Clinical trial NCT04675151
Conditions: Presbyopia
Keywords: Nyxol Presbyopia Pilocarpine
Sponsor: Ocuphire Pharma, Inc.
Start date: 2021-02-15; Completion date: 2021-06-30
Enrollment: 150 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objectives of this study are:

To evaluate the efficacy of Nyxol + Pilocarpine to improve DCNVA in subjects with presbyopia

Detailed description:

Arms: arm 1: 1 drop of Nyxol (Treatment 1) and 1 drop of Pilocarpine (Treatment 2) arm 2: 1 drop of Nyxol (Treatment 1) arm 3: 1 drop of Pilocarpine (Treatment 2) arm 4: 1 drop of Placebo (Treatment 1)
Interventions: intervention 1: Phentolamine Ophthalmic Solution 0.75% intervention 2: Pilocarpine intervention 3: Placebo
Intervention descriptions: intervention 1: 0.75% phentolamine ophthalmic solution (Nyxol), a non-selective alpha-1 and alpha-2 adrenergic antagonist intervention 2: Pilocarpine ophthalmic solution intervention 3: Topical sterile ophthalmic solution

Protocol documents:
Prot_000.pdf: 
 
 
 
CONFIDENTIAL 
 
Clinical Study Phase 2 Protocol 
OPI-NYXP-201 
VEGA-1 
 
Randomized, Placebo-Controlled, Double-Masked Study of the Safety and 
Efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) with Low-Dose 
(0.4%) Pilocarpine Eye Drops in Subjects with Presbyopia 
 
Ocuphire Pharma, Inc. 
37000 Grand River Avenue, Suite 120 
Farmington Hills, MI 48335 
 
 
 
 
 
Version: 
01 
Original: 
November 30, 2020 
Amendment 1: 
 
n/a 
 
 
CONFIDENTIAL 
This document contains Ocuphire Pharma, Inc. (Ocuphire) information that is confidential, a 
trade secret and/or proprietary in nature. It is loaned to you for your confidential use on behalf of 
Ocuphire and is not to be photocopied, disclosed or transmitted to any other person or party who 
is not covered by a Confidential Disclosure Agreement with Ocuphire. As the Principal 
Investigator you are responsible for the safekeeping and return of this document to Ocuphire 
upon request. You will be sent updated information and/or amendments as they become 
available.




Ocuphire Pharma, Inc. 
 
Clinical Protocol 
OPI-NYXP-201 
 
VEGA-1 
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INVESTIGATOR’S AGREEMENT 
 
OPI-NYXP-201 
VEGA-1 
 
Randomized, Placebo-Controlled, Double-Masked Study of the Safety and Efficacy of Nyxol 
(0.75% Phentolamine Ophthalmic Solution) with Low-Dose (0.4%) Pilocarpine Eye Drops in 
Subjects with Presbyopia 
 
Version: 
 
 
01 
Original: 
 
 
November 30, 2020 
 
Investigator Agreement: 
I, the undersigned, have reviewed this protocol and I agree to conduct this protocol in accordance 
with Good Clinical Practice, the ethical principles set forth in the Declaration of Helsinki and 
with the U.S. Code of Federal Regulations governing the protection of human subjects 
(21 CFR 50), Institutional Review Boards (21 CFR 56) and the obligations of clinical 
investigators (21 CFR 312). 
 
Signature: ____________________________ Date: _________________________ 
 
Printed Name: _________________________ 
 
 
 


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PROCEDURES IN CASE OF EMERGENCY 
 
EMERGENCY CONTACT INFORMATION 
Role in Study 
Name 
Contact Information 
Clinical Study 
Leader 
Roselyn Judd 
Director of Clinical Operations  
Oculos Development Services, LLC 
 
Email: 
 
Medical Monitor 
Charles Slonim, MD 
Chief Medical Officer 
Oculos Development Services, LLC 
 
Email: 
 
 
 
 


