difference at 08:00AM and 10:00AM at Visit 2. 
4. Corrected visual acuity in each eye +1.0 logMAR (Snellen equivalent to 20/200) or better 
by ETDRS in each eye. 
5. Central corneal thickness of ≥ 400 and ≤ 620 µm in each eye. 
6. Able and willing to give signed informed consent and follow study instructions. 
7. In the opinion of the Investigator, the subject is able to safely discontinue current ocular 
hypotensive medication(s), when a washout is required. 
4.2 
Exclusion Criteria 
Subjects with any of the following will not be allowed to participate in this study: 
Ophthalmic:  
1. Known hypersensitivity to any kinase inhibitors, any excipient or preservative of the 
formulation or to topical anesthetics or fluorescein. 
2. Unmedicated IOP of > 36 mm Hg in either eye at any time point at Visit 2. 
3. History of intraocular implant for IOP treatment, glaucoma filtering surgery, placement, or 
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4. History of laser IOP lowering surgery within 6 months of Visit 1 in the study eye.   
• Note: Laser peripheral iridotomy for narrow angle is allowed if performed 3 months or 
more prior to Visit 1. 
5. Ocular trauma within the past six months, or surgery involving the ocular surface 
(including cataract surgery) or non-IOP-lowering laser treatment within the past three 
months of Visit 1 in the study eye. 
• Refractive surgery in the study eye (including but not limited to radial keratotomy, 
Photorefractive Keratectomy [PRK], Laser-Assisted in situ Keratomileusis [LASIK], 
etc.). 
6. Refractive surgery planned during study participation in either eye. 
7. Have 
received 
intravitreal 
corticosteroid 
injection 
within 
6 
months 
or 
subtenon/subconjunctival steroid injection within 3 months of Visit 1 in either eye. 
8. History or evidence of ocular infection, inflammation, clinically significant blepharitis, 
conjunctivitis, or of herpes simplex keratitis within 2 months of Visit 1 in either eye. 
9. Unable or unwilling to avoid wearing of contact lenses from Visit 1 through the study.   
10. Currently using 3 or more ocular hypotensive medications prior to screening in either eye 
(combination medications count as 2 medications). 
11. Ocular medication of any kind within 28 days of Visit 2 in either eye other than: 
• Ocular hypotensive medications which must be washed out according to the provided 
schedule 
• Ocular Medication as part of an eye exam 
• Lubricating drops for dry eye 
12. Clinically significant ocular disease in either eye (e.g. corneal edema, uveitis, severe 
keratoconjunctivitis sicca) which might interfere with the study. 
13. Pseudoexfoliative, pigmentary, congenital, developmental or secondary glaucoma (e.g. 
neovascular, uveitic, pigmentary, lens-induced, steroid-induced, trauma induced or 
glaucoma associated with increased episcleral venous pressure) in either eye. 
14. Closed angle forms of glaucoma in either eye 
• Shaffer grade < 3 (in 1 or more quadrants) 
• Gonioscopy within 3 months prior to Visit 1 is acceptable. 
15. Cup-disc ratio of 0.7 or greater in either eye. 
16. Nerve fiber layer thickness assessment by Optical Coherence Tomography (OCT) 
documents thinning of the NFL into the abnormal range for either eye.  
• If, in the opinion of the investigator, the thinning of the NFL may be attributed to other 
ocular features (including but not limited to optic nerve drusen, myopia, chorioretinal 
scars), then the subject is allowed. 
17. Subjects with visual fields with substantial glaucomatous loss (e.g., Bjerrum scotoma, 
blind spot enlargement) for whom washout and potential randomization would be unsafe. 


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18. In addition to the above criteria, glaucomatous damage in either eye so severe that washout 
of ocular hypotensive medications for up to 5 weeks and treatment for 3 weeks (for subjects 
randomized to vehicle control) is not judged safe in the opinion of the Investigator.  
• Note: Subjects may be changed to a another IOP lowering therapy with a shorter 
washout period as long as the appropriate washout is met prior to Visit 2. 
19. Any abnormality preventing reliable applanation tonometry of either eye. 
General/Systemic: 
20. Laboratory tests indicative of any clinically significant disease in the opinion of the 
Investigator. 
21. Clinically significant systemic disease (e.g., uncontrolled diabetes, myasthenia gravis, 
hepatic, renal, cardiovascular or endocrine disorders) which might interfere with the study 
in the opinion of the Investigator. 
22. Participation in any study involving an investigational product (IP) within the past 30 days 
of Visit 1. 
23. Changes of systemic beta blockers during the study that in the opinion of the Investigator 
could have a substantial effect on IOP within 30 days prior to Visit 1 or anticipated during 
the study. 
24. Have received systemic corticosteroids (oral, injectable, inhaled) within 30 days of Visit 1 
or planned use during the study.   
• Note: Topical dermatologic steroids are allowed if used for less than 3 consecutive days 
on no more than 3 separate areas of the body within 30 days of Visit 1 and the dosage 
is not expected to increase during the study.   
• Intranasal steroids are allowed if used less than or 4 times per week within 30 days of 
Visit 1 and the dosage is not expected to increase during the study. 
25. History of substance abuse within 1 year of Visit 1 
26. Subjects that are employees or relatives of employees at the clinical site. 
27. Women of childbearing potential who are pregnant, nursing, planning a pregnancy, or not 
using a medically acceptable form of birth control.  
• Females of childbearing potential with a positive urine pregnancy test (UPT) result at 
Visit 1 or Visit 2 are excluded. 
• An adult woman is considered to be of childbearing potential unless she is one year 
post-menopausal or three months post-surgical sterilization.   
4.3 
Lifestyle Considerations 
Substance abuse is not permitted during the study and subjects with a history of substance abuse 
1 year prior to Visit 1 will be excluded from the study. 


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Subjects are required to refrain from wearing contact lenses for the duration of the study. Subjects 
not willing or able to refrain from wearing contact lenses will be excluded from the study.  
Subjects should not have any planned ocular surgeries while participating in the study (Visit 1 – 
Visit 6). Investigators should discuss with the Medical Monitor should an ocular surgery become 
necessary while a subject is participating in the study. 
4.4 
Screen Failures 
Screen failures are defined as subjects who consent to participate in the clinical study but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set 
of screen failure information is required to ensure transparent reporting of screen failure subjects, 
to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and 
to respond to queries from regulatory authorities. Minimal information includes demography, 
screen failure details, eligibility criteria, and any serious adverse event. 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of 
the following may be rescreened.: 
• Prohibited medications from which the subject may discontinue for the purposes of 
eligibility and / or participating in the study (with approval from the Investigator based on 
clinical safety). 
• Exclusionary medical history status at the time of screening that will change (e.g., ocular 
infection, surgical history duration). 
Rescreened subjects should be assigned a new subject number.   
4.5 
Strategies for Recruitment and Retention 
It is anticipated that approximately 90 subjects will need to be screened to achieve 60 qualified 
completed subjects (20 per arm) for the study. Subjects will be mostly recruited from the patient 
population at participating study centers in the US. Subject retention is not considered a major 
issue for this study due to the relatively short duration.  


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5 
Study Treatment(s) or Intervention(s) 
5.1 
Investigational Product(s) 
Three (3) investigational products (IPs) will be administered during this study: 
• VVN539 ophthalmic solution 0.02% (3mL in 5 mL bottles) 
• VVN539 ophthalmic solution 0.04% (3mL in 5 mL bottles) 
• VVN539 ophthalmic solution vehicle (3mL in 5 mL bottles) 
5.1.1 
Description 
VVN539 ophthalmic solution will be supplied to study sites in 5mL low-density polyethylene 
LPDE multidose droppers filled with at least 3 mL solution. Table 1 describes the composition 
and general information about VVN539 supplied for this study. The VVN539 vehicle control has 
the same composition as VVN539 ophthalmic solution except that it does not contain VVN539. 
Additional detail can be found in the Investigator’s Brochure. 
Table 1: VVN539 Ophthalmic Solution General Information 
Dosage Form 
Sterile solution 
Active Ingredient 
0.02% or 0.04% VVN539 
Inactive Ingredients 
Benzalkonium chloride, Polyvinylpyrrolidone (PVP) K90, Poloxamer 407, 
glycerin, boric acid, sodium borate, water 
Route of Administration 
Topical ocular administration 
Physical Description 
Isosmotic, clear solution 
pH 
Approximately 5.0 
Pharmacokinetics 
VVN539 metabolite and nitric oxide active moieties dosed q.d. or b.i.d 
Stability 
12 months expiry for samples stored at 5°C temperature 
 
5.1.2 
Dosage and Administration 
Eligible subjects will be randomized in a 1:1:1 ratio to receive either 0.02% or 0.04% VVN539 or 
vehicle control. The IP will be administered as 1 drop either q.d. or b.i.d in the inferior conjunctival 
fornix in each eye.  
The first dose of VVN539 will be administered in clinic after the 8:00AM IOP has been measured 
at Visit 3. The subjects will then receive the bottle of IP and will self-administer the IP in the 
morning (suggested timing between 7:00AM and 9:00AM) each day for 5 days. The study site 
should call the subject the day prior to Visit 4 to remind them to NOT dose in the morning at home 
on the day of the visit. The last day of q.d. morning dosing will be Visit 4 when subjects will be 
dosing in clinic after the 8:00AM IOP has been measured.  


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Starting the day after Visit 4, subjects will self-administer the IP in the evening (suggested timing 
between 7:00PM and 9:00PM) each day for 6 days. The last q.d. evening dose will be administered 
in the evening Visit 5 after the last IOP has been measured.  
Starting the day after Visit 5, subjects will self-administer the IP b.i.d (suggested timing between 
7:00AM and 9:00AM for the morning dose and between 7:00PM and 9:00PM for the evening 
dose) each day for 6 days. The study site should call the subject the day prior to Visit 6 to remind 
them to NOT dose in the morning at home on the day of the visit. The last morning dose will be 
administered in clinic after the 8:00AM IOP dose has been measured. Subjects will return the IP 
at Visit 6 and will resume their standard of care treatment. 
Subjects should be instructed to administer missed doses as soon as possible, as long as it is not 
administered within 12 hours of the next scheduled dosing during the q.d. dosing periods or not 
within 4 hours of the next scheduled dosing during the b.i.d dosing period. Missed doses will be 
recorded as deviations in the electronic case report form (eCRF). 
5.2 
Preparation/Storage/Handling/Accountability 
5.2.1 
Acquisition and Accountability 
VVN539 ophthalmic solution and vehicle will be provided to study sites in identical bottles so that 
masking is maintained. Shipment records will be verified by comparing the shipment inventory 
sheet to the actual quantity received at the site. Accurate records of receipt and disposition of the 
IP (e.g. dates, quantity, subject number, date dispensed, date returned) must be maintained by the 
study site. At the end of the study, all used and unused IP will be returned to VivaVision (or 
designee), following approval by VivaVision. The study monitor (or designee) will verify IP 
accountability prior to return of the IP. All accountability procedures must be completed before 
the study is complete or before the study site is closed, whichever comes first.  
5.2.2 
Product Packaging and Labeling 
Each randomization kit contains one dropper bottle of IP sufficient for 14-18 days of q.d. and 7-9 
days of b.i.d. dosing. Double masked labels will minimally contain the following information: 
• Protocol number 
• Sponsor’s name  
• Kit number 
• Instructions for use 
• Storage temperature 
• Required statement(s) per regulatory agency 


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5.2.3 
Product Storage and Stability 
IP and active comparator should be stored at controlled refrigerated temperatures from 2°C to 8°C 
and protected from light. Once dispensed to the subject, IP bottles may be stored by the subject in 
the refrigerator (approximately 2-8°C/35-46°F) or at ambient room temperature until use. 
5.3 
Measures to Minimize Bias: Randomization and Blinding 
Subjects will be randomized to a treatment assignment. A randomization schedule will be 
generated by qualified personnel independent of the project team and study conduct and 
maintained in a secure and limited-access location separate from the Investigators and members 
of the project team. Treatment will not be blocked by study site which essentially stratifies the 
treatment by study site. Treatment assignments will be masked to the Sponsor, subjects, 
Investigators, and investigative staff, until completion of the study and the final database is locked. 
Appropriate precautions will be taken to prevent unauthorized access to the randomization scheme. 
Both doses of VVN ophthalmic solution as well as vehicle will be provided in identical dropper 
bottles to prevent inadvertent unmasking. If unmasking is required, the integrity of the study 
assessments and objectives will be maintained by limiting access to the unmasked data. Unless the 
subject’s safety requires otherwise and if time permits, the decision to unmask an individual 
subject’s treatment assignment is to be made jointly by the Investigator and medical monitor after 
consultation with the Sponsor, thus leaving the masking of the remaining subjects intact. 
5.4 
Treatment Adherence 
Adherence to the treatment schedule will be assessed by site staff by querying subjects at Visits 4-
6 whether they missed any doses in the dosing period preceding the visit and if so, whether 2 or 
more doses were missed. Subjects should be counseled on the importance of adhering to the dosing 
scheduled. Missed doses will be recorded as deviations in the eCRF. 
5.5 
Concomitant Therapy 
All medications that the subject has taken 2 months prior to Visit 1 and / or is taking through Visit 
6 (or discontinuation from the study) will be recorded in the subject chart and the eCRF. The 
generic name of the drug, dose, route of administration, duration of treatment (including start and 
stop dates), frequency, indication and whether or not the medication was taken due to an AE will 
be recorded for each medication. 
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the case report form are 
concomitant prescription medications, over-the-counter medications and supplements.  


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The medications and therapies listed in Table 2 are exclusionary for the study and / or are 
prohibited during study participation. Investigators should discuss with the Medical Monitor 
should any of these medications or procedures become medically necessary for a subject during 
study participation. 
Table 2: Medications and Procedures Not Permitted 
Medication / Procedure 
Eye 
Limitation 
Intraocular implant for IOP treatment, glaucoma filtering 
surgery, placement, or removal of minimally invasive 
glaucoma implant (MIGS) 
Study Eye Exclusionary any time prior to Visit 1 
Laser IOP lowering surgery in the study eye 
Study Eye Exclusionary if performed within 6 months 
of Visit 1 
Laser peripheral iridotomy for narrow angle in study eye 
Study Eye Exclusionary if performed within 3 months 
of Visit 1 
Ocular trauma in the study eye 
Study Eye Exclusionary if occurred within 6 months of 
Visit 1 
Surgery involving the ocular surface (including cataract 
surgery) or non-IOP-lowering laser in the study eye 
Study Eye Exclusionary if performed within 3 months 
of Visit 1 
Refractive surgery (including but not limited to radial 
keratotomy, PRK, LASIK) 
Study Eye Exclusionary any time prior to Visit 1 and 
prohibited during study participation 
Intravitreal corticosteroid injection 
Either Eye Exclusionary if performed within 6 months 
of Visit 1 
Subtenon/subconjunctival steroid injection 
Either Eye Exclusionary if performed within 3 months 
of Visit 1 
Ocular infection, inflammation, clinically significant 
blepharitis, conjunctivitis, or of herpes simplex keratitis 
Either Eye Exclusionary if occurred within 2 months of 
Visit 1 
Contact lens use 
Either Eye Prohibited during study participation 
Ocular medication of any kind other than: 
• Ocular hypotensive medications which must be 
washed out according to the provided schedule 
• Ocular Medication as part of an eye exam 
• Lubricating drops for dry eye 
Either Eye Exclusionary within 28 days of Visit 1 and 
prohibited during study participation 
Ocular hypotensive medication 
Either Eye 
Subjects on ocular hypotensive medication 
must wash out prior to Visit 2 
See section 5.5.1 for use of ocular 
hypotensive medication other than IP during 
study participation   
Changes of systemic beta blockers that in the opinion of 
the investigator could have a substantial effect on IOP 
N/A 
Exclusionary within 30 days of Visit 1 and 
prohibited during study participation 
Systemic corticosteroids (oral, injectable, inhaled) within 
30 days of Visit 1 or planned use during the study. 
• Topical dermatologic steroids are allowed if used 
for less than 3 consecutive days on no more than 3 
separate areas of the body and the dosage is not 
expected to increase during the study. 
• Intranasal steroids are allowed if used less than or 
4 times per week and the dosage is not expected to 
increase during the study. 
N/A 
Exclusionary within 30 days of Visit 1 and 
prohibited during study participation 


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Medication / Procedure 
Eye 
Limitation 
Substance abuse 
N/A 
Exclusionary within one year of Visit 1 and 
prohibited during study participation 
 
5.5.1 
Management of Elevated IOP During Treatment 
Any subject who has an elevated IOP that the PI considers unsafe for the subject may be rescued 
and placed on appropriate alternate therapy. The choice of rescue medication is at the Investigator's 
discretion. Rescue medication should be entered into the eCRF with annotation indicating that the 
medication was used for rescue.   
Rescued subjects will be considered as treatment failures, and the need for rescue therapy will not 
be considered an adverse event (AE). Rescued subjects will stop the investigational product and 
should not be withdrawn from the study. 
The following guidance is provided to Investigators for rescue and the decision to rescue is at the 
discretion of the investigator’s judgement: 
• Ocular hypotensive medication (other than IP) is prohibited during study participation 
unless the subject is rescued. 
• If the subject’s IOP is > 36 mm HG, the Investigator may recheck the IOP within 3 day of 
the visit and rescue medication may be considered if the IOP remains elevated 
• Rescued subjects should discontinue the investigational product. 
• Rescued subjects should continue in the study for safety follow-up after receiving the 
rescue medication. 
 