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ABBREVIATIONS AND TERMS 
Abbreviation 
Full term 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ARP 
All Randomized Population 
BCDVA 
best-corrected distance visual acuity  
BCIVA 
best-corrected intermediate visual acuity 
BP 
blood pressure 
°C 
degrees Celsius 
CCLRU 
Cornea and Contact Lens Research Unit 
CFR 
Code of Federal Regulations 
CI 
confidence interval 
CRA 
clinical research associate 
CRO 
clinical research organization 
DCNVA 
 
DLD 
distance-corrected near visual acuity 
 
dim light vision disturbances (also referred to as night vision disturbances 
or NVD) 
eCRF 
electronic Case Report Form 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
°F 
degrees Fahrenheit 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HR 
heart rate 
IB 
Investigators’ Brochure 
ICH 
International Council for Harmonisation 
IND 
Investigational New Drug  
IOP 
intraocular pressure 
IRB 
Institutional Review Board 
IUD 
intrauterine device 


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LCVA 
low-contrast visual acuity 
LDP 
Low-Dose (0.4%) Pilocarpine Ophthalmic Solution 
LDPE 
low-density polyethylene 
LSM 
least-squares mean 
MedDRA 
Medical Dictionary for Regulatory Activities 
mITT 
Modified Intention-to-Treat 
NVD 
night vision disturbances 
Nyxol 
0.75% Phentolamine Ophthalmic Solution or 1% Phentolamine Mesylate 
Ophthalmic Solution  
Nyxol + LDP 
Nyxol dosed in the evening with LDP dosed during the day 
OD 
oculus dexter (right eye) 
OR 
odds ratio 
OTC 
over-the-counter 
OU 
oculus uterque (both eyes) 
PD 
pupil diameter 
POS 
Phentolamine Ophthalmic Solution 
PP 
Per Protocol 
SAE 
serious adverse event 
SOC 
system organ class 
SNV 
severe night vision disturbances 
SP 
Safety Population 
TEAE 
treatment-emergent adverse event 
US 
United States 
USP 
United States Pharmacopeia 
VA 
visual acuity 
WHODrug 
World Health Organization Drug Dictionary 
 


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1. STUDY SUMMARY 
Study Number 
OPI-NYXP-201 
Clinical Phase 
Phase 2 
Type of Study 
Randomized, placebo-controlled, double-masked study of the 
safety and efficacy of Nyxol® Eye Drops (0.75% Phentolamine 
Ophthalmic Solution [POS]) with Low-Dose (0.4%) Pilocarpine 
Ophthalmic Solution (LDP) in subjects with presbyopia 
Name of Investigational 
Product 
Nyxol Eye Drops – 0.75% POS (Treatment 1) 
0.4% LDP (Treatment 2) 
Duration of Study  
Approximately 5 to 9 days, including Visit 1 (Screening/Baseline), 
Visit 2 (Treatment), and Visit 3 (Safety Follow-Up Call) 
Rationale 
Presbyopia is an age-related condition typically starting at around 
40 years of age. It is caused by the inability of the aging lens to 
dynamically change shape, curvature, and dioptric power in an 
effort to focus images of nearby objects onto the retina. Presbyopia 
has a significantly negative impact on quality of life, interfering 
with reading, use of computers or hand-held devices, and seeing 
the dashboard of a car. There are no current pharmacological 
therapies approved, but there is some evidence that decreasing 
pupil diameter (PD), especially to a size of 1.6 to 2 mm to create a 
“pinhole” effect, can improve visual acuity (VA) by increasing the 
depth of focus. 
The combination of Nyxol (0.75% POS) and 0.4% LDP (Nyxol + 
LDP) could be a potential treatment option for presbyopia. 
Specifically, Nyxol would be dosed once daily at or near bedtime 
and LDP dosed in the morning. Nyxol + LDP dosing can 
potentially reduce PD to ≤ 2.0 mm, which has the potential to 
improve near VA due to a “pinhole” effect. 
Phentolamine is a non-selective alpha-1 and alpha-2 adrenergic 
receptor antagonist that inhibits contraction of the iris dilator 
muscle, resulting in a smaller pupil size. 
Pilocarpine hydrochloride is a cholinergic, parasympathomimetic 
agent which acts through stimulation of muscarinic receptors on 
smooth muscles such as the iris and secretory glands. Pilocarpine 
reduces pupil size through stimulating contraction of the iris 
sphincter muscle. 
In a Phase 2 trial, a statistically significant number of presbyopic 
subjects treated with Nyxol demonstrated ≥ 1 line of improvement 


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in distance-corrected near visual acuity (DCNVA) compared to 
Placebo-treated subjects, with a trend for a 2-line improvement at 
all timepoints. In these studies, Nyxol was generally well tolerated 
in the eye (most common complaint was mild-to-moderate ocular 
hyperemia) and there were no clinically meaningful systemic 
effects, as measured by heart rate (HR) and blood pressure (BP). 
Study Objectives 
The objectives of this study are: 
• To evaluate the efficacy of Nyxol + LDP to improve 
DCNVA compared to Placebo alone in subjects with 
presbyopia 
• To evaluate the efficacy of Nyxol + LDP to prevent < 1 
line of loss of BCDVA 
• To evaluate the efficacy of Nyxol + LDP to improve 
DCNVA compared to Nyxol alone or LDP alone   
• To evaluate the ability of Nyxol + LDP to produce the 
“pinhole” pupil size (approximately 1.6-2 mm) 
• To evaluate the efficacy of Nyxol + LDP to improve 
BCIVA and BCDVA 
• To evaluate the effect of iris color on the efficacy of Nyxol 
+ LDP  
• To evaluate the ocular and systemic safety of Nyxol + LDP 
and each component individually 
The Sponsor intends to use this Phase 2 study to evaluate Nyxol + 
LDP for the chronic indication of “temporary treatment of 
presbyopia.” 
Design 
This is a placebo-controlled, double-masked, multiple-dose, Phase 
2 study in approximately 152 randomized subjects with presbyopia 
(approximately 140 evaluable for efficacy), evaluated for safety 
and efficacy following administration of masked Nyxol or Placebo 
(Treatment 1) at or near bedtime for 3 to 4 days, with 
administration of LDP or No Treatment (Treatment 2) at the next 
visit (Visit 2) in the morning. Measurements will be made at 
multiple visits and timepoints, and analysis will consist of 
comparison across 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Subjects will be randomized 4:3:3:4 into the above groups. 
Randomization will be stratified 1:1 by iris color. 


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After randomization, masked Treatment 1 study medications 
(Nyxol or Placebo) will be dispensed. Treatment 1 study 
medication will be taken by the subject at or near bedtime 
(approximately 8PM-10PM). The treatment will start the night of 
Visit 1 (Screening/Baseline) or at the appropriate evening in order 
for Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2. 
Treatment 2 (LDP or No Treatment) is unmasked and will be 
administered at Visit 2 by a designated, unmasked, Site Staff 
member, distinct from the Site Staff member recording 
measurements/assessments. 
Treatment 1 (Nyxol or Placebo) will be administered to both eyes 
(OU) by the subject.  
Treatment 2 (LDP or No Treatment) will be administered OU by 
the Site Staff. 
The study eye is defined as the eye with worse Baseline DCNVA. 
In the case where both eyes have the same Baseline DCNVA, the 
study eye will be the right eye (OD). The non-study eye will be the 
fellow eye. The study eye and fellow eye will both be evaluated at 
all assessments.  
Subject Population  
Approximately 152 randomized subjects with presbyopia 
(approximately 140 evaluable for efficacy) 
Inclusion Criteria 
1. Males or females ≥ 40 and ≤ 64 years of age. 
2. Able to comply with all protocol-mandated procedures 
independently and to attend all scheduled office visits. 
3. Able and willing to give signed informed consent. 
4. Able to self-administer study medication throughout the study 
period. 
5. BCDVA of 0.0 LogMAR (20/20 Snellen equivalent) or better in 
each eye under photopic conditions. 
6. DCNVA of 0.4 LogMAR (20/50 Snellen equivalent) or worse 
under photopic conditions in each eye and binocularly. 
7. Subjects who depend on reading glasses or bifocals in which 
their binocular best-corrected near VA is 0.1 LogMAR (20/25 
Snellen equivalent) or better. 
Exclusion Criteria 
Ophthalmic (in either eye): 
1. Use of any topical prescription or over-the-counter (OTC) 
ophthalmic medications of any kind within 7 days of Screening 
until study completion, with the exception of lid scrubs with 