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6 
Study Discontinuation/Subject Withdrawal 
6.1 
Discontinuation of Study Treatment or Intervention 
IP may be discontinued by a subject at any time. In addition, the Investigator or Sponsor can 
discontinue a subject from further IP administration for other reasons related to the best interest of 
the subject.  
6.2 
Subjects Discontinuation/Withdrawal from the Study 
Subjects are free to withdraw from participation in the study at any time upon request. An 
Investigator may discontinue or withdraw a subject from the study for the following reasons:  
• IOP measurement of 36 mmHg or higher in either eye 
• Severe ocular irritation, burning, pain, or hyperemia 
• Study terminated by the Sponsor, Food and Drug Administration (FDA), or other 
regulatory authorities  
• Any woman who becomes pregnant while participating in the study. Information on the 
pregnancy and outcome will be requested. 
• Significant study treatment/intervention non-compliance  
• If any clinical adverse event, laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the subject 
• Disease progression which requires discontinuation of the study intervention 
• If the subject meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
The reason for subject discontinuation or withdrawal from the study will be recorded on the case 
report form. Subjects who sign the informed consent form and are randomized but do not receive 
the study intervention may be replaced. Those who sign the informed consent form, and are 
randomized and receive the study treatment, and subsequently withdraw, or are withdrawn or 
discontinued from the study, will not be replaced.   
Additionally, the trial or parts of the trial may be discontinued by the Sponsor or at the 
recommendation of the Investigator after consultation with VivaVision Biotech, Inc. This may be 
based on a significant number of AEs of a similar nature that warrant such action. 
In the event study discontinuation of a randomized subject is necessary, the Investigator should 
make every attempt to have the subject complete all Visit 6 assessments. If a non-serious AE is 
unresolved at the time of the subject's final study visit, an effort will be made to follow up until 


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the AE is resolved or stabilized, the subject is lost to follow-up, or there is some other resolution 
of the event. The Investigator should make every attempt to follow all SAEs to resolution. 
6.3 
Lost to Follow up 
A subject will be considered lost to follow-up if he or she fails to return for scheduled visits and is 
unable to be contacted by the study site staff.   
The following actions must be taken if a subject fails to return to the clinic for a required study 
visit: 
• The site will attempt to contact the subject and reschedule the missed visit within 1-2 days 
and counsel the subject on the importance of maintaining the assigned visit schedule and 
ascertain if the subject wishes to and/or should continue in the study. Subjects should 
continue the current dosing regimen until the rescheduled visit. 
• Before a subject is deemed lost to follow-up, the Investigator or designee will make every 
effort to regain contact with the subject (where possible, 3 telephone calls and, if necessary, 
a certified letter to the subject’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the subject’s medical record or study file. 
• Should the subject continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up.   
 


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7 
Study Procedures 
Written informed consent and Health Insurance Portability and Accountability Act (HIPAA) 
authorization will be obtained from all subjects prior to any study-related procedures being 
performed.  The Schedule of Procedures and Assessments (Appendix 1) lists the procedures that 
should occur at each study visit.  Details for the study assessments can be found in Appendices 3-
12. 
7.1 
Visit Descriptions 
7.1.1 
Screening (Visit 1, Day –35 to -1) 
The following procedures will be performed on all subjects at the screening visit. Ophthalmic 
procedures should be performed in the order suggested below: 
• Explain the purpose and conduct of the study to the subject, answer the subject’s questions, 
and obtain written informed consent and HIPAA authorization. 
• Obtain information including: demographics, concomitant medications, ocular and 
systemic medical and medication history and surgical history. 
• Determine study eligibility based on Inclusion/Exclusion criteria. 
• Best corrected visual acuity (BCVA) 
• Perimetry  
o Visual field within 3 months of Visit 1 is acceptable, provided that it is reliable 
meets the requirement for automated threshold visual field (e.g., 30-2 or 24-2 
Humphrey). Unreliable entry fields must be repeated prior to study drug 
dispensation. 
• OCT 
o OCT within 3 months of Visit 1 is acceptable. 
• Slit-Lamp Biomicroscopy 
• IOP measurement 
o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be taken. The mean will be taken if two measurements are used, for 3 measurements 
record the median. 
• Pachymetry 
• Gonioscopy 
o Gonioscopy performed within 3 months of Visit 1 is acceptable 
• Dilated ophthalmoscopy 


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o Determine cup-disc ratio 
• Blood pressure & heart rate 
• Blood chemistry, hematology 
• Urine pregnancy test (UPT) for women of childbearing potential 
o An adult woman is considered to be of childbearing potential unless she is one year 
post-menopausal or three months post-surgical sterilization. 
• Instruct subjects who were on ocular hypotensive medication prior to the visit to start 
washout based on the table below.  Subjects may be changed to a another IOP lowering 
therapy with a shorter washout period as long as the appropriate washout is met prior to 
Visit 2. 
Ocular Hypotensive Medication Class 
Minimum washout period 
Prostaglandins 
β-adrenoceptor antagonists 
Kinase inhibitors 
4 weeks 
Adrenergic agonists 
2 weeks 
Muscarinic agonists 
Carbonic anhydrase inhibitors 
5 days 
7.1.2 
Randomization (Visit 2, Day 0) 
The following procedures will be performed on Day 0 (randomization). Ophthalmic procedures 
should be performed in the order suggested below: 
• Determine study eligibility based on Inclusion/Exclusion criteria. 
• BCVA 
• Biomicroscopy 
• Diurnal IOP 
o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Readings at: 
 8:00AM (±30 mins) 
 10:00AM (±30 mins) 
 4:00PM (±30 mins) 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be obtained. The mean will be taken if two measurements are used, for 3 
measurements record the median. 
• Blood pressure & heart rate 
• Randomization 
• Urine pregnancy test (UPT) for women of childbearing potential 


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o An adult woman is considered to be of childbearing potential unless she is one year 
post-menopausal or three months post-surgical sterilization. 
• Assess subject ability to instill drops in eye using artificial tears. Subjects should be 
retrained on the correct way to instill drops if they are not able to do so. 
• Update medical and ocular history, as needed 
• Update concomitant medications, as needed 
7.1.3 
Visit 3 (Day 1) 
The following procedures will be performed at Visit 3 (Day 1). Ophthalmic procedures should be 
performed in the order suggested below:  
• BCVA 
• Biomicroscopy 
• IOP 
o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Reading at: 
 8:00AM (±30 mins) 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be obtained. The mean will be taken if two measurements are used, for 3 
measurements record the median. 
• Administer first dose of study drug in clinic after IOP is taken. 
• Dispense study drug 
• Instruct subject to dose daily in the morning (suggested timing between 7:00AM and 
9:00AM). 
• Blood pressure & heart rate 
• Update concomitant medications, as needed 
• Adverse event assessment 
7.1.4 
Visit 4 (Day 7 + 2 days) 
Visit 4 needs should be scheduled 5 days after Visit 3 in order to ensure at least 7 days of q.d. 
dosing in the morning.  
The following procedures will be performed at Visit 4 (Day 7). Ophthalmic procedures should be 
performed in the order suggested below:  
• BCVA 
• Biomicroscopy 
• Diurnal IOP 


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o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Readings at: 
 8:00AM (±30 mins) 
 10:00AM (±30 mins) 
 4:00PM (±30 mins) 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be obtained. The mean will be taken if two measurements are used, for 3 
measurements record the median. 
• Administer last q.d. morning dose in clinic after 8:00AM IOP is taken. 
• Collect used study drug and dispense a new bottle. 
• Instruct subject to start dosing daily the day after the visit in the evening (suggested timing 
between 7:00PM and 9:00PM). 
• Blood pressure & heart rate 
• Update concomitant medications, as needed 
• Adverse event assessment 
7.1.5 
Visit 5 (Day 14 + 2 days) 
Visit 5 needs should be scheduled 6days after Visit 4 in order to ensure at least 7 days of q.d. 
dosing in the PM. The following procedures will be performed at Visit 5 (Day 14). Ophthalmic 
procedures should be performed in the order suggested below: 
• BCVA 
• Biomicroscopy 
• Diurnal IOP 
o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Readings at: 
 8:00AM (±30 mins) 
 10:00AM (±30 mins) 
 4:00PM (±30 mins) 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be obtained. The mean will be taken if two measurements are used, for 3 
measurements record the median. 
• Collect used study drug and dispense a new bottle.  
• Instruct subject to instill the last daily evening dose in the evening after the visit (suggested 
timing between 7:00PM and 9:00PM). 


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• Instruct subject to start dosing twice daily the day after the visit in the morning (suggested 
timing between 7:00AM and 9:00AM for the morning dose and between 7:00PM and 
9:00PM for the evening dose). 
• Blood pressure & heart rate 
• Update concomitant medications, as needed 
• Adverse event assessment 
7.1.6 
End of Study (Visit 6, Day 21 + 2 days) 
Visit 6 needs should be scheduled 6 days after Visit 5 in order to ensure at least 6 days of b.i.d. 
dosing.  
The following procedures will be performed at the End of Study visit (Visit 6) on Day 21. 
Ophthalmic procedures should be performed in the order suggested below: 
• BCVA 
• Biomicroscopy 
• Diurnal IOP 
o Measured using Goldmann applanation tonometer and a two-person reading 
method. 
o Readings at: 
 8:00AM (±30 mins) 
 10:00AM (±30 mins) 
 4:00PM (±30 mins) 
o Measure twice in each eye; if results differ by >1 mm Hg, a third measurement will 
be obtained. The mean will be taken if two measurements are used, for 3 
measurements record the median. 
• Administer last dose of b.i.d. study drug in clinic after 8:00AM IOP is taken. 
• Pachymetry 
• Dilated ophthalmoscopy 
o After last IOP measurement is taken. 
• Blood pressure & heart rate 
• Blood chemistry, hematology 
• UPT for women of childbearing potential 
o An adult woman is considered to be of childbearing potential unless she is one year 
post-menopausal or three months post-surgical sterilization. 
• Collect used study drug. 
• Update concomitant medications, as needed 
• Adverse event assessment 


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o If a non-serious AE is unresolved at the time of the subject's final study visit, an 
effort will be made to follow up until the AE is resolved or stabilized, the subject 
is lost to follow-up, or there is some other resolution of the event.  
o The Investigator should make every attempt to follow all SAEs to resolution. 
7.1.7 
Unscheduled Visits 
Any visits or procedures performed beyond those specified within the protocol must be 
documented in the Unscheduled Visit pages of the eCRF. Subjects IOP should be checked at all 
unscheduled visits. Unscheduled visits may include but are not limited to reporting AEs, changes 
in concomitant medications, or ophthalmic assessments as deemed appropriate by an appropriately 
qualified physician. 
7.1.8 
Early Termination 
In the event that a subject exits or is terminated from the study prior to the End of Study visit (Visit 
6), every attempt will be made to ensure that the subject completes all Visit 6 assessments. If a 
non-serious AE is unresolved at the time of the subject's final study visit, an effort will be made to 
follow up until the AE is resolved or stabilized, the subject is lost to follow-up, or there is some 
other resolution of the event. The Investigator should make every attempt to follow all SAEs to 
resolution. 
 


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8 
Study Assessments 
The Schedule of Procedures and Assessments (Appendix 1) provides a list of study assessments 
and evaluations to be performed and the timing of each.   
8.1 
Efficacy Evaluations 
Efficacy assessments will be conducted at the time points indicated on the Schedule of Procedures 
and Assessments (Appendix 1) and as detailed in the sections below.   
8.1.1 
IOP 
IOP will be measured per the instructions in Appendix 5. 
8.2 
Safety Evaluations 
Safety assessments will be performed at the time points indicated on the Schedule of Procedures 
and Assessments (Appendix 1) and as detailed in the sections below.   
• AE monitoring 
• BCVA (Appendix 3) 
• Slit-Lamp Biomicroscopy (Appendix 4) 
• Central corneal thickness (Appendix 7) 
• Dilated ophthalmoscopy (Appendix 6) 
• Vital signs (heart rate and blood pressure) (Appendix 12) 
• Clinical laboratory tests (blood chemistry, hematology) (Appendix 11) 
8.2.1 
Adverse Events (AEs) and Serious Adverse Events (SAEs) 
AEs will be monitored throughout the study.  Subjects will be encouraged to report any adverse 
findings during the study whether or not they are related to IP. These can be collected either in an 
unsolicited fashion without any prompting or in response to a general question such as: “Have you 
noticed anything different since you started the study; began the IP, etc?”  
All AEs will be captured on the appropriate eCRF.  Information to be collected at minimum 
includes event description, onset, assessment of severity, relationship to IP, and outcome.  
The Investigator will record all AEs with start dates occurring any time after informed consent is 
obtained until 7 (for nonserious AEs) or 30 days (for SAEs) after the last day of study participation. 
At each study visit, the Investigator will inquire about the occurrence of AE/SAEs since the last 
visit. Events will be followed for outcome information until resolution or stabilization.  


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Any medical condition that is present at the time that the subject is screened will be considered as 
baseline and not reported as an AE. However, if the study subject’s condition deteriorates at any 
time during the study, it will be recorded as an AE.   
8.2.1.1 
Definitions 
An AE is any untoward medical occurrence in a patient or clinical study subject, temporally 
associated with the use of study intervention, whether or not considered related to the study 
intervention.  An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use 
of study intervention. 
An AE or suspected adverse reaction is considered “serious” if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes:  
• Death,  
• A life-threatening AE,  
• Inpatient hospitalization or prolongation of existing hospitalization,  
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or  
• A congenital anomaly or birth defect.  
Important medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may jeopardize 
the subject and may require medical or surgical intervention to prevent one of the outcomes listed 
in this definition.  
8.2.1.2 
Classification of Adverse Events 
Severity of Adverse Events 
The severity of all AEs will be assessed by the Investigator and graded as follows:  
• Mild: requires minimal or no treatment and do not interfere with the subject’s daily 
activities; 
• Moderate: results in a low level of inconvenience or concern and may cause some 
interference with functioning; 
• Severe: interrupts a subject’s usual daily activity and may require systemic drug therapy 
or other treatment. Severe events are usually potentially life-threatening or incapacitating. 
The term “severe” does not necessarily equate to “serious”.  
Relationship of Adverse Events 


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All AEs must have their relationship to study intervention assessed by the Investigator who 
examines and evaluates the subject based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical 
study, the IP must always be suspect. 
• Unrelated: no reasonable possibility that the administration of the IP caused the event, no 
temporal relationship between the IP and event onset, or an alternate etiology has been 
established. 
• Related: is known to occur with the IP, is a reasonable possibility that the IP caused the 
AE, or there is a temporal relationship between the IP and event. Reasonable possibility 
means that there is evidence to suggest a causal relationship between the IP and the AE. 
Expectedness 
An AE or suspected adverse reaction is considered “unexpected” if it is not listed in the 
Investigator’s Brochure (IB), package insert, or device labeling or is not listed at the specificity or 
severity that has been observed; or, if an IB is not required or available, is not consistent with the 
risk information described in the protocol, as amended. “Unexpected,” as used in this definition, 
also refers to AEs or suspected adverse reactions that are mentioned in the IB, package insert, or 
device labeling as occurring with a class of drugs (or other medical products) or as anticipated 
from the pharmacological properties or other characteristics of the IP, but are not specifically 
mentioned as occurring with the particular IP under investigation. 
The Investigator will be responsible for determining whether an AE is unexpected, i.e., if the 
nature, severity, or frequency of the event is not consistent with the risk information previously 
described for the IP.   
8.2.1.3 
Adverse Event Reporting Requirements 
According to federal regulations, an Investigator must immediately (within 24 hours) report to the 
Sponsor or a designee any SAE, whether or not considered drug related, including those listed in 
the protocol or IB and must include an assessment of whether there is a reasonable possibility that 
the drug caused the event.  
According to federal regulations, the Sponsor must notify the FDA and all participating 
Investigators as soon as possible, but in no case later than 15 calendar days after the Sponsor 
determines that a potential serious risk arising from a clinical study qualifies for reporting.  Sponsor 
must report any suspected adverse reaction that is both serious and unexpected. The Sponsor must 
report an AE as a suspected adverse reaction only if there is evidence to suggest a causal 
relationship between the drug and the adverse event (See 21 CFR 312.32(c)(1)).  