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OTC products (e.g., OCuSOFT® lid scrub, SteriLid®, baby 
shampoo, etc.). 
2. Use of any OTC artificial tears (preserved or unpreserved) at 
least once per day within 7 days of Screening until study 
completion. 
3. Current use of any topical ophthalmic therapy for dry eye (e.g., 
Restasis, Xiidra, etc.). 
4. Tear break-up time of < 5 seconds or corneal fluorescein 
staining (≥ grade 2 in the inferior zone or ≥ grade 1 in the 
central zone using the National Eye Institute scale). 
5. Clinically significant ocular disease (e.g., cataract, glaucoma, 
corneal edema, uveitis, retinal degeneration, loss of visual 
field, any macular pathology) that might interfere with the 
study as deemed by the Investigator. 
6. Recent or current evidence of ocular infection or inflammation 
in either eye (such as current evidence of clinically significant 
blepharitis, conjunctivitis, keratitis, etc.). Subjects must be 
symptom free for at least 7 days. 
7. Any history of herpes simplex or herpes zoster keratitis. 
8. History of diabetic retinopathy or diabetic macular edema. 
9. Known allergy, hypersensitivity, or contraindication to any 
component of the phentolamine, pilocarpine, or vehicle 
formulations.  
10. History of cauterization of the punctum or punctal plug 
(silicone or collagen) insertion or removal. 
11. Ocular trauma, ocular surgery (e.g., intraocular lenses), ocular 
laser treatment within the 6 months prior to Screening. Any 
subject with multifocal intraocular lenses are excluded. 
12. History of any traumatic (surgical or nonsurgical) or 
non-traumatic condition affecting the pupil or iris (e.g., 
irregularly shaped pupil, neurogenic pupil disorder, iris 
atrophy, iridotomy, iridectomy, iritis, etc.). 
13. Unwilling or unable to discontinue use of contact lenses at 
Screening until study completion. 
14. Conjunctival hyperemia ≥ grade 2 on the Cornea and Contact 
Lens Research Unit (CCLRU) 4-point scale. 
Systemic: 
15. Known hypersensitivity or contraindication to alpha- and/or 
beta-adrenoceptor antagonists (e.g., chronic obstructive 
pulmonary disease or bronchial asthma; abnormally low BP or 


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HR; second- or third-degree heart blockage or congestive heart 
failure). 
16. Known hypersensitivity or contraindication to any systemic 
cholinergic parasympathomimetic agents. 
17. Clinically significant systemic disease (e.g., uncontrolled 
diabetes, myasthenia gravis, cancer, hepatic, renal, endocrine, 
or cardiovascular disorders) that might interfere with the study 
as deemed by the Investigator. 
18. Initiation of treatment with, or any changes to, the current 
dosage, drug, or regimen of any systemic adrenergic or 
cholinergic drugs within 7 days prior to Screening or during 
the study. 
19. Participation in any investigational study within 30 days prior 
to Screening.  
20. Females of childbearing potential who are pregnant, nursing, 
planning a pregnancy, or not using a medically acceptable form 
of birth control. Acceptable methods include the use of at least 
one of the following: intrauterine device (IUD), hormonal 
(oral, injection, patch, implant, ring), barrier with spermicide 
(condom, diaphragm), or abstinence. A female is considered to 
be of childbearing potential, unless she is 1 year 
postmenopausal or 3 months post-surgical sterilization. All 
females of childbearing potential including those with 
post-tubal ligation must have a negative urine pregnancy test 
result at Visit 1 (Screening/Baseline).  
21. Resting HR outside the specified range of 50 to 110 beats per 
minute following at least a 5-minute rest period in the sitting 
position at the Screening Visit 1. HR may be repeated only 
once if outside the specified range, following another 5-minute 
rest period in the sitting position. 
22. Hypertension with resting diastolic BP > 105 mmHg or 
systolic BP > 160 mmHg following at least a 5-minute rest 
period in the sitting position at the Screening Visit 1. BP may 
be repeated only once if outside the specified range, following 
another 5-minute rest period in the sitting position.  
Screening/Baseline 
Visit (Visit 1) 
Individuals who are potential subjects are identified by the study 
center to schedule the Screening Visit. The Screening Visit shall 
occur the same day as the Baseline Visit.  
Once a subject arrives at the study center, a member of the Site 
Staff will interview the individual as to their qualifications for 
participation in the study, and if the subject wishes to continue, the 
informed consent form is signed, and a subject number is assigned.  