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Furthermore, the Sponsor must also notify FDA of any unexpected fatal or life-threatening 
suspected adverse reaction as soon as possible but in no case later than 7 calendar days after the 
Sponsor's initial receipt of the information (See 21 CFR 312.32(c)(2)). 
8.2.1.4 
Pregnancy 
All pregnancies are to be reported from the time informed consent is signed until 14 days after the 
last study visit.  
Any report of pregnancy for any female study subject must be reported within 24 hours to the 
Sponsor or its delegate using the Pregnancy Report Form. The pregnant female study subject must 
be withdrawn from the study. 
Every effort should be made to gather information regarding the pregnancy outcome and condition 
of the infant. It is the responsibility of the Investigator to obtain this information within 30 calendar 
days after the initial notification and approximately 30 calendar days after delivery. 
Pregnancy complications such as spontaneous abortion/miscarriage or congenital abnormality are 
considered SAEs and must be reported to the Sponsor or its delegate using the Serious Adverse 
Event Form. Note: An elective abortion is not considered an SAE. 
In addition to the above, if the Investigator determines that the pregnancy meets serious criteria, it 
must be reported as an SAE to the Sponsor or its delegate. 
 


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9 
Statistical Considerations 
Continuous measures will be summarized descriptively by the mean, standard deviation, median, 
minimum, and maximum values. Categorical measures will be summarized by the frequency and 
percentage of subjects. 
A separate Statistical Analysis Plan (SAP) will be prepared prior to unmasking of study data. 
Baseline for computing change from baseline for IOP at each time point will be time-matched. 
9.1 
Statistical Hypothesis 
The null hypothesis is that VVN539 has an equivalent effect compared to vehicle for mean IOP at 
each diurnal time point (8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14), and Visit 6 
(Day 21). The alternative hypothesis is that VVN539 is superior to vehicle for mean IOP at these 
time points. 
9.2 
Sample Size Determination 
With a sample size in each group of 20, the study will have 80% power to detect a difference of 
3.0 mm Hg between an VVN539 dose compared to vehicle at each diurnal time point assuming a 
common standard deviation of 3.3 mm Hg, α = 0.05 (two-tailed). 
There is no correction for multiplicity for multiple time points or comparisons for high or low 
VVN539 doses.  
9.3 
Analysis Populations 
The Full Analysis Set (FAS) consists of all subjects who were randomized. Subjects will be 
analyzed in the group to which they were randomized. This set will be used for the analysis of all 
efficacy endpoints as the primary analysis.  
The Per Protocol (PP) Analysis Set will include all subjects who completed study-required 
treatment and who followed the protocol without significant deviations. The determination of 
significant protocol violations will be made prior to locking the final database and unmasking. 
Subjects will be analyzed in the group to which they were randomized.  
The Safety Analysis Set will include all subjects who took at least one dose of investigational 
product as indicated on the dosing record. Subjects will be analyzed in the group according to the 
treatment received. All safety variables will be analyzed using the Safety Analysis Set and only 
observed data will be included (i.e., missing data will remain missing for the safety analysis). 


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9.4 
Statistical Analyses 
The statistical analysis of the study will be performed on the data through Day 21 and will be 
performed after all subjects have either completed the Day 21 visit or discontinued early from the 
study and after the study database has been cleaned, verified, and locked. It is planned that the data 
from all clinical sites that participate in this study will be combined so that the target sample size 
will be available for analysis. 
9.4.1 
Baseline Descriptive Analyses 
Demographic characteristics including age (years), sex, race, ethnicity, and baseline disease 
characteristics will be summarized by cohort and overall. Medical history (coded using the most 
recent version of MedDRA), and prior and concomitant medications (coded using World Health 
Organization Dictionary) will be summarized by cohort and overall. 
The numbers of subjects who were enrolled and completed each visit of the study will be provided, 
as well as the reasons for all enrollment discontinuations, grouped by major reason (e.g., lost to 
follow-up, adverse event, poor compliance, rescue due to lack of efficacy). A list of discontinued 
subjects, protocol deviations, and subjects excluded from the analysis sets will be provided as well. 
Exposure and compliance to study treatment will be summarized by treatment and overall. 
9.4.2 
Efficacy Analyses 
Efficacy analyses will be based on the study eye using the FAS.  
9.4.2.1 
Primary Efficacy Analyses 
Primary Estimands: The primary estimands are the treatment differences between VVN539 and 
vehicle at 8:00 AM, 10:00 AM, and 4:00 PM at each of Days 7, 14, and 21 in the study eye using 
the FAS. The primary estimands treat subjects as if they do not receive rescue/prohibited therapy. 
Target Population: Subjects with open-angle glaucoma or ocular hypertension that meet the 
study entry criteria.  
Endpoint: Observed IOP measurements at each time point in the study eye.  
Treatment Condition(s): Treatment condition is based on the randomized treatment group.  
Population-level Summaries: The difference in the mean IOP at the nine time points of interest 
and their corresponding p-values and 95% confidence intervals.  


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Intercurrent Events and Strategies to Address Intercurrent Events  
• Discontinuation of study therapy with continued participation in the study without receipt 
of prohibited/rescue therapy 
o Treatment Policy – no imputation; use observed data  
• Receipt of prohibited/rescue therapy in the study eye  
o Hypothetical Approach – data for visits after the receipt of rescue therapy will be 
set to missing and will be analyzed assuming missing at random (MAR) using a 
longitudinal model (mixed model repeated measures, MMRM)  
• Missing data without withdrawal or with withdrawal regardless of reason 
o Hypothetical Approach: visits that are missing data will be analyzed assuming 
MAR using MMRM  
Time-matched longitudinal models adjusting for baseline and including a random effect for center 
will be used to test time-specific comparisons.  
Data from Days 7, 14, and 21 will be analyzed using MMRM with an unstructured covariance 
assumed for each treatment with treatment, visit, and visit by treatment interaction as a fixed 
effects, baseline measurement as a covariate, and a random effect for site.  
Sample code for the model run separately for 8AM, 10 AM, and 4 PM:  
PROC MIXED DATA=indata;  
CLASS site visit treatment;  
MODEL IOP = baseline visit treatment visit*treatment / solution DDFM=KR;  
REPEATED visit / SUBJECT=subject TYPE=UN group = treatment;  
RANDOM site;  
LSMEANS treatment*visit / slice=visit CL;  
RUN;  
 
Sensitivity analyses include a time-matched last observation carried forward (LOCF) of the last 
available IOP measurement as well as analysis of covariance (ANCOVA) analyses at each visit. 
Additional sensitivity analyses will be performed on the PP Analysis Set as necessary. 
9.4.2.2 
Secondary Efficacy Analyses 
Analyses of change from baseline and observed and change from baseline for diurnal IOP will be 
performed in a manner similar to the primary efficacy analysis. 
9.4.3 
Safety Analyses 
Safety analyses will be performed on all subjects in the Safety Analysis Set. The assessment of safety 
will be based on the summary of ocular and non-ocular treatment emergent adverse events (TEAEs), 
BCVA, central corneal thickness, vital signs, clinical laboratory tests, and ophthalmic exams using slit-


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lamp biomicroscopy and dilated ophthalmoscopy. Summaries will be provided by treatment group, 
and for ocular assessments separately by study-eye and non-study-eye. 
9.4.3.1 
Adverse Events 
AEs will be coded using MedDRA (most current version) and categorized by system organ class using 
preferred terms. Separate summaries of AEs related to treatment (as reported by the Investigator) and 
by severity will be presented. The number of deaths and SAEs will also be presented, and events 
leading to discontinuation from the study will be listed and tabulated. 
9.4.3.2 
Clinical Laboratory Tests 
Summary statistics for observed and change from baseline for serum chemistry and hematology 
laboratory tests will be presented. 
9.4.3.3 
Other Safety Evaluations 
Summary statistics for observed and change from baseline for BCVA, central corneal thickness, and 
vital signs will be presented. Abnormalities in slit-lamp biomicroscopy and dilated ophthalmoscopy 
will be summarized by frequency and percentage. 
9.5 
Interim Analysis 
Not applicable. 
9.6 
Subgroup Analyses 
Subgroup analyses will be described in the SAP. 
9.7 
Exploratory Analyses 
Not applicable. 
9.8 
Missing or Unused Data 
Missing data for IOP will be imputed using LOCF methodology as a sensitivity analysis for the primary 
endpoint and diurnal IOP. Otherwise, observed data will be analyzed at each visit and analyzed using 
MMRM which assumes MAR. 
9.9 
Tabulation of Individual Subject Data 
All data collected in this study will be presented in individual subject data listings for all subjects. 
 


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10 
Supporting Documentation and Operational Considerations 
10.1 
Regulatory Issues, Ethical Concerns, and Study Oversight 
10.1.1 
Informed Consent Process 
10.1.1.1 
Consent/Assent Documents 
Consent forms describing in detail the study intervention, study procedures, and risks are given to 
the subject and written documentation of informed consent is required prior to starting 
intervention/administering study intervention.   
10.1.1.2 
Consent Procedures and Documentation 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be 
Investigational Review Board (IRB)-approved and the subject will be asked to read and review the 
document. The Investigator will explain the research study to the subject and answer any questions 
that may arise. A verbal explanation will be provided in terms suited to the subject’s 
comprehension of the purposes, procedures, and potential risks of the study and of their rights as 
research subjects. Subjects will have the opportunity to carefully review the written consent form 
and ask questions prior to signing. The subjects should have the opportunity to discuss the study 
with their family or surrogates or think about it prior to agreeing to participate. The subject will 
sign the informed consent document prior to any procedures being done specifically for the study. 
Subjects must be informed that participation is voluntary and that they may withdraw from the 
study at any time, without prejudice. A copy of the informed consent document will be given to 
the subjects for their records. The informed consent process will be conducted and documented in 
the source document (including the date), and the form signed, before the subject undergoes any 
study-specific procedures. The rights and welfare of the subjects will be protected by emphasizing 
to them that the quality of their medical care will not be adversely affected if they decline to 
participate in this study.  
10.1.2 
Study Discontinuation and Closure 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or 
termination, will be provided by the Sponsor to the Investigators and regulatory authorities. If the 
study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform 
study subjects and the reviewing IRB. Study subjects will be contacted, as applicable, and be 
informed of changes to study visit schedule.   


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Circumstances that may warrant termination or suspension may include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to subjects 
• Demonstration of efficacy that would warrant stopping 
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination that the primary endpoint has been met 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the Sponsor, IRB and/or FDA.   
10.1.3 
Confidentiality and Privacy 
Subject confidentiality and privacy will be strictly held in trust by the participating Investigators, 
their staff, and the Sponsor(s) and their interventions. This confidentiality will be extended to cover 
testing of biological samples and genetic tests in addition to the clinical information relating to 
subjects. Therefore, the study protocol, documentation, data, and all other information generated 
will be held in strict confidence. No information concerning the study or the data will be released 
to any unauthorized third party without prior written approval of the Sponsor.   
All research activities will be conducted in as private a setting as possible.   
The Study Monitor, other authorized representatives of the Sponsor, representatives of the IRB, 
regulatory agencies or pharmaceutical company supplying study product will be able to inspect all 
documents and records required to be maintained by the Investigator, including but not limited to, 
medical records (i.e., office, clinic, or hospital) and pharmacy records for the subjects in this study. 
The clinical study site will permit access to such records.   
The study subject’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by the reviewing IRB, Institutional policies, or Sponsor 
requirements.   
Study subject research data, which is for purposes of statistical analysis and scientific reporting, 
will be transmitted to and stored at Lexitas Pharma Services, Inc. This will not include the subject’s 
contact or identifying information. Rather, individual subjects and their research data will be 
identified by a unique study identification number. The study data entry and study management 
systems used by clinical sites and by Lexitas research staff will be secured and password protected. 
At the end of the study, all study databases will be de-identified and archived at Lexitas.   


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10.1.4 
Key Roles and Study Governance 
The Manual of Procedures (MOP) will include a list of study team roles and responsibilities of 
those involved in the conduct, management, or oversight of the trial. 
10.1.5 
Clinical Monitoring 
Lexitas Pharma Services, Inc. will conduct the clinical monitoring for this study. A clinical 
Monitoring Plan is to be used, which will describe in detail who will conduct the monitoring, at 
what frequency monitoring will be done, at what level of detail monitoring will be performed and 
the distribution of monitoring reports. 
10.1.6 
Quality Assurance and Quality Control 
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.   
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical 
trial is conducted and data are generated and biological specimens are collected, documented 
(recorded), and reported in compliance with the protocol, International Council for Harmonisation 
(ICH) Good Clinical Practice (GCP), and applicable regulatory requirements (e.g., Good 
Laboratory Practice [GLP], Good Manufacturing Practices [GMP]).   
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the Sponsor, and inspection by local and 
regulatory authorities.   
10.1.7 
Data Handling and Record Keeping 
10.1.7.1 
Data Collection and Management Responsibilities 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the 
site’s Investigator. The Investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported.   
All source documents should be completed in a neat, legible manner to insure accurate 
interpretation of data. Data recorded in the eCRF derived from source documents should be 
consistent with the data recorded on the source documents.   
Clinical data (including AEs, concomitant medications, and expected adverse reactions data) will 
be entered into IBM Clinical Development, a 21 CFR Part 11-compliant data capture system 
provided by Lexitas Pharma Services, Inc. The data system includes password protection and 


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internal quality checks, such as automatic range checks, to identify data that appear inconsistent, 
incomplete, or inaccurate. Clinical data will be entered directly from the source documents.   
10.1.7.2 
Study Records Retention 
Study documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing applications 
in an ICH region or until at least 2 years have elapsed since the formal discontinuation of clinical 
development of the study intervention. These documents should be retained for a longer period, 
however, if required by local regulations. No records will be destroyed without the written consent 
of the Sponsor, if applicable. It is the responsibility of the Sponsor to inform the Investigator when 
these documents no longer need to be retained.   
10.1.8 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, ICH GCP, or Manual 
of Procedures (MOP) requirements. The noncompliance may be either on the part of the subject, 
the Investigator, or the study site staff. As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly.   
These practices are consistent with ICH GCP:  
• 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 5.1 Quality Assurance and Quality Control, section 5.1.1  
• 5.20 Noncompliance, sections 5.20.1, and 5.20.2. 
It is the responsibility of the site’s Investigator to use continuous vigilance to identify and report 
deviations. All deviations must be addressed in study source documents, reported to the reviewing 
IRB (as applicable per the IRB policies), the Contract Research Organization and/or Sponsor. The 
site Investigator is responsible for knowing and adhering to the reviewing IRB requirements. 
Further details about the handling of protocol deviations will be included in the MOP.   
10.1.9 
Publication and Data Sharing Policy 
This study will comply with the Clinical Trials Registration and Results Information Submission 
rule. As such, this trial will be registered at ClinicalTrials.gov, and results information from this 
trial will be submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish 
results in peer-reviewed journals. The institution and Investigators participating in this trial shall 
have no right to publish or present the results of this study without the prior written consent of the 
Sponsor.   


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11 
References 
Cavet ME, DeCory HH. The Role of Nitric Oxide in the Intraocular Pressure Lowering Efficacy 
of Latanoprostene Bunod: Review of Nonclinical Studies. J Ocul Pharmacol Ther 2018;34 (1-
2):52-60. 
Garcia-Calvo M, et al. Purification and reconstitution of the high-conductance, calcium activated 
potassium channel from tracheal smooth muscle. J Biol Chem. 1994;269(1994):676–82. 
Honjo M, Inatani M, Kido N, et al. Effects of protein kinase inhibitor, HA1077, on intraocular 
pressure and outflow facility in rabbit eyes. Arch Ophthalmol 2001;119(8):1171–1178.  
Kahook MY, Serle JB, Mah FS, et al. Long-term Safety and Ocular Hypotensive Efficacy 
Evaluation of Netarsudil Ophthalmic Solution. Am J Ophthalmol 2019;200:130-137. 
Kopczynski C, Epstein D. Emerging trabecular outflow drugs. J Ocul Pharmacol Ther. 2014; 30(2-
3):85-7. 
Medeiros FA, Martin KR, Peace J, Scassellati Sforzolini B, Vittitow JL, Weinreb RN. Comparison 
of Latanoprostene Bunod 0.024% and Timolol Maleate 0.5% in Open-Angle Glaucoma or Ocular 
Hypertension: The LUNAR Study. Am J Ophthalmol. 2016;168:250-9. 
Rao PV, Pattabiraman PP, Kopczynski C. Role of the Rho GTPase/Rho kinase signaling pathway 
in pathogenesis and treatment of glaucoma: bench to bedside research. Exp Eye Res 2017;158:23–
32. 
Serle JB, Katz LJ, McLaurin E, et al. Two Phase 3 Clinical Trials Comparing the Safety and 
Efficacy of Netarsudil to Timolol in Patients With Elevated Intraocular Pressure: Rho Kinase 
Elevated IOP Treatment Trial 1 and 2 (ROCKET-1 and ROCKET-2). Am J Ophthalmol 
2018;186:116-127. 
Shaffer RN. Primary glaucomas. Gonioscopy, ophthalmoscopy and perimetry. Trans Am Acad 
Ophthalmol Otolaryngol. 1960 Mar-Apr;64:112-27.  
Singh IP, Fechtner RD, Myers JS, Kim T, Usner DW, McKee H, Sheng H, Lewis RA, Heah T, 
Kopczynski CC. Pooled Efficacy and Safety Profile of Netarsudil Ophthalmic Solution 0.02% in 
Patients With Open-angle Glaucoma or Ocular Hypertension. J Glaucoma. 2020 Oct;29(10):878-
884.  
Tanihara H, Inoue T, Yamamoto T, Kuwayama Y, Abe H, Suganami H, et al. Additive Intraocular 
Pressure-Lowering Effects of the Rho Kinase Inhibitor Ripasudil (K-115) Combined With Timolol 


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or Latanoprost: A Report of 2 Randomized Clinical Trials. JAMA Ophthalmol. 2015;133(7):755-
61. 
Tian B, Kaufman PL. Effects of the Rho kinase inhibitor Y- 27632 and the phosphatase inhibitor 
calyculin A on outflow facility in monkeys. Exp Eye Res 2005;80(2):215–225. 
Torfard KE, Ahlner J. Mechanisms of action of nitrates Cardiovasc Drugs Ther. 1994;8(5):701-
17. 
Tokushige H, Inatani M, Nemoto S, et al. Effects of topical administration of Y-39983, a selective 
rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys. Invest 
Ophthalmol Vis Sci 2007;48(7):3216–3222.  
Wang R-F, Williamson JE, Kopczynski C, Serle JB. Effect of 0.04% AR-13324, a ROCK and 
norepinephrine transporter inhibitor, on aqueous humor dynamics in normotensive monkey eyes. 
J Glaucoma 2015;24(1):51–54. 
 


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12 
Appendices 
12.1 
Appendix 1: Schedule of Procedures and Assessments 
Study Period 
Screening 
Washout1 
Baseline 
Treatment Period 
End of 
Treatment 
Visit No. 
1 
 
2 
3 
4 
5 
6 
Visit Window 
Day-35 to -1 
Day 0 
Day 1 
Day 7 (+2) 
Day 14 (+2) 
Day 21 (+2) 
Informed consent2  
X 
 
 
 
 
 
 
Ocular and medical history 
X 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
BCVA 
X 
X 
X 
X 
X 
X 
Biomicroscopy3 
X 
X 
X 
X 
X 
X 
IOP 
X 
 
 
X4, 
 
 
 
Diurnal IOP5 
 
 
X 
 
X 
X 
X 
Cup-to-disc ratio 
X 
 
 
 
 
 
X 
Dilated ophthalmoscopy 
X 
 
 
 
 
 
X6 
Pachymetry7 
X 
 
 
 
 
X 
Gonioscopy8 
X 
 
 
 
 
 
 
1 Washout period: Prostaglandins, β-adrenoceptor antagonists, kinase inhibitors (4 weeks), adrenergic agonists (2 weeks), muscarinic agonists and carbonic anhydrase inhibitors (5 
days) 
2 Informed consent will be obtained prior to the conduct of any study specified or associated procedures or evaluations. 
3 Biomicroscopy of anterior segment includes corneal epithelium, bulbar and lower conjunctiva, and lens.  
4 Measured at 8:00AM 
5 Measured at 8:00AM, 10:00AM and 4:00PM 
6 After the last IOP measurement 
7 Central corneal thickness will be measured at entry for IOP interpretation. Pachymetry performed within 3 months of screening is acceptable. Ultrasound and optical pachymetry 
is permissible provided that the same methodology is used at Visits 1 and 6. 
8 Gonioscopy performed within 3 months of Visit 1 is acceptable. 


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Study Period 
Screening 
Washout1 
Baseline 
Treatment Period 
End of 
Treatment 
Visit No. 
1 
 
2 
3 
4 
5 
6 
Visit Window 
Day-35 to -1 
Day 0 
Day 1 
Day 7 (+2) 
Day 14 (+2) 
Day 21 (+2) 
Visual field9 
X 
 
 
 
 
 
Optical Coherence 
Tomography 
X10 
 
 
 
 
 
 
Blood chemistry, hematology 
X 
 
 
 
 
 
X 
BP, HR 
X 
X 
X 
X 
X 
X 
Pregnancy test11 
X 
X 
 
 
 
X 
Randomization 
 
X 
 
 
 
 
Study drug administration in 
clinic 
 
 
 
X12 
X13 
 
X13 
Dispense investigational 
product 
 
 
X 
X 
X 
 
Collect study drug 
 
 
 
X 
X 
X 
Prior/Concomitant medications 
X 
X 
X 
X 
X 
X 
Adverse event assessment 
 
 
X 
X 
X 
X 
Abbreviation key: BCVA=best corrected visual acuity; BP=blood pressure; HR=heart rate; IOP=intraocular pressure 
 
 
 
9 Visual field within 3 months of Visit 1 is acceptable, provided that it is reliable and meets the requirement for automated threshold visual field (e.g., 30-2 or 24-2 Humphrey). 
Unreliable entry fields must be repeated prior to study drug dispensation. 
10 Optical Coherence Tomography within 3 months of Visit 1 is acceptable. 
11 Women of childbearing potential only 
12 After 8:00AM IOP measurement 
13 After 8:00AM and prior to 10:00AM IOP measurement 


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12.2 
Appendix 2: Abbreviations and Definition of Terms 
AE 
Adverse Event 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
ANCOVA 
Analysis of Covariance 
BCVA 
Best Corrected Visual Acuity 
b.i.d 
Twice a Day (Bis in Die) 
BP 
Blood Pressure 
CFR 
Code of Federal Regulations  
CONSORT 
Consolidated Standards of Reporting Trials 
CS 
Clinically Significant 
eCRF 
Electronic Case Report Form 
FAS 
Full Analysis Set 
FDA 
Food and Drug Administration 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
GMP 
Good Manufacturing Practice 
hCG 
Human Chorionic Gonadotropin 
Hct 
Hematocrit 
HDL 
High-Density Lipoprotein 
Hgb 
Hemoglobin 
HIPAA 
Health Insurance Portability and Accountability Act 
HR 
Heart Rate 
IB 
Investigator’s Brochure 
ICH 
International Council for Harmonisation 
IND 
Investigational New Drug 


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IOP 
Intraocular Pressure 
IP 
Investigational Product 
IRB 
Institutional Review Board 
LASIK 
Laser-Assisted in situ Keratomileusis 
LDH 
Lactate Dehydrogenase 
LDL 
Low-Density Lipoprotein 
LOCF 
Last Observation Carried Forward 
logMAR 
Logarithm of the Minimum Angle of Resolution 
LPDE 
Low-Density Polyethylene 
MAR 
Missing at Random 
MCH 
Mean Corpuscular Hemoglobin 
MCHC 
Mean Corpuscular Hemoglobin Concentration 
MCV 
Mean Corpuscular Volume 
MIGS 
Minimally Invasive Glaucoma Surgery 
MMRM 
Mixed Model Repeated Measures 
MOP 
Manual of Procedures 
NCE 
New Chemical Entity 
NCS 
Not Clinically Significant 
NO 
Nitric Oxide 
OAG 
Open Angle Glaucoma 
OCT 
Optical Coherence Tomography 
OHT 
Ocular Hypertension 
OU 
Both Eyes (Oculus Uterque) 
PI 
Principal Investigator 
PK 
Pharmacokinetic 
POAG 
Primary Open Angle Glaucoma 
PP 
Per Protocol 
PRK 
Photorefractive Keratectomy 


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PVP 
Polyvinylpyrrolidone 
QC 
Quality Control 
q.d 
Once a Day (Quaque Die) 
RBC 
Red Blood Cells 
ROCK 
Rho Kinase 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SOP 
Standard Operating Procedure 
TEAE 
Treatment Emergent Adverse Events 
UPT 
Urine Pregnancy Test 
US 
United States 
VA 
Visual Acuity 
WBC 
White Blood Cells 
 
 
 


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12.3 
Appendix 3: Visual Acuity 
Distance visual acuity (VA) will be assessed as outlined in Appendix 1 using ETDRS visual acuity 
charts either retro-illuminated or frontally illuminated (60-watt bulb or well-lit room, respectively). 
It is recommended that the ETDRS Chart 1 be used for testing the right eye and the Chart 2 be 
used for testing the left eye.  
BCVA testing should precede slit lamp examination, intraocular pressure measurement, the 
administration of topical anesthetic agents or any examination requiring contact with the eye.  
12.3.1 
Manifest Refraction 
Visual acuity can be tested using the subject's prescription eyeglasses for distance correction, 
provided the prescription is less than one year old. Sites should measure the eyeglasses and record 
the readings at Visit 1 in case that subjects forget to bring their eyeglasses to later visits.  
Manifest refraction should be performed per the sites standard procedures if the subject's eyeglass 
prescription is more than one year old or if the subject forgets to bring their eyeglasses to Visit 1. 
This refraction should then be used for all following visits. 
12.3.2 
Best Corrected Visual Acuity 
Visual Acuity will be assessed at each visit in monocularly in both eyes, testing the right eye first 
and then the left eye. 
The subject should be positioned according to the elevation of the chart (either seated or standing) 
so that the chart is at a comfortable viewing angle. The subject should attempt to read each letter, 
line-by-line, left to right, beginning with line 1 at the top of the chart. The subject should be told 
that the chart has letters only, no numbers. If the subject reads a number, they should be reminded 
that the chart contains no numbers, and the examiner should then request a letter instead of the 
number. The subject should be asked to read slowly, about 1 letter per second, to achieve the best 
identification of each letter. The subject is not to proceed to the next letter until they have given a 
definite response. If the subject changes a response before he has read aloud the next letter, then 
the change must be accepted. 
Maximum effort should be made to identify each letter on the chart; the subject should be 
encouraged to guess. When it becomes evident that no further meaningful readings can be made, 
the examiner should stop the test. The number of letters missed or read incorrectly should be noted. 
In order to provide standardized and well-controlled assessments of visual acuity during the study, 
the same lighting conditions must be used consistently throughout the study  


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Calculations: LogMAR VA = Baseline value + (n x 0.02)  
• where:  the baseline value is the LogMAR number of the last line read (at least 1 letter read 
correctly in this line), and “n” is the total number of letters missed up to and including the 
last line read, and “0.02” is the value for each letter. 
12.4 
Appendix 4: Biomicroscopy 
The slit-lamp biomicroscopy exam will be performed to examine eye structures at each study visit 
for each eye. Areas assessed will include the following: anterior chamber, eyelids, conjunctiva, 
cornea, iris, pupils, lashes and lens.  
Note: the same Investigator should assess and score anterior chamber cells and flare across all 
study visits for an individual subject. 
It should be performed with a slit lamp (halogen illumination system is required) using a beam 
width and intensity that provide optimal evaluation of the anterior segment.  
This procedure will be performed in the same manner for all subjects observed at the Investigator’s 
site. The site will record all ABNORMAL findings in the source document and the Investigator 
will evaluate the ABNORMAL findings as Not Clinically Significant (NCS) or Clinically 
Significant (CS). CS and NCS ABNORMAL findings will be recorded in the source 
documentation. However, only ABNORMAL CS descriptions from Visits 3-6 will be captured in 
the eCRF as AE. 
The scoring scales below will be used: 
Anterior Chamber Cells  
 
0 = No cells seen  
1 = 1 - 5 cells  
2 = 6 - 15 cells  
3 = 16 - 30 cells  
4 = greater than 30 cells 
Anterior Chamber Flare  
0 = None  
1 = Mild (trace to clearly noticeable, visible) 
2 = Moderate (without plastic aqueous humor)  
3 = Marked (with plastic aqueous humor)  
4 = Severe (with fibrin deposits and/or clots) 
Eyelid 
• 
Eyelid Hyperemia 
• 
Eyelid Edema 
0 = None  
1 = Mild  
2 = Moderate 
3 = Severe  
Conjunctiva 
• 
Hyperemia 
• 
Edema (Chemosis) 
• 
Conjunctival Discharge/Exudate 
0 = None  
1 = Mild  
2 = Moderate 
3 = Severe 


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Cornea 
• 
Corneal Edema 
0 = None  
1 = Mild  
2 = Moderate 
3 = Severe 
Iris 
Brown 
Blue 
Hazel 
Grey 
Green 
Black 
Other 
Pupil 
0 = Normal 
1 = Abnormal 
Lashes 
0 = Normal 
1 = Abnormal 
Lens 
0 = Normal 
1 = Abnormal 
 
12.5 
Appendix 5: Intraocular Pressure 
IOP measurement should be conducted after the biomicroscopy exam is completed and prior to 
pupil dilation, as applicable.   
IOP measurements will be performed utilizing a Goldmann applanation tonometer affixed to a slit 
lamp according to the Investigator’s standard procedure (two-person reading method). 
Measurements will be taken with the subject seated. The subject and slit lamp should be adjusted 
so that the subject’s head is firmly positioned on the chin rest and against the forehead rest without 
leaning forward or straining. All pressure will be recorded in mmHg. Two consecutive 
measurements are taken in each eye and recorded as the mean of the two measurements. If the two 
measurements differ by >1.0 mmHg, a third measurement is performed, and the median is 
recorded. 
At Visit 1, IOP will be measured once during screening visit. 
At Visit 3, IOP will be measured once at 8:00AM (±30 minutes) prior to the first q.d. AM dosing.  
At Visits 2, 4, 5, and 6 diurnal IOP will be measured:  
• At 8:00AM (±30 minutes), 10:00AM (±30 minutes) and 4:00PM (±30 minutes) 
• At Visits 4 and 6, the 8:00AM measurement will be taken prior to the AM dosing. 
12.6 
Appendix 6: Dilated Ophthalmoscopy 
Dilated ophthalmoscopy will include assessment of the retina for any abnormal findings, optic 
nerve head for pallor and cupping (cup to disc ratio) for each eye. The status of the vitreous 


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(including haze), retina (including vessels), macula, optic nerve will be determined as Normal or 
Abnormal. The cup-to-disc ratio will be recorded with two decimal points (e.g., 0.80). After the 
Ophthalmoscopy procedure, the Investigator will determine if findings are within normal limits or 
are abnormal. For abnormal findings at Visit 1, the Investigator will determine whether or not the 
abnormality would exclude the subject from study participation. 
12.7 
Appendix 7: Pachymetry 
Corneal thickness evaluation (in microns) will be performed using a calibrated ultrasonic or optical 
pachymeter, provided that the same method is used for all measurements. Measurements will be 
taken in the central cornea after instilling topical anesthetic. A total of three readings will be 
obtained for each eye. Care should be taken to place the probe perpendicular to the corneal surface 
and to not a compress the cornea. 
Pachymetry is performed at Visit 1 and Visit 6.  
• At Visit 1, pachymetry within 3 months of the visit is acceptable.  
• At Visit 6, Pachymetry is to be performed after the last IOP measurement. 
12.8 
Appendix 8: Gonioscopy 
Gonioscopy will be performed at Visit 1 using the Shaffer system (Shaffer, 1960) to grade the 
angle anatomy as follows:  
Angle 
Grade 
closed 
0 
10 – 15o 
I 
15 – 25o 
II 
25 – 35o 
III 
>35 o 
IV 
• Gonioscopy performed within 3 months of Visit 1 is acceptable. 
12.9 
Appendix 9: Visual Field 
Visual fields are performed at Visit using a 30-2 or 24.2 Humphrey perimeter. SITA Standard is 
preferred, SITA Fast is also allowed. Visual fields must be reliable, defined as: 
• Fixation losses ≤ 33% 
• False positives ≤ 33% 
• False negatives ≤ 33% 