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The start of Screening includes an explanation of the study, a 
medical and ophthalmic history, and demographics. 
 
 
 
 This shall be followed by a urine pregnancy test for 
females of childbearing potential (Exclusion Criterion #20), and 
HR/BP (Exclusion Criteria #21-22).  
The subject will then undergo several VA measurements, 
including photopic BCDVA as well as 
 
DCNVA (
 In addition, subjects will 
undergo measurement of photopic best-corrected intermediate 
visual acuity (BCIVA) and mesopic BCDVA. 
The screening assessment will also include an ophthalmic 
examination that includes assessment of PD, biomicroscopy, dry 
eye examination with tear break-up time testing and corneal 
fluorescein staining (
), intraocular pressure 
(IOP) measurement (using a Tono-Pen), and direct or indirect 
ophthalmoscopy without dilation.  
In addition, conjunctival hyperemia will be assessed visually with 
a 4-point grading scale using images from the CCLRU 
 
).  
If all eligibility criteria are met, the subject will be randomized 
into the study.  
After randomization, masked Treatment 1 study medications 
(Nyxol or Placebo) will be dispensed.  
All treatments and assessments will be administered OU. 
Visit 2 Treatment + 
Assessments 
On the morning of Visit 2, subjects will undergo certain 
assessments immediately prior to receiving Treatment 2 at 
0 minutes: 
• Urine pregnancy test for females of childbearing potential 
• HR/BP  
• Concomitant medications  
• PD 
• DCNVA (
), BCDVA (
 
), BCIVA 
) – 
 
Treatment 2 (LDP or No Treatment) is unmasked and will be 
administered on Visit 2 by a designated, unmasked, Site Staff 
member, distinct from the Site Staff member recording 
measurements/assessments.  


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After instillation of Treatment 2 (LDP or No Treatment), subjects 
will undergo certain assessments at multiple timepoints after Time 
0 (from 30 minutes to 6 hours): 
• PD 
• DCNVA (
), BCDVA (
 
), BCIVA 
) – 
 
• Conjunctival hyperemia 
• Adverse events (AEs) 
• Subjective ocular tolerability 
Biomicroscopy, IOP, and ophthalmoscopy will be performed at the 
6-hour timepoint. 
Follow-Up Call (Visit 3) Safety Follow-Up Call (Visit 3; 1-3 days after Treatment Visit 2) 
subject assessments: 
• Concomitant medications  
• AEs 
Number of 
Investigational Sites 
Approximately 20 sites 
Estimated Total 
Sample Size 
Approximately 152 randomized subjects, with approximately 140 
subjects evaluable for efficacy 
Sample Size 
Justification 
A sample size of approximately 140 subjects (40 for Nyxol + LDP 
and Placebo alone arms and 30 for each of the other 2 arms) who 
are evaluable for efficacy is needed for the study. The primary 
treatment comparison will be Nyxol + LDP compared to Placebo 
alone. Using α = 0.05 significance and a two-tailed test, 76 total 
subjects (38 subjects each in the Nyxol + LDP and Placebo alone 
arms) 
 
 
 
 
  
Subjects will be randomized 4:3:3:4 to Nyxol + LDP, Nyxol + No 
Treatment 2 (Nyxol alone), Placebo + LDP, and Placebo + No 
Treatment 2 (Placebo alone), respectively. 
It is assumed that there will be approximately 7% drop-out 
between Visit 1 and end of Visit 2. To account for this drop-out, a 
total of approximately 152 subjects will be randomized into the 
study. 