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The gaze track and blind spot should be turned on in order to calculate the fixation losses. Visual 
fields should be performed with a non-dilated pupil, unless, in the opinion of the Investigator, the 
pupil is so miotic that dilation is required. 
• Visual fields performed within 3 months of Visit 1 are acceptable. 
12.10 Appendix 10: Optical Coherence Tomography (OCT) 
OCT will be used at the screening visit to determine the nerve fiber layer thickness at Visit 1. 
12.11 Appendix 11: Blood Chemistry, Hematology 
Laboratory requisition forms must be completed, and samples must be clearly labelled with the 
subject number, protocol number, site/center number and visit date. Refer to the Central 
Laboratory Manual for details for the collection, preparation and shipment of samples and 
reference ranges. 
Laboratory reports must be reviewed and signed by the Investigator prior to Visit 2 and results 
cannot be indicative of any clinically significant or uncontrolled disease in the opinion of the 
Investigator.  If any blood is not resulted, repeat laboratory collection must be performed and 
results reviewed by the Investigator prior to Visit 2. 
Approximately 10 mL of blood will be obtained at Visit 1 and Visit 6. Fasting will not be 
required, therefore specific values may be out of the typical fasting range.  
Abnormal laboratory values noted as clinically significant at Visit 6 that are changes from the 
Screening values are documented as AEs and is to be followed-up as appropriate. 
The following serum chemistry and hematology parameters will be measured: 
Chemistry 
Hematology 
Alanine aminotransferase (ALT) 
Absolute/percent basophil count 
Albumin 
Absolute/percent eosinophil count 
Alkaline phosphatase (ALP) 
Absolute/percent lymphocyte count 
Aspartate aminotransferase (AST) 
Absolute/percent monocyte count 
Bilirubin (total, direct, indirect) 
Absolute/percent neutrophil count 
Bun 
Differential WBC 
Calcium 
Hematocrit (Hct) 
Cholesterol (total, High-Density 
Lipoprotein (HDL) and Low-Density 
Lipoprotein (LDL)) 
Hemoglobin (Hgb) 
Carbon dioxide 
Mean corpuscular hemoglobin (MCH) 
Chloride 
Mean corpuscular hemoglobin conc. (MCHC) 
Creatinine, serum 
Mean corpuscular volume (MCV) 


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Glucose 
Platelet count 
Lactate dehydrogenase (LDH) 
Red blood cells (RBC) 
Potassium 
White blood cells (WBC) 
Sodium 
 
Total protein 
 
Triglycerides 
 
12.12 Appendix 12: Heart Rate & Blood Pressure 
12.12.1 Heart Rate 
Measure the heart rate after the subject has been seated in a quiet room for at least 5 minutes. 
Instruct the subject that they should refrain from talking during the procedure. Measure the pulse 
rate over a 30-second period by palpitation at the wrist, and multiply by 2. An electronic device 
may be used instead. The method of measurement (manual or electronic) must be documented in 
the subject records. Repeat measurements should be taken for out-of-range values. Confirmed 
heart rate readings outside the normal range must be evaluated by the Investigator for clinical 
significance. 
12.12.2 Blood Pressure 
Measure the blood pressure after heart rate (while the subject is in a resting state). Subject should 
be seated comfortably, with both feet resting on the floor; subject’s arm should not be 
constrained by any clothing. Use a sphygmomanometer with appropriate size cuff with a 
stethoscope. A digital device may be used instead. All measurements will be obtained from the 
same arm using the same cuff size for all study visits. Repeat measurements should be taken for 
significantly high or low measurements. Confirmed BP readings outside the normal range must 
be evaluated by the Investigator for clinical significance. 
12.13 Appendix 13: Pregnancy Test 
A urine human chorionic gonadotropin (hCG) pregnancy test is performed at Visits 1, 2, and 6 
for adult women of childbearing potential. An adult woman is considered to be of childbearing 
potential unless she is one year post-menopausal or three months post-surgical sterilization.   
 
 


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12.14 Appendix 14: Investigator Agreement 
 
A phase 2, double-masked, randomized, vehicle-controlled, dose-
response study assessing the safety and ocular hypotensive efficacy of 
VVN539 in subjects with primary open angle glaucoma or ocular 
hypertension. 
 
 
Version No.: 2.0 
Issue Date: 07/Jun/2022 
 
 
I have read the clinical study protocol and understand it.  I agree to conduct the study as outlined 
in this document and in accordance with Good Clinical Practice Guidelines, all local and federal 
requirements and regulations, and in compliance with those precepts set forth in the Declaration 
of Helsinki with respect to the use of human subjects in clinical studies and investigations.   
Further, I agree to maintain the confidentiality of all information received or developed in 
connection with this protocol.  
 
 
Signature of Investigator: 
 
 
 
Name (printed) 
Signature 
Date 
 
 


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12.15 Appendix 15: Compliance Statement 
The study will be conducted in accordance with International Council for Harmonisation Good 
Clinical Practice (ICH GCP), with the United States (US) Code of Federal Regulations (CFR) 
applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, 
and/or 21 CFR Part 812), and as stipulated in the Declaration of Helsinki with respect to the use 
of human subjects in clinical studies and investigations.  
The Principal Investigator (PI) will assure that no deviation from, or changes to the protocol will 
take place without prior agreement from the Investigational New Drug (IND) Sponsor, funding 
agency and documented approval from the Institutional Review Board (IRB), except where 
necessary to eliminate an immediate hazard(s) to the study subjects. All personnel involved in the 
conduct of this study have completed Human Subjects Protection and ICH GCP Training.  
The protocol, informed consent form(s), recruitment materials, and all subject materials will be 
submitted to the IRB for review and approval.  Approval of both the protocol and the consent form 
must be obtained before any subject is enrolled.  Any amendment to the protocol will require 
review and approval by the IRB before the changes are implemented to the study.  All changes to 
the consent form will be IRB approved; a determination will be made regarding whether a new 
consent needs to be obtained from subjects who provided consent, using a previously approved 
consent form. 
Sponsor’s Representative(s): 
 
 
 
Name (printed) 
Signature 
Date 
Medical Monitor(s): 
 
 
 
Name (printed) 
Signature 
Date 
Principal Investigator(s): 
 
 
 
Name (printed) 
Signature 
Date 
 
 


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12.16 Appendix 16: Protocol Amendments 
 
Date 
Description 
Substantive/Non-Substantive 
07 Jun 2022 
Protocol Amendment 1 
(Version 2.0) 
This is a substantiative amendment; changes were 
made based on FDA comments on 25 May 2022 to 
minimize the risk of study subjects randomized to 
vehicle for potential glaucomatous loss. Detail can 
be found in the corresponding Summary of 
Changes document. 
14 Apr 2022 
Original Protocol 
(Version 1.0) 
N/A 
 
 
 


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Last page 
 

SAP_001.pdf: 
NCT05451329 
Study VVN559-CS201 
Statistical Analysis Plan 
 
A phase 2, double-masked, randomized, vehicle-controlled, dose-response study assessing the 
safety and ocular hypotensive efficacy of VVN539 in subjects with primary open angle 
glaucoma or ocular hypertension. 
 
Dated 21 December 2022 


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    VVN539-CS-201 
Statistical Analysis Plan 
 
 
 
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Statistical Analysis Plan for A phase 2, double-masked, randomized, 
vehicle-controlled, dose-response study assessing the safety and ocular 
hypotensive efficacy of VVN539 in subjects with primary open angle 
glaucoma or ocular hypertension 
 
 
 
Protocol No: 
VVN539-CS-201 
Protocol Date: 
2022-06-07 (Amendment 1) 
Sponsor:  
VivaVision Biotech, Inc. 
304-866 Halei Rd, Pudong District, 
Shanghai, China 
SAP Version: 
2.0 (Amended) 
SAP Date: 
2022-12-21 (Final) 
Prepared by: 
 
 
 
 
 
Confidentiality Statement  
This document contains information from VivaVision Biotech, Inc. and is of confidential, trade secret, and/or proprietary nature 
and may not be disclosed unless such disclosure is required by federal or state law or regulation. It is loaned to you for your 
confidential review on behalf of VivaVision Biotech, Inc. only and is not to be photocopied, disclosed, or transmitted to any other 
person or party without the advance written approval of VivaVision Biotech, Inc. 
 
 


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SIGNATURE PAGE 
 
 
 
 


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TABLE OF CONTENTS 
LIST OF TABLES .......................................................................................................................... 5 
LIST OF FIGURES ........................................................................................................................ 6 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ................................................. 7 
PROTOCOL SYNOPSIS.............................................................................................................. 10 
9.5. 
Efficacy and Safety Variables ............................................................................................ 15 
9.5.1. 
Efficacy and Safety Measurements Assessed and Flow Chart ................................... 15 
9.5.1.1. 
Visit and Procedure Schedule ............................................................................. 15 
9.5.1.2. 
Demographics and Baseline Characteristics ....................................................... 15 
9.5.1.3. 
Efficacy Assessments .......................................................................................... 16 
9.5.1.4. 
Safety Assessments ............................................................................................. 17 
9.5.2. 
Appropriateness of Measurements ............................................................................. 21 
9.5.3. 
Primary Efficacy Variable(s) ...................................................................................... 21 
9.5.4. 
Drug Concentration Measurements ............................................................................ 21 
9.6. 
Data Quality Assurance ..................................................................................................... 22 
9.6.1. 
Training and Education of Investigators and Study Site Personnel ........................... 22 
9.6.2. 
Monitoring of Study Sites .......................................................................................... 22 
9.6.3. 
Data Entry and Verification of Database Used for Analysis and Reporting .............. 22 
9.6.4. 
Clinical Study Report ................................................................................................. 23 
9.6.5. 
Inter-Laboratory Standardization Methods ................................................................ 23 
9.7. 
Statistical Methods Planned in the Protocol and Determination of Sample Size .............. 24 
9.7.1. 
Statistical and Analytical Plans .................................................................................. 24 
9.7.1.1. 
Analysis Populations ........................................................................................... 26 
9.7.1.2. 
Analysis of Subject Disposition .......................................................................... 26 
9.7.1.3. 
Analysis of Demographic and Baseline Characteristics ..................................... 27 
9.7.1.4. 
Analysis of Study Medication Adherence and Exposure .................................... 28 
9.7.1.5. 
Analysis of Efficacy ............................................................................................ 29 
9.7.1.6. 
Analysis of Safety ............................................................................................... 31 
9.7.2. 
Determination of Sample Size .................................................................................... 33 
9.8. 
Changes in the Conduct of the Study or Planned Analyses ............................................... 34 
9.8.1. 
Protocol Amendments ................................................................................................ 34 


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9.8.2. 
Changes from Protocol-Specified Analyses ............................................................... 34 
REFERENCES ............................................................................................................................. 35 
APPENDIX I: SCHEDULE OF ASSESSMENTS....................................................................... 36 
 
 


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LIST OF TABLES 
Table 1: Schedule of Assessments ................................................................................................ 36 
 
 
 
 


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LIST OF FIGURES 
Figure 1: Study Schematic ............................................................................................................ 15 
 
 
 


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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
AE 
Adverse event 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANCOVA 
Analysis of Covariance 
AST 
Aspartate aminotransferase 
BCVA 
Best Corrected Visual Acuity 
b.i.d. 
Twice a Day (Bis in Die) 
BP 
Blood Pressure 
CFR 
Code of Federal Regulations  
CRF 
Case report form 
CRO 
Contract research organization 
CS 
Clinically significant 
eCRF 
Electronic case report form 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FAS 
Full Analysis Set 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
GMP 
Good Manufacturing Practice 
Hct 
Hematocrit 
HDL 
High-Density Lipoprotein 
Hgb 
Hemoglobin 
HR 
Heart Rate 
ICH 
International Conference on Harmonisation  
IOP 
Intraocular Pressure 
IP 
Investigational product 
LASIK 
Laser-Assisted in situ Keratomileusis  
LDH 
Lactate Dehydrogenase 


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LDL 
Low-Density Lipoprotein 
LOCF 
Last observation carried forward 
LogMAR 
Logarithm of the Minimum Angle of Refraction 
MAR 
Missing at random 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean Corpuscular Volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
MIGS 
Minimally Invasive Glaucoma Surgery 
MMRM 
Mixed-model repeated measures 
NCS 
Non-clinically significant 
OD 
Oculus dexter (right eye) 
OHT 
Ocular Hypertension 
OS 
Oculus sinister (left eye) 
OU 
Oculus uterque (both eyes) 
PI 
Principal Investigator 
POAG 
Primary Open Angle Glaucoma 
PP 
Per Protocol 
PRK 
Photorefractive Keratectomy 
PT 
Preferred term 
QC 
Quality control 
q.d. 
Once a Day (Quaque Die) 
RBC 
Red blood cell count 
SAE 
Serious adverse event 
SAP 
Statistical Analysis Plan 
SD 
Standard deviation 
SOC 
System organ class 
SOP 
Standard Operating Procedure 


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TEAE 
Treatment emergent adverse event 
UPT 
Urine pregnancy test 
US 
United States 
WBC 
White blood cell count 
WHODrug 
World Health Organization Drug Dictionary 
 
 
 


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PROTOCOL SYNOPSIS 
 
Title: 
A phase 2, double-masked, randomized, vehicle-controlled, dose-response 
study assessing the safety and ocular hypotensive efficacy of VVN539 in 
subjects with primary open angle glaucoma or ocular hypertension. 
Phase: 
2 
Design/Conduct: 
Prospective, parallel-comparison, multi-center, double-masked, randomized, 
vehicle-controlled dose-response study assessing the safety and ocular 
hypotensive efficacy of VVN539 in subjects with primary open angle 
glaucoma (POAG) or ocular hypertension (OHT) in the United States (US). 
Objective(s): 
Efficacy: To evaluate the ocular hypotensive efficacy of 2 concentrations of 
VVN539. 
Safety: To evaluate the ocular and systemic safety of 2 concentrations of 
VVN539 
Endpoint(s): 
Primary Efficacy: Mean intraocular pressure (IOP) at each diurnal time point 
(8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14) and Visit 6 (Day 
21). 
Secondary Efficacy: Mean change in IOP from Visit 2 (Baseline) to Visit 4 
(Day 7), Visit 5 (Day 14) and visit 6 (Day 21). 
Population studied: 
Approximately 60 (20 per cohort) evaluable adult males or females diagnosed 
with OHT or POAG. 
Inclusion criteria: 
1. 18 years of age or older. 
2. Diagnosis of POAG or OHT in both eyes that are untreated, or if 
treated, in the opinion of the investigator are well controlled on 2 or 
fewer ocular hypotensive medications prior to Visit 1.  
• 
Combination medications count as 2 medications 
3. Unmedicated IOP of ≥ 22 mm Hg and ≤ 36 mm Hg in the study eye, 
with no more than 5 mm Hg inter-eye difference at 08:00AM and 
10:00AM at Visit 2. 
4. Corrected visual acuity in each eye +1.0 logMAR (Snellen equivalent 
to 20/200) or better by ETDRS in each eye. 
5. Central corneal thickness of ≥ 400 and ≤ 620 µm in each eye. 
6. Able and willing to give signed informed consent and follow study 
instructions. 
7. In the opinion of the Investigator, the subject is able to safely 
discontinue current ocular hypotensive medication(s), when a 
washout is required. 
Exclusion criteria: 
Ophthalmic:  
1. Known hypersensitivity to any kinase inhibitors, any excipient or 
preservative of the formulation or to topical anesthetics or fluorescein. 