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Primary Efficacy 
Endpoint 
The primary efficacy endpoint is the percent of subjects with ≥ 15 
letters of improvement in photopic binocular DCNVA on Visit 2 at 
1 hour with Nyxol + LDP compared to Placebo alone. The 
improvement in binocular DCNVA for each subject is relative to 
the subject’s own Baseline value (Visit 1). 
Secondary Efficacy 
Endpoints 
Secondary efficacy endpoints (for the study eye, the fellow eye, 
and binocular) will include: 
• 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
   
 
 
 
 
 
   
• 
 
 
 
   
 
 
 
 
 
  
 
 
 
   
 
 
 
 


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, Placebo + 
  
 
 
 
 


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Measurements: 
• DCNVA Reading/Near will be measured under 
 
 
 
  
• BCDVA will be measured under photopic conditions by a 
high-contrast Standard Early Treatment Diabetic 
Retinopathy Study (ETDRS) i
 
  
• BCIVA will be measured under photopic conditions by a 
 
 
 
 
• PD will be measured with a 
  
All of the efficacy endpoints will be analyzed overall and by 
at all timepoints. Subjects will also be analyzed by 
study eye, fellow eye, and binocular. 
All of the efficacy endpoints will also be analyzed by Modified 
Intention-to-Treat (mITT) and Per Protocol (PP) populations. 
Safety Endpoints 
The primary safety measures are conjunctival hyperemia, 
subjective ocular tolerability (Visit 2), and AEs.  
Analysis for conjunctival hyperemia includes change from 
Baseline (Visit 1) in the conjunctival hyperemia grading scale 
(CCLRU images) at each visit, for study eye and fellow eye. Any 
increase of 2 units in conjunctival hyperemia between consecutive 
visits is considered an AE. 
Other safety measures include IOP, biomicroscopy, 
ophthalmoscopy, and systemic safety as measured by HR and BP. 
Urine pregnancy tests for females of childbearing potential will be 
conducted. 
Measurements: 
• Conjunctival hyperemia will be assessed visually with a 
grading scale (0-3) using images from the CCLRU  
• Subjective ocular tolerability will be measured on a 4-
point scale (0-3) 
• IOP will be measured with the Tono-Pen 


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Study Medications, 
Dose and Mode of 
Administration 
Treatment 1 (Masked) 
Nyxol® Eye Drops (0.75% Phentolamine Ophthalmic Solution): 
Treatment 1 study medication will be taken by the subject at or near 
bedtime (approximately 8PM-10PM) starting the night of Visit 1 
(Screening/Baseline) or at the appropriate evening in order for 
Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2. 
Placebo (Nyxol vehicle): 
One drop of Placebo will be administered in each eye daily at or 
near bedtime (approximately 8PM-10PM) starting the night of Visit 
1 (Screening/Baseline) or at the appropriate evening in order for 
Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2.  
Subjects will be given enough Nyxol or Placebo for 4 doses. 
Treatment 2 (Unmasked) 
LDP (Low-Dose [0.4%] Pilocarpine Ophthalmic Solution): 
One drop of LDP will be administered in each eye by Site Staff at 
Time 0 minutes during Visit 2. 
No Treatment: 
No drop of LDP will be administered by Site Staff at Time 0 
minutes during Visit 2. 
Duration of Subject 
Participation 
and Study 
The total length of subject participation is approximately 5 to 9 
days (accommodating for weekends) with 2 clinic visits and one 
telephone call follow-up as summarized below: 
• Screening/Baseline Visit 1 (Day 1)  
• Treatment Visit 2 (Day 4 to 6)  
• Follow-up Call Visit 3 (Day 5 to 9)  
The execution of the entire study (first subject screen through last 
randomized subject completed) is expected to take approximately 
6 to 9 months. 