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2. Unmedicated IOP of > 36 mm Hg in either eye at any time point at 
Visit 2. 
3. History of intraocular implant for IOP treatment, glaucoma filtering 
surgery, placement, or removal of minimally invasive glaucoma 
implant (MIGS) in the study eye.   
4. History of laser IOP lowering surgery within 6 months of Visit 1 in 
the study eye.   
• 
Note: Laser peripheral iridotomy for narrow angle is allowed if 
performed 3 months or more prior to Visit 1. 
5. Ocular trauma within the past six months, or surgery involving the 
ocular surface (including cataract surgery) or non-IOP-lowering laser 
treatment within the past three months of Visit 1 in the study eye. 
6. Refractive surgery in the study eye (including but not limited to radial 
keratotomy, Photorefractive Keratectomy [PRK], Laser-Assisted in 
situ Keratomileusis [LASIK], etc.). 
• 
Refractive surgery planned during study participation in either 
eye. 
7. Have received intravitreal corticosteroid injection within 6 months or 
subtenon/subconjunctival steroid injection within 3 months of Visit 1 
in either eye. 
8. History or evidence of ocular infection, inflammation, clinically 
significant blepharitis, conjunctivitis, or of herpes simplex keratitis 
within 2 months of Visit 1 in either eye. 
9. Unable or unwilling to avoid wearing of contact lenses from Visit 1 
through the study.   
10. Currently using 3 or more ocular hypotensive medications prior to 
screening in either eye (combination medications count as 2 
medications). 
11. Ocular medication of any kind within 28 days of Visit 2 in either eye 
other than: 
• 
Ocular hypotensive medications which must be washed out 
according to the provided schedule 
• 
Ocular Medication as part of an eye exam 
• 
Lubricating drops for dry eye 
12. Clinically significant ocular disease in either eye (e.g. corneal edema, 
uveitis, severe keratoconjunctivitis sicca) which might interfere with 
the study. 
13. Pseudoexfoliative, 
pigmentary, 
congenital, 
developmental 
or 
secondary glaucoma (e.g. neovascular, uveitic, pigmentary, lens-
induced, steroid-induced, trauma induced or glaucoma associated 
with increased episcleral venous pressure) in either eye. 
14. Closed angle forms of glaucoma in either eye 
• 
Shaffer grade < 3 (in 1 or more quadrants) 
• 
Gonioscopy within 3 months prior to Visit 1 is acceptable. 
15. Cup-disc ratio of 0.7 or greater in either eye. 


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16. Nerve fiber layer thickness assessment by Optical Coherence 
Tomography (OCT) documents thinning of the NFL into the abnormal 
range for either eye.  
• 
If, in the opinion of the investigator, the thinning of the NFL may 
be attributed to other ocular features (including but not limited to 
optic nerve drusen, myopia, chorioretinal scars), then the subject 
is allowed. 
17. Subjects with visual fields with substantial glaucomatous loss (e.g., 
Bjerrum scotoma, blind spot enlargement) for whom washout and 
potential randomization would be unsafe. 
18. In addition to the above criteria, glaucomatous damage in either eye 
so severe that washout of ocular hypotensive medications for up to 5 
weeks and treatment for 3 weeks (for subjects randomized to vehicle 
control) is not judged safe in the opinion of the Investigator.  
• 
Note: Subjects may be changed to a another IOP lowering therapy 
with a shorter washout period as long as the appropriate washout 
is met prior to Visit 2. 
19. Any abnormality preventing reliable applanation tonometry of either 
eye. 
General/Systemic: 
20. Laboratory tests indicative of any clinically significant disease in the 
opinion of the Investigator. 
21. Clinically significant systemic disease (e.g., uncontrolled diabetes, 
myasthenia gravis, hepatic, renal, cardiovascular or endocrine 
disorders) which might interfere with the study in the opinion of the 
Investigator. 
22. Participation in any study involving an investigational product within 
the past 30 days of Visit 1. 
23. Changes of systemic beta blockers during the study that in the opinion 
of the Investigator could have a substantial effect on IOP within 30 
days prior to Visit 1 or anticipated during the study. 
24. Have received systemic corticosteroids (oral, injectable, inhaled) 
within 30 days of Visit 1 or planned use during the study.   
• 
Note: Topical dermatologic steroids are allowed if used for less 
than 3 consecutive days on no more than 3 separate areas of the 
body within 30 days of Visit 1 and the dosage is not expected to 
increase during the study.   
• 
Intranasal steroids are allowed if used less than or 4 times per 
week within 30 days of Visit 1 and the dosage is not expected to 
increase during the study. 
25. History of substance abuse within 1 year of Visit 1 
26. Subjects that are employees or relatives of employees at the clinical 
site. 
27. Women of childbearing potential who are pregnant, nursing, planning 
a pregnancy, or not using a medically acceptable form of birth control.  
• 
Females of childbearing potential with a positive urine pregnancy 
test (UPT) result at Visit 1 or Visit 2 are excluded. 


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• 
An adult woman is considered to be of childbearing potential 
unless she is one year post-menopausal or three months post-
surgical sterilization.  
Investigational product(s): 
VVN539 ophthalmic solution 0.02% (3mL in 5 mL bottles) 
VVN539 ophthalmic solution 0.04% (3mL in 5 mL bottles) 
VVN539 ophthalmic solution vehicle (3mL in 5 mL bottles) 
Dosing regimen: 
Both eyes (OU), q.d. (morning) for 7 days, then q.d. (evening) for 7 days, and 
then b.i.d. for 7 days. 
Assessments/Evaluations: 
Efficacy:  
• 
IOP for efficacy will be measured by Goldmann applanation 
tonometry at 8:00AM, 10:00AM and 4:00PM at Visits 2, 4, 5 and 6. 
Safety: 
• 
Adverse event monitoring 
• 
Best Corrected Visual Acuity (ETDRS) 
• 
IOP 
• 
Biomicroscopy of anterior segment  
• 
Central corneal thickness  
• 
Cup-disc ratio  
• 
Ophthalmoscopy (dilated) 
• 
Heart rate and blood pressure (BP) 
• 
Blood chemistry, hematology 
Duration of study: 
There are a total of 6 clinical visits over 8 weeks (up to 5 weeks of washout 
and 3 weeks of study treatment) 
Statistical methods: 
With a sample size in each group of 20, the study will have 80% power to detect 
a difference of 3.0 mm Hg between an VVN539 dose compared to vehicle at 
each diurnal time point assuming a common standard deviation of 3.3 mm Hg, 
α = 0.05 (two-tailed). There is no correction for multiplicity for multiple time 
points or comparisons for high or low VVN539 doses. 
 
The primary endpoint for this study is to compare mean IOP at each diurnal 
time point (8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14) and 
Visit 6 (Day 21) for two doses of VVN539 compared to vehicle using data from 
the study eye. Time-matched longitudinal models adjusting for baseline and 
including a random effect for center will be used to test time-specific 
comparisons. Data from Days 7, 14, and 21 will be analyzed using mixed-
model repeated measures (MMRM) with an unstructured covariance assumed 
for each treatment with treatment, visit, and visit by treatment interaction as a 
fixed effects, baseline measurement as a covariate, and a random effect for site. 
Sensitivity analyses include a time-matched last observation carried forward 
(LOCF) of the last available IOP measurement as well as analysis of covariance 
(ANCOVA) analyses at each visit. Additional sensitivity analyses will be 
performed on the PP Analysis Set as necessary. 


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Analyses of change from baseline and observed and change from baseline for 
diurnal IOP will be performed in a manner similar to the primary efficacy 
analysis. 
 
Safety analyses will be performed on all subjects in the Safety Analysis Set. 
The assessment of safety will be based on the summary of ocular and non-
ocular treatment emergent adverse events (TEAEs), Best Corrected Visual 
Acuity (BCVA), central corneal thickness, heart rate and blood pressure, 
clinical laboratory tests, and ophthalmic exams using slit-lamp biomicroscopy 
and dilated ophthalmoscopy. Summaries will be provided by treatment group, 
and for ocular assessments separately by study-eye and non-study-eye. 
 
 
 
 
 
 


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9.5. 
EFFICACY AND SAFETY VARIABLES 
9.5.1. 
Efficacy and Safety Measurements Assessed and Flow Chart 
The primary objective of this study is to evaluate the ocular hypotensive efficacy of 2 
concentrations of VVN539 in subjects with ocular hypertension (OHT) or primary open angle 
glaucoma (POAG). The secondary objective is to evaluate the ocular and systemic safety of the 2 
concentrations in this subject population. 
Figure 1 summarizes the design of the study. 
Figure 1: Study Schematic 
9.5.1.1. 
Visit and Procedure Schedule 
See Table 1 in Appendix I for a complete visit and procedure schedule. 
9.5.1.2. 
Demographics and Baseline Characteristics 
9.5.1.2.1. Demographics and Disease Characteristics 
Demographic characteristics such as age (years), sex, race, and ethnicity and the normality of nerve 
fiber layer thickness will be collected at Visit 1. Baseline diurnal intraocular pressure (IOP) will 
be collected at Visit 2. 
9.5.1.2.2. Medical and Surgical History 
Ocular and non-ocular medical history will be collected at Visits 1 and 2. 


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9.5.1.2.3. Prior and Concomitant Medications 
All medications that the subject has taken 2 months prior to Visit 1 and/or is taking through Visit 
6 (or discontinuation from the study) will be recorded in the subject chart and the eCRF. The 
generic name of the drug, dose, route of administration, duration of treatment (including start and 
stop dates), frequency, indication and whether or not the medication was taken due to an AE will 
be recorded for each medication. 
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the case report form are 
concomitant prescription medications, over-the-counter medications and supplements. 
9.5.1.2.4. Prohibited and Rescue Medications 
Table 2 of the protocol lists medications and procedures that are exclusionary for the study and/or 
are prohibited during study participation. 
Any subject who has an elevated IOP that the PI considers unsafe for the subject may be rescued 
and placed on appropriate alternate therapy. The choice of rescue medication is at the 
Investigator’s discretion. Rescue medication should be entered into the eCRF with annotation 
indicating that the medication was used for rescue.  
Rescued subjects will be considered as treatment failures, and the need for rescue therapy will not 
be considered an adverse event (AE). Rescued subjects will stop the investigational product and 
should not be withdrawn from the study.  
The following guidance is provided to Investigators for rescue and the decision to rescue is at the 
discretion of the investigator’s judgement:  
• Ocular hypotensive medication (other than IP) is prohibited during study participation 
unless the subject is rescued.  
• If the subject’s IOP is > 36 mmHg, the Investigator may recheck the IOP within 3 day of 
the visit and rescue medication may be considered if the IOP remains elevated.  
• Rescued subjects should discontinue the investigational product.  
• Rescued subjects should continue in the study for safety follow-up after receiving the 
rescue medication.  
9.5.1.3. 
Efficacy Assessments 
9.5.1.3.1. Primary Efficacy Assessment(s) 
The study has a single primary assessment that compares the mean IOP in the study eye at each 
diurnal time point (8AM, 10AM, and 4PM) at Visits 4, 5, and 6 for two doses of VVN539 
compared to vehicle. 
9.5.1.3.2. Secondary Efficacy Assessments 
Secondary efficacy assessments will examine: 


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• Change from baseline (Visit 2) in IOP at each diurnal time point to Visits 4, 5, and 6; 
• Change from baseline (Visit 2) in diurnal IOP to Visits 4, 5, and 6. Diurnal IOP is the 
average of 8AM, 10AM, and 4PM measurements at a given visit. 
9.5.1.3.3. Description of Efficacy Assessments 
IOP measurements will be performed utilizing a Goldmann applanation tonometer affixed to a slit 
lamp according to the Investigator’s standard procedure (two-person reading method). 
Measurements will be taken with the subject seated. All pressure will be recorded in mmHg. Two 
consecutive measurements are taken in each eye and recorded as the mean of the two 
measurements. If the two measurements differ by >1.0 mmHg, a third measurement is performed, 
and the median is recorded. 
At Visit 1, IOP will be measured once during screening visit. 
At Visit 3, IOP will be measured once at 8:00AM (±30 minutes) prior to the first q.d. AM dosing. 
At Visits 2, 4, 5, and 6 diurnal IOP will be measured: 
• At 8:00AM (±30 minutes), 10:00AM (±30 minutes) and 4:00PM (±30 minutes) 
• At Visits 4 and 6, the 8:00AM measurement will be taken prior to the AM dosing. 
9.5.1.4. 
Safety Assessments 
The safety of VVN539 ophthalmic solution will be evaluated using the following assessments. 
1. Adverse event (AE) monitoring 
2. Best corrected visual acuity (BCVA) 
3. Slit-lamp biomicroscopy 
4. Central corneal thickness 
5. Dilated ophthalmoscopy 
6. Heart rate and blood pressure 
7. Clinical laboratory tests (blood chemistry, hematology) 
9.5.1.4.1. Adverse Events 
AEs will be monitored throughout the study. Subjects will be encouraged to report any adverse 
findings during the study whether or not they are related to IP. These can be collected either in an 
unsolicited fashion without any prompting or in response to a general question such as: “Have you 
noticed anything different since you started the study; began the IP, etc?” 
All AEs will be captured on the appropriate eCRF. Information to be collected at minimum 
includes event description, onset, assessment of severity, relationship to IP, and outcome. 
The Investigator will record all AEs with start dates occurring any time after informed consent is 
obtained until 7 (for nonserious AEs) or 30 days (for SAEs) after the last day of study participation. 
At each study visit, the Investigator will inquire about the occurrence of AE/SAEs since the last 
visit. Events will be followed for outcome information until resolution or stabilization. 


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Any medical condition that is present at the time that the subject is screened will be considered as 
baseline and not reported as an AE. However, if the study subject’s condition deteriorates at any 
time during the study, it will be recorded as an AE. 
An AE is any untoward medical occurrence in a patient or clinical study subject, temporally 
associated with the use of study intervention, whether or not considered related to the study 
intervention. An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use 
of study intervention. 
An AE or suspected adverse reaction is considered “serious” if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death, 
• A life-threatening AE, 
• Inpatient hospitalization or prolongation of existing hospitalization, 
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or 
• A congenital anomaly or birth defect. 
Important medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may jeopardize 
the subject and may require medical or surgical intervention to prevent one of the outcomes listed 
in this definition. 
The severity of all AEs will be assessed by the Investigator and graded as follows: 
• Mild: requires minimal or no treatment and do not interfere with the subject’s daily 
activities; 
• Moderate: results in a low level of inconvenience or concern and may cause some 
interference with functioning; 
• Severe: interrupts a subject’s usual daily activity and may require systemic drug therapy 
or other treatment. Severe events are usually potentially life-threatening or incapacitating. 
The term “severe” does not necessarily equate to “serious”. 
All AEs must have their relationship to study intervention assessed by the Investigator who 
examines and evaluates the subject based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical 
study, the IP must always be suspect. 
• Unrelated: no reasonable possibility that the administration of the IP caused the event, no 
temporal relationship between the IP and event onset, or an alternate etiology has been 
established. 


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• Related: is known to occur with the IP, is a reasonable possibility that the IP caused the 
AE, or there is a temporal relationship between the IP and event. Reasonable possibility 
means that there is evidence to suggest a causal relationship between the IP and the AE. 
9.5.1.4.2. Best Corrected Visual Acuity (BCVA) 
The BCVA will be measured on both eyes in LogMAR at every study visit using Early Treatment 
Diabetic Retinopathy Study (ETDRS) charts. LogMAR is calculated as Baseline value + (n × 
0.02); where the baseline value is the LogMAR number of the last line read (at least one letter read 
correctly in this line), “n” is the total number of letters missed up to and including the last line 
read, and “0.02” is the value for each letter. 
9.5.1.4.3. Slit-Lamp Biomicroscopy 
The slit-lamp biomicroscopy exam will be performed to examine eye structures for each eye at 
each study visit. 
This procedure will be performed in the same manner for all subjects observed at the Investigator’s 
site. The site will record all ABNORMAL findings in the source document and the Investigator 
will evaluate the ABNORMAL findings as Not Clinically Significant (NCS) or Clinically 
Significant (CS). CS and NCS ABNORMAL findings will be recorded in the source 
documentation. However, only ABNORMAL CS descriptions from Visits 3-6 will be captured in 
the eCRF as AE. 
Findings for the anterior chamber (cells and flare), eyelid (hyperemia and edema), conjunctiva 
(hyperemia, edema (chemosis), discharge/exudate), cornea (edema), iris color, pupil, lashes, and 
lens will be collected. 
9.5.1.4.4. Central Corneal Thickness 
Corneal thickness evaluation (in microns) will be performed using a calibrated ultrasonic or optical 
pachymeter, provided that the same method is used for all measurements. Measurements will be 
taken in the central cornea after instilling topical anesthetic. A total of three readings will be 
obtained for each eye. Care should be taken to place the probe perpendicular to the corneal surface 
and to not a compress the cornea. 
Pachymetry is performed at Visit 1 and Visit 6. 
• At Visit 1, pachymetry within 3 months of the visit is acceptable. 
• At Visit 6, Pachymetry is to be performed after the last IOP measurement. 
9.5.1.4.5. Dilated Ophthalmoscopy 
Dilated ophthalmoscopy will be performed at Visit 1 and Visit 6 and will include assessment of 
the retina for any abnormal findings and optic nerve head for pallor and cupping (cup to disc ratio). 
The status of the vitreous (including haze), retina (including vessels), macula, optic nerve will be 
determined as Normal or Abnormal. The cup-to-disc ratio will be recorded with two decimal points 
(e.g., 0.80). 


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9.5.1.4.6. Heart Rate and Blood Pressure 
Heart rate and blood pressure will be measured at each study visit. 
Heart rate will be measured after the subject has been seated for at least 5 minutes. The pulse rate 
will be measured over a 30-second period by palpitation at the wrist, and multiplied by 2. An 
electronic device may be used instead. The method of measurement (manual or electronic) must 
be documented in the subject records. Repeat measurements should be taken for out-of-range 
values. Confirmed heart rate readings outside the normal range must be evaluated by the 
Investigator for clinical significance. 
Blood pressure will be measured after heart rate (while the subject is in a resting state). A 
sphygomomanometer with appropriate size cuff with a stethoscope will be used. A digital device 
may be used instead. All measurements will be obtained from the same arm using the same cuff 
size for all study visits. Repeat measurements should be taken for significantly high or low 
measurements. Confirmed BP readings outside the normal range must be evaluated by the 
Investigator for clinical significance. 
9.5.1.4.7. Clinical Laboratory Tests (Blood Chemistry, Hematology) 
Approximately 10 mL of blood will be obtained at Visit 1 and Visit 6. Fasting will not be required, 
therefore specific values may be out of the typical fasting range. 
Abnormal laboratory values noted as clinically significant at Visit 6 that are changes from the 
Screening values are documented as AEs and is to be followed-up as appropriate. 
The following serum chemistry and hematology parameters will be measured: 
Chemistry 
Hematology 
Alanine aminotransferase (ALT) 
Absolute/percent basophil count 
Albumin 
Absolute/percent eosinophil count 
Alkaline phosphatase (ALP) 
Absolute/percent lymphocyte count 
Aspartate aminotransferase (AST) 
Absolute/percent monocyte count 
Bilirubin (total, direct, indirect) 
Absolute/percent neutrophil count 
BUN 
Differential WBC 
Calcium 
Hematocrit (Hct) 
Cholesterol (total, High-Density  
Hemoglobin (Hgb) 
Lipoprotein (HDL) and Low-Density  
Mean corpuscular hemoglobin (MCH) 
Lipoprotein (LDL)) 
Mean corpuscular hemoglobin conc. (MCHC) 
Carbon dioxide 
Mean corpuscular volume (MCV) 
Chloride 
Platelet count 
Creatinine, serum 
Red blood cells (RBC) 
Glucose 
White blood cells (WBC) 
Lactate dehydrogenase (LDH) 
 
Potassium 
 


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Sodium 
 
Total protein 
 
Triglycerides 
 
9.5.2. 
Appropriateness of Measurements 
All assessments used in this study are widely used and generally recognized as reliable, accurate, 
and relevant. 
9.5.3. 
Primary Efficacy Variable(s) 
The primary efficacy endpoint for this study is to compare mean IOP at each diurnal time point 
(8AM, 10AM, and 4PM) at Visit 4 (Day 7), Visit 5 (Day 14), and Visit 6 (Day 21) for two doses 
of VVN539 compared to vehicle using data from the study eye. 
To claim superiority, the two-sided 95% confidence interval estimates of the treatment differences 
in mean IOP between VVN539 and vehicle must exclude zero for all 9 time points. 
9.5.4. 
Drug Concentration Measurements 
No drug concentration measurements will be made for this study. 


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9.6. 
DATA QUALITY ASSURANCE 
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical 
trial is conducted and data are generated and biological specimens are collected, documented 
(recorded), and reported in compliance with the protocol, International Council for Harmonisation 
(ICH) Good Clinical Practice (GCP), and applicable regulatory requirements (e.g., Good 
Laboratory Practice [GLP], Good Manufacturing Practices [GMP]). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the Sponsor, and inspection by local and 
regulatory authorities. 
9.6.1. 
Training and Education of Investigators and Study Site Personnel 
 
An Investigator Meeting will be held before study initiation and attended by the Investigators 
and/or Sub-Investigators from each study site, study coordinators from each study site, if possible, 
and personnel from the sponsor and the Contract Research Organization (CRO), Lexitas Pharma 
Services, Inc. (hereafter referred to as Lexitas). The purpose of this meeting is to ensure the 
Investigators and study coordinators are trained and instructed on the proper conduct of the clinical 
trial and ensure that all participants are aware of their obligations set out by the protocol, ICH 
guidelines, GCP guidelines, and other applicable regulatory requirements. 
9.6.2. 
Monitoring of Study Sites 
Lexitas Pharma Services, Inc. will conduct the clinical monitoring for this study. A clinical 
Monitoring Plan is to be used, which will describe in detail who will conduct the monitoring, at 
what frequency monitoring will be done, at what level of detail monitoring will be performed and 
the distribution of monitoring reports. 
9.6.3. 
Data Entry and Verification of Database Used for Analysis and 
Reporting 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the 
site’s Investigator. The Investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. 
All source documents should be completed in a neat, legible manner to insure accurate 
interpretation of data. Data recorded in the eCRF derived from source documents should be 
consistent with the data recorded on the source documents. 
Clinical data (including AEs, concomitant medications, and expected adverse reactions data) will 
be entered into IBM Clinical Development, a 21 CFR Part 11-compliant data capture system 
provided by Lexitas Pharma Services, Inc. The data system includes password protection and 


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internal quality checks, such as automatic range checks, to identify data that appear inconsistent, 
incomplete, or inaccurate. Clinical data will be entered directly from the source documents. 
9.6.4. 
Clinical Study Report 
The final clinical study report will be reviewed, audited, and approved by medical, clinical, 
statistical, and regulatory staff from the sponsor, and key study contributors from Lexitas. 
9.6.5. 
Inter-Laboratory Standardization Methods 
Not applicable. A single, qualified, and certified central clinical laboratory will be used to analyze 
and report clinical laboratory data. 


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9.7. 
STATISTICAL METHODS PLANNED IN THE PROTOCOL AND 
DETERMINATION OF SAMPLE SIZE 
This section of the analysis plan describes the analyses explicitly mentioned in the protocol as well 
as additional analyses not explicitly mentioned in the protocol but planned prior to breaking the 
treatment mask. Section 9.8 describes any changes to analyses that were explicitly mentioned in 
the protocol or statistical analysis plan. 
9.7.1. 
Statistical and Analytical Plans 
General Conventions 
Summary statistics for the data collected during this study will be presented to give a general 
description of the patients studied. Data from all sites will be combined in the computation of these 
descriptive summaries. Categorical variables will be summarized by the frequency and percentage 
of patients in each category. Continuous variables will be summarized using N, mean, standard 
deviation (SD), median, minimum, and maximum values. 
Number of subjects, minimums, and maximums will be calculated to the same number of decimal 
places as the source data. Means, medians, standard deviations, standard errors, and quartiles will 
be calculated to one more decimal place than the source data. Percentages will be calculated to the 
nearest one decimal place and will use the number of non-missing responses as the denominator, 
unless otherwise noted. Zero count cells will be displayed as “0” with percentage of (0%). Unless 
otherwise noted, summaries will be performed by the treatment group and presented in the order 
of: VVN539, 0.04%; VVN539, 0.02%; Vehicle. 
Statistical tests will be presented as two-sided p-values rounded to four decimal places; p-values 
less than 0.0001 will be presented as '<0.0001' and p-values = 1 will be presented as '>0.9999' in 
all tables. Unless otherwise indicated, statistical testing will be carried out at the α = 0.05 
significance level. 
Baseline values will be defined as the last scheduled or unscheduled measurement prior through 
Visit 2. Ocular measurements will use the most recent measurement for each eye. 
Numeric laboratory data may be recorded at limits of detection (with a ‘<’ or ‘>’ sign, i.e. < 0.1 or 
> 0.1). To summarize the data, the original value will be converted to one unit less or more at the 
level of measured precision (e.g. 0.4 in the case of < 0.5 and to 0.6 in the case of > 0.5). The actual 
values will be presented in the data listings. 
All data collected in this study will be presented in individual patient data listings for all patients. 
Computations for all results will be performed using SAS (Version 9.4, SAS/STAT 15.2) computer 
software package (SAS Institute, Inc, 2013, 2020), unless otherwise specified. 
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For analysis of efficacy endpoints, analysis of covariance (ANCOVA) or longitudinal models will 
model the observed value or change from baseline of each endpoint as the dependent variable with 
the baseline measurement of the corresponding endpoint as a covariate and treatment group as a 
main effect. Longitudinal models will include a covariate for visit as well as an interaction term 
for visit and treatment. 
Subgroups 
Not applicable. 
Multiplicity 
Time-matched longitudinal models adjusting for baseline and including a random effect for center 
will be used to test time-specific superiority comparisons as well as the overall superiority 
hypothesis using 95% confidence intervals. To claim study success for the primary endpoint based 
on the model, the two-sided 95% confidence interval estimates of the treatment differences in 
mean IOP between VVN539 and vehicle must exclude zero for all 9 time points (8AM, 10AM, 
and 4PM at Visits 4, 5, and 6). No adjustment to the individual confidence intervals is required 
since all criteria need to be met to claim superiority. 
Comparisons will be made first between VVN539, 0.04% versus vehicle. If the treatment 
differences for all 9 time points are statistically significant, testing will proceed to VVN539, 0.02% 
versus vehicle. 
Missing Data and Outliers 
Every attempt will be made to capture all study data. The primary analysis will be analyzed using 
observed data assuming missing at random (MAR) using a longitudinal model (mixed model 
repeated measures, MMRM). Sensitivity analyses of the primary endpoint will be performed 
utilizing time-matched last observation carried forward (LOCF) of the last available IOP 
measurement. 
Safety analyses will use observed data only. 
Visit Windows 
The nominal visits listed in the CRF will be used in the summaries. In general, unscheduled visits 
will not be summarized in tables unless otherwise noted. 
Missing Dates 
Missing dates that occur for prior or concomitant medications or adverse events will be queried 
for a date. If no date is obtained, the following imputation rules will apply: 
• For start dates, if the given year (or year-month) is the same as study drug administration, 
the start date will be imputed as study drug administration date; otherwise, missing month-
day (or day) will be imputed as ’01-01’ (or ’01’). 


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• For stop dates, missing months will be imputed as ‘12’ and missing days will be imputed 
as the last day of the month. If this creates a date after discontinuation/completion, the date 
of discontinuation/completion will be used. 
Imputed dates will only be used to classify events or medications, such as occurring before or after 
the start of treatment. Imputed dates will only be used in tables. Listings will display the collected 
date data. 
Interim Analysis 
There is no unmasked efficacy or futility interim analysis for this study. 
9.7.1.1. 
Analysis Populations 
9.7.1.1.1. Populations 
The Full Analysis Set (FAS) consists of all subjects who were randomized. Subjects will be 
analyzed in the group to which they were randomized. This set will be used for the analysis of all 
efficacy endpoints as the primary analysis. 
The Per Protocol (PP) Analysis Set will include all subjects who completed study-required 
treatment and who followed the protocol without significant deviations. The determination of 
significant protocol violations, including the use of prohibited medications, will be made prior to 
locking the final database and unmasking. Subjects will be analyzed in the group to which they 
were treated. 
The Safety Analysis Set will include all subjects who took at least one dose of investigational 
product as indicated on the dosing record. Subjects will be analyzed in the group according to the 
treatment received. All safety variables will be analyzed using the Safety Analysis Set and only 
observed data will be included (i.e., missing data will remain missing for the safety analysis). 
A listing of subjects excluded from the analysis sets will be provided. 
9.7.1.1.2. Analysis Eyes 
The study eye is defined as meeting all inclusion criteria and no exclusion criteria, and with the 
higher IOP at 8:00AM at Visit 2. Should both eyes have the same IOP and meet all inclusion 
criteria and no exclusion criteria, then the right eye will be the study eye. 
Efficacy analyses will focus on the study eye only, though supportive analyses will also be 
presented by the non-study eye, irrespective of qualification. Ophthalmic safety analyses will be 
presented by the study eye and non-study eye. 
9.7.1.2. 
Analysis of Subject Disposition 
The number of subjects randomized at each site will be summarized by treatment group and 
overall. 


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Subjects’ enrollment and disposition during the study will be summarized by treatment group and 
overall based on Randomized Subjects. The reasons for discontinuation will be displayed in the 
order as they appear on the eCRF. 
Summary tables will include the following. The percentages will be calculated based on the 
number of subjects randomized (with reasons for discontinuation based on the number of subjects 
discontinued). 
• Number of patients screened; 
• Number and percentage of subjects randomized; 
• Number and percentage of subjects randomized with any protocol deviation; 
• Number and percentage of subjects treated; 
• Number and percentage of subjects treated with any protocol deviation; 
• Number and percentage of subjects treated who completed the study through Visit 6 (Day 
21); 
• Number and percentage of subjects treated who discontinued from the study; 
• The reasons for study discontinuation; 
• Number and percentage of subjects withdrawn from study drug due to elevated IOP and 
subsequent rescue treatment; 
• Number and percentage of subjects attending each visit. 
A listing of subjects who do not meet all inclusion criteria or meet exclusion criteria will be 
provided. A table of major protocol deviations will be presented using the FAS. 
9.7.1.3. 
Analysis of Demographic and Baseline Characteristics 
9.7.1.3.1. Demographics and Disease Characteristics 
Demographic and baseline disease characteristics including age (years), age group (18-<45, ≥45), 
sex, race, ethnicity, iris color, study eye (OD/OS), normality of nerve fiber layer thickness, 
baseline diurnal IOP of the study eye, and baseline IOP at 8:00 AM, 10:00 AM, and 4:00 PM will 
be summarized descriptively by treatment group and overall using the FAS, Per Protocol, and 
Safety Analysis Sets. For categorical parameters, the percentages will be calculated overall and 
based on the number of subjects in each treatment group based on non-missing observations. 
Ocular baseline characteristics for central corneal thickness (average of three measurements), 
gonioscopy, and visual field will be summarized by study eye and non-study eye. 
9.7.1.3.2. Medical History 
Medical history will be coded using the Medical Dictionary for Regulatory Activities (MedDRA 
Version 24.1, Mozzicato, 2009). The frequency and percentage of subjects with any medical 
history will be summarized by treatment group using the FAS. System organ class (SOC) will be 
sorted alphabetically and preferred term (PT) within each SOC will be sorted by overall 
descending order of frequency according to the following rules in order: 


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1. Descending frequency within VVN539, 0.04%; 
2. Descending frequency within VVN539, 0.02%; 
3. Descending frequency within Vehicle; 
4. PT in alphabetical order. 
The medical history will include both the ocular and the general (non-ocular) history. Ocular 
medical history and general (non-ocular) medical history will be summarized separately. Ocular 
and non-ocular medical histories are identified according to which CRF the event is recorded. 
Ocular medical history will be summarized separately for study eye, non-study eye, and either eye. 
9.7.1.3.3. Prior and Concomitant Medications 
Prior and concomitant medications will be coded using the World Health Organization Drug 
Dictionary (WHODrug) (Version 2021-09, Lagerlund et al., 2020) for anatomical therapeutic 
chemical (ATC) classification and preferred drug name. 
The frequency and percentage of subjects with coded medications will be summarized by treatment 
group using the Safety Analysis Set. A subject who used multiple medications will be counted 
only once for each ATC and preferred drug name. Therapeutic Subgroup is sorted alphabetically, 
and preferred term is sorted by descending frequency overall within each Level 3 term according 
to: 
1. Descending frequency within VVN539, 0.04%; 
2. Descending frequency within VVN539, 0.02%; 
3. Descending frequency within Vehicle; 
4. PT in alphabetical order. 
Prior and concomitant medications will be summarized separately. Ocular medications are defined 
as those medications for which an eye has been specified (OD, OS, or OU). Ocular and non-ocular 
medications will be summarized separately. Ocular medications will be summarized separately for 
study eye, non-study eye, and either eye. 
Prior medications are defined as any medications that started and stopped prior to the date of first 
dose of double-masked study drug. Concomitant medications are defined as any medications that 
(1) start prior to the date of first dose of double-masked study drug and stop or are ongoing at or 
after the date of first dose of double-masked study drug; or (2) start at or after the date of first dose 
of double-masked study drug. Medications started after Visit 6 or withdrawal from the study are 
not considered concomitant. 
9.7.1.4. 
Analysis of Study Medication Adherence and Exposure 
Treatment adherence and exposure will be assessed by the data from the Study Drug 
Administration and Treatment Adherence eCRFs and will be summarized by treatment group 
using the Safety Analysis Set. 


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Treatment adherence was assessed by 3 questions: Did subject return (clinic staff), and two subject 
reported measures: Did subject miss any doses, and if so, how many doses missed.  Descriptive 
statistics will be presented for these measures by visit by treatment group. 
Duration of exposure of double-masked study drug will be calculated as the total number of days 
from the first double-masked dose date to the last double-masked dose date plus 1 (one) regardless 
of temporary dose interruptions. Other dosing durations are calculated as follows: 
• For q.d. morning dosing, duration of exposure is calculated as Visit 4 date - Visit 3 date + 
1. If the date of last dose is between Visit 3 and Visit 4 date (inclusive), the Visit 4 date is 
replaced with the date of last dose. 
• For q.d. evening dosing, duration of exposure will be calculated as Visit 5 date - Visit 4 
date. If the date of last dose is between Visit 4 + 1 date and Visit 5 date (inclusive), the 
Visit 5 date is replaced with the date of last dose. 
• For b.i.d. dosing, duration of exposure will be calculated as Visit 6 - Visit 5 date. If the 
date of last dose is between Visit 5 + 1 date and Visit 6 date (inclusive), the Visit 6 date is 
replaced with the date of last dose. 
9.7.1.5. 
Analysis of Efficacy 
Summary descriptive statistics will be presented for all study visits at which efficacy data are 
collected. Efficacy analyses will be presented for the study eye with supportive analyses presented 
for the non-study eye. 
In general, efficacy analyses will be performed using the FAS and the observed data at each Visit 
for the study eye. Only data prior to rescue therapy will be summarized and imputation strategies 
for missing data will only consider data prior to rescue therapy. The primary and secondary 
endpoint analysis will also be performed using the Per Protocol Analysis Set. 
For subjects who discontinue the study prior to the Visit 6, data collected using Early Termination 
eCRFs will not be summarized separately unless the termination occurred on a scheduled visit 
(within the visit windows of a particular visit). 
ANCOVA and longitudinal models for continuous outcomes will be fit using the MIXED 
procedure. Categorical analyses will be conducted using the FREQ procedure. 
9.7.1.5.1. Primary Efficacy Analysis 
Primary Estimands: The primary estimands are the treatment differences between VVN539 and 
vehicle at 8:00 AM, 10:00 AM, and 4:00 PM at each of Days 7, 14, and 21 in the study eye using 
the FAS. The primary estimands treat subjects as if they do not receive rescue therapy. 
Target Population: Subjects with open-angle glaucoma or ocular hypertension that meet the study 
entry criteria. 
Endpoint: Observed IOP measurements at each time point in the study eye. 


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Treatment Condition(s): Treatment condition is based on the randomized treatment group. 
Population-level Summaries: The difference in the mean IOP at the nine time points of interest 
and their corresponding p-values and 95% confidence intervals. 
Intercurrent Events and Strategies to Address Intercurrent Events 
• Discontinuation of study therapy with continued participation in the study without receipt 
of rescue therapy 
o Treatment Policy – no imputation; use observed data 
• Receipt of rescue therapy in the study eye 
o Hypothetical Approach – data for visits after the receipt of rescue therapy will be 
set to missing and will be analyzed assuming missing at random (MAR) using a 
longitudinal model (mixed model repeated measures, MMRM) 
• Missing data without withdrawal or with withdrawal regardless of reason 
o Hypothetical Approach: visits that are missing data will be analyzed assuming 
MAR using MMRM 
Time-matched longitudinal models adjusting for baseline and including a random effect for center 
will be used to test time-specific superiority comparisons as well as the overall superiority 
hypothesis using 95% confidence intervals. To claim study success for the primary endpoint based 
on the model, the two-sided 95% confidence interval estimates of the treatment differences in 
mean IOP between VVN539 and vehicle must exclude zero for all 9 time points (8AM, 10AM, 
and 4PM at Visits 4, 5, and 6). Comparisons will be made first between VVN539, 0.04% versus 
vehicle. If the treatment differences for all 9 time points are statistically significant, testing will 
proceed to VVN539, 0.02% versus vehicle. 
Data from Days 7, 14, and 21 will be analyzed using MMRM with an unstructured covariance 
assumed for each treatment with treatment, visit, and visit by treatment interaction as a fixed 
effects, baseline measurement as a covariate, and a random effect for site. If the model fails to 
converge using this covariance structure, heterogenous Toeplitz, or compound symmetry will be 
implemented in this order until convergence is reached. 
Sample code for the model run separately for 8AM, 10 AM, and 4 PM: 
PROC MIXED DATA=indata;  
CLASS subject site visit treatment;  
MODEL IOP = baseline visit treatment visit*treatment / solution DDFM=KR;  
REPEATED visit / SUBJECT=subject TYPE=UN group = treatment;  
RANDOM site;  
LSMEANS visit*treatment / slice = visit CL diff;  
RUN;  
Sensitivity analyses include a time-matched last observation carried forward (LOCF) of the last 
available IOP measurement as well as analysis of covariance (ANCOVA) analyses at each visit 
with baseline measurement as a covariate. Percent of baseline and percent change from baseline 


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will also be performed as a sensitivity of the primary analysis. Additional sensitivity analyses will 
be performed on the PP Analysis Set as necessary. 
9.7.1.5.2. Secondary Efficacy Analyses 
Analyses of change from baseline in IOP at each time point and of observed and change from 
baseline in diurnal IOP will be performed in a manner similar to the primary efficacy analysis. 
Percent of baseline and percent change from baseline of diurnal IOP will also be performed. 
9.7.1.6. 
Analysis of Safety 
Safety will be evaluated by AEs, BCVA, slit-lamp biomicroscopy, central corneal thickness, 
dilated ophthalmoscopy, heart rate and blood pressure, and clinical laboratory tests (blood 
chemistry, hematology). 
The Safety Analysis Set will be used for all safety analyses. All data, including data after rescue, 
will be summarized as observed and no data imputation will be performed. No statistical treatment 
group comparisons will be performed, unless otherwise specified. Analyses will be presented by 
study eye and non-study eye, if applicable. 
For by visit summaries, data collected using Early Termination eCRFs will not be summarized 
unless the termination occurred within the visit windows of a particular visit. 
All visits will be included in listings. 
9.7.1.6.1. Adverse Events 
AEs are coded using MedDRA Version 24.1. Treatment-emergent adverse events (TEAE) are 
defined as events that start on or after the date of first dose of double-masked study drug up to and 
including the last dose of double-masked study medication. Ocular AEs are defined as those events 
for which an eye has been specified (OD, OS, or OU). 
Ocular and non-ocular AEs will be summarized separately. Ocular AEs will be presented by study 
eye and non-study eye and overall (OU) if an event occurs in either eye. 
In all summaries of AEs, percentages are calculated based on the number of subjects in each 
treatment group of the Safety Analysis Set. 
Overall summaries of AEs by treatment will include: 
• the number of AEs and SAEs reported; 
• the number and percentage of subjects who experienced any AE; 
• the number and percentage of subjects who experienced any serious adverse event (SAE) 
and the reason for seriousness; 
• the number and percentage of subjects with any AE by worst severity and worst 
relationship. 
The overall summaries of TEAEs will also include: 


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• the number and percentage of subjects with any TEAEs leading to discontinuation of 
double-masked study drug; 
• the number and percentage of subjects with any TEAEs leading to study termination. 
Summaries of the frequency and percentage of subjects with AEs by SOC and preferred term by 
treatment group will include: 
• All AEs by SOC and preferred term; 
• All AEs by SOC, preferred term, and maximum severity; 
• All AEs by SOC, preferred term, and maximum relationship. 
System organ class (SOC) will be sorted alphabetically, and preferred term (PT) within each SOC 
will be sorted by overall descending order of frequency according to the following rules in order: 
1. Descending frequency within VVN539, 0.04% 
2. Descending frequency within VVN539, 0.02% 
3. Descending frequency within Vehicle; 
4. PT in alphabetical order. 
Subjects are counted only once for each SOC and PT. In summaries of maximum severity and 
maximum relationship, subjects with multiple occurrences of events will only be counted once at 
the maximum severity/relationship per SOC and PT. 
Any treatment-emergent AEs that have a missing severity will be presented as severe in the 
summary table but will be presented with a missing severity in the data listing. Any treatment-
emergent AEs that have a missing relationship will be presented as “Related” in the summary table 
but will be presented with a missing relationship in the data listing. 
All AEs are displayed in listings. In addition, separate listings will be provided for: 
• Subjects with any treatment-emergent adverse event leading to study drug discontinuation 
or study termination; 
• Subjects with any serious adverse event (treatment-emergent or otherwise); 
• Subject deaths. 
9.7.1.6.2. Best Corrected Visual Acuity (BCVA) 
Descriptive summaries of the observed values of logMAR at each scheduled visit as well as the 
change from baseline at each post-baseline visit will be presented for both eyes. A categorical 
analysis of subjects who lose 0.3 lines ETDRS in either eye at each visit or final visit will be 
conducted.  
9.7.1.6.3. Slit-Lamp Biomicroscopy 
The frequency and percentage of subjects with observed values of each category as well as the 
categorical shift from baseline at each post-baseline visit will be tabulated at each scheduled visit 
for both eyes for the following measures of interest: anterior chamber cell, anterior chamber flare, 


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and conjunctive hyperemia.  The percentages are calculated based on the number of subjects at 
each visit in each treatment group of the Safety Analysis Set. 
9.7.1.6.4. Dilated Ophthalmoscopy 
The frequency and percentage of subjects with observed values of each categorical response at 
Visits 1 and 6 as well as the categorical shift from baseline at Visit 6 will be tabulated for both 
eyes. The percentages are calculated based on the number of subjects at each visit in each treatment 
group of the Safety Analysis Set.  
Descriptive summaries of the observed cup-to-disc ratio at Visits 1 and 6 as well as the change 
from baseline at Visit 6 will be presented for both eyes. 
9.7.1.6.5. Heart Rate and Blood Pressure 
Descriptive summaries of the observed values for heart rate and blood pressure at each scheduled 
visit as well as the change from baseline at each post-baseline visit will be presented.  
Repeat measurements of cardiovascular outcomes will be taken for out-of-range measurements. 
The most recent (latest measurement) at each visit will be summarized in tables. 
9.7.1.6.6. Clinical Laboratory Tests (Blood Chemistry, Hematology) 
Descriptive summaries of the observed test results at Visits 1 and 6 as well as the change from 
baseline at Visit 6 will be presented for blood chemistry and hematology labs. The frequency and 
percentage of subjects with observed values of Low, Normal, High at baseline and Visit 6 will be 
tabulated. The percentages are calculated based on the number of subjects at each visit in each 
treatment group of the Safety Analysis Set. 
It is expected that all samples will be analyzed by a central laboratory. If a local laboratory is used 
(such as for an unscheduled visit), the results will be included in the listings of laboratory data, but 
will not be included in descriptive summaries. 
The results of pregnancy tests for women of childbearing potential will be presented in a listing. 
9.7.2. 
Determination of Sample Size 
With a sample size in each group of 20, the study will have 80% power to detect a difference of 
3.0 mmHg between an VVN539 dose compared to vehicle at each diurnal time point assuming a 
common standard deviation of 3.3 mmHg, α = 0.05 (two-tailed). 
There is no correction for multiplicity for multiple time points or comparisons for high or low 
VVN539 doses. 


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9.8. 
CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED 
ANALYSES 
9.8.1. 
Protocol Amendments 
Amendment 1 of protocol made the following notable changes: 
• Revised exclusion criteria to minimize the risk of study subjects randomized to vehicle 
for potential glaucomatous loss; 
• Allowed investigators to switch subjects who were previously treated with an ocular 
hypotensive medication with a 4 week washout period to a ocular hypotensive medication 
with a shorter washout period provided the appropriate washout is met prior to Visit 2. 
9.8.2. 
Changes from Protocol-Specified Analyses 
Version 2 of the statistical analysis plan clarifies the inconsistent presentation of analyses with 
regards to rescue and prohibited medications. This version clarifies that data prior to rescue 
treatment will be used for efficacy analyses. Minor corrections to sample proc mixed code were 
made as well. 
 
 
 
 
 


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REFERENCES 
1. Mozzicato P. (2009). MedDRA: An overview of the medical dictionary for regulatory 
activities. Pharmaceutical Medicine 23: 65-75. 
2. Lagerlund O, Strese S, Fladvad M & Lindquist M. (2020). WHODrug: A global, 
validated and updated dictionary for medicinal information. Therapeutic Innovation & 
Regulatory Science 54: 1116–1122. 
3. SAS Institute Inc. What’s New in Base SAS® 9.4 and SAS® Viya®. (2013). SAS 
Institute Inc., Cary, NC, USA.  
4. SAS Institute Inc. (2020). SAS/STAT User’s Guide. SAS Institute Inc., Cary, NC, USA.  
 


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APPENDIX I: SCHEDULE OF ASSESSMENTS 
 
Table 1: Schedule of Assessments 
Study Period 
Screening 
Washout1 
Baseline 
Treatment Period 
End of 
Treatment 
Visit No. 
1 
 
2 
3 
4 
5 
6 
Visit Window 
Day-35 to -1 
Day 0 
Day 1 
Day 7 (+2) 
Day 14 (+2) 
Day 21 (+2) 
Informed consent2  
X 
 
 
 
 
 
 
Ocular and medical history 
X 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
BCVA 
X 
X 
X 
X 
X 
X 
Biomicroscopy3 
X 
X 
X 
X 
X 
X 
IOP 
X 
 
 
X4, 
 
 
 
Diurnal IOP5 
 
 
X 
 
X 
X 
X 
Cup-to-disc ratio 
X 
 
 
 
 
 
X 
Dilated ophthalmoscopy 
X 
 
 
 
 
 
X6 
Pachymetry7 
X 
 
 
 
 
X 
Gonioscopy8 
X 
 
 
 
 
 
 
1 Washout period: Prostaglandins, β-adrenoceptor antagonists, kinase inhibitors (4 weeks), adrenergic agonists (2 weeks), muscarinic agonists and carbonic anhydrase inhibitors (5 
days) 
2 Informed consent will be obtained prior to the conduct of any study specified or associated procedures or evaluations. 
3 Biomicroscopy of anterior segment includes corneal epithelium, bulbar and lower conjunctiva, and lens.  
4 Measured at 8:00AM 
5 Measured at 8:00AM, 10:00AM and 4:00PM 
6 After the last IOP measurement 
7 Central corneal thickness will be measured at entry for IOP interpretation. Pachymetry performed within 3 months of screening is acceptable. Ultrasound and optical pachymetry 
is permissible provided that the same methodology is used at Visits 1 and 6. 
8 Gonioscopy performed within 3 months of Visit 1 is acceptable. 


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Study Period 
Screening 
Washout1 
Baseline 
Treatment Period 
End of 
Treatment 
Visit No. 
1 
 
2 
3 
4 
5 
6 
Visit Window 
Day-35 to -1 
Day 0 
Day 1 
Day 7 (+2) 
Day 14 (+2) 
Day 21 (+2) 
Visual field9 
X 
 
 
 
 
 
Optical Coherence 
Tomography 
X10 
 
 
 
 
 
 
Blood chemistry, hematology 
X 
 
 
 
 
 
X 
BP, HR 
X 
X 
X 
X 
X 
X 
Pregnancy test11 
X 
X 
 
 
 
X 
Randomization 
 
X 
 
 
 
 
Study drug administration in 
clinic 
 
 
 
X12 
X13 
 
X13 
Dispense investigational 
product 
 
 
X 
X 
X 
 
Collect study drug 
 
 
 
X 
X 
X 
Prior/Concomitant medications 
X 
X 
X 
X 
X 
X 
Adverse event assessment 
 
 
X 
X 
X 
X 
Abbreviation key: BCVA=best corrected visual acuity; BP=blood pressure; HR=heart rate; IOP=intraocular pressure 
 
 
9 Visual field within 3 months of Visit 1 is acceptable, provided that it is reliable and meets the requirement for automated threshold visual field (e.g., 30-2 or 24-2 Humphrey). 
Unreliable entry fields must be repeated prior to study drug dispensation. 
10 Optical Coherence Tomography within 3 months of Visit 1 is acceptable. 
11 Women of childbearing potential only 
12 After 8:00AM IOP measurement 
13 After 8:00AM and prior to 10:00AM IOP measurement