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TABLE OF CONTENTS 
SPONSOR SIGNATURE & CONTACTS .......................................................................................................... 2 
INVESTIGATOR’S AGREEMENT.................................................................................................................... 3 
PROCEDURES IN CASE OF EMERGENCY ................................................................................................... 4 
ABBREVIATIONS AND TERMS ....................................................................................................................... 5 
1. 
STUDY SUMMARY .................................................................................................................................... 7 
2. 
INTRODUCTION ...................................................................................................................................... 20 
2.1. 
Investigational Products ................................................................................................................... 20 
2.2. 
Findings From Nonclinical and Clinical Studies............................................................................ 20 
2.3. 
Design Justification........................................................................................................................... 23 
2.4. 
Route of Administration, Dosage Regimen, and Treatment Period ............................................. 24 
2.5. 
Compliance ........................................................................................................................................ 24 
2.6. 
Study Population .............................................................................................................................. 25 
3. 
OBJECTIVES AND PURPOSE ............................................................................................................... 25 
4. 
STUDY DESIGN ........................................................................................................................................ 26 
4.1. Primary and Secondary Endpoints ........................................................................................................ 26 
4.2. Description and Schedule of Visits and Procedures .............................................................................. 29 
4.3. 
Measures Taken to Minimize/Avoid Bias ....................................................................................... 31 
4.4. Study Medications.................................................................................................................................... 31 
4.4.1. Packaging and Labeling ..................................................................................................................... 33 
4.4.2. Storage of Study Medication and Dispensing .................................................................................... 34 
4.4.3. Study Medication Administration ...................................................................................................... 34 
4.4.4. Study Medication Accountability ....................................................................................................... 34 
4.5. 
Expected Duration of Subject Participation................................................................................... 34 
4.6. Randomization and Procedure for Breaking the Code ........................................................................ 35 
4.7. Collection of Data..................................................................................................................................... 35 
4.8. Completed Subject ................................................................................................................................... 35 
4.9. Non-completing Subject .......................................................................................................................... 35 
4.9.1. Study Medication Discontinuation ..................................................................................................... 36 
4.9.2. Reasons for Withdrawal From Study ................................................................................................. 36 
4.9.3. Entire Study Terminated .................................................................................................................... 36 
4.9.4. Actions After Discontinuation ........................................................................................................... 36 
4.10. 
Completed Study .............................................................................................................................. 36 
4.11. Procedure After the Completion of the Study ..................................................................................... 37 
5. 
SUBJECT INCLUSION AND EXCLUSION CRITERIA ..................................................................... 37 
5.1 
Subject Inclusion Criteria ................................................................................................................ 37 
5.2 
Subject Exclusion Criteria ............................................................................................................... 37 
6. 
TREATMENT OF SUBJECTS ................................................................................................................ 39 
6.1. Treatment Adherence .............................................................................................................................. 39 
6.2. Concomitant Medications ....................................................................................................................... 39 
7. 
ASSESSMENT OF EFFICACY ............................................................................................................... 40 
7.1. Specification of the Efficacy Parameters ............................................................................................... 40 
7.2. Assessing, Recording, and Analyzing of Efficacy Parameters ............................................................. 40 
7.2.1. Visit 1 (Screening/Baseline) – Day 1 ................................................................................................. 40 
7.2.2. Visit 2 (Treatment 2) – Day 4 ............................................................................................................ 41 
7.2.3. Visit 3 (Safety Follow-Up Call) – Day 5 ........................................................................................... 42 
7.2.4. Unscheduled Visits............................................................................................................................. 42 
7.2.5. Visit Variation .................................................................................................................................... 42 
8. 
ASSESSMENT OF SAFETY .................................................................................................................... 42 
8.1. Specification of Safety Parameters ......................................................................................................... 42 
8.2. 
Assessing, Recording, and Analyzing Safety Parameters ............................................................. 43 
8.3. Adverse Events and Serious Adverse Events......................................................................................... 43 
8.3.1. Adverse Event Definitions ................................................................................................................. 43 


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8.3.2. Follow-Up of Subjects After Adverse Events .................................................................................... 47 
9. 
STATISTICS .............................................................................................................................................. 47 
9.1. Sample Size ............................................................................................................................................... 47 
9.2. Analysis Populations ................................................................................................................................ 48 
9.3. Statistical Methods ................................................................................................................................... 48 
9.3.1. General Considerations ...................................................................................................................... 48 
9.3.2. Demographic and Baseline Characteristics ........................................................................................ 48 
9.3.3. Subject Disposition ............................................................................................................................ 48 
9.3.4. Medical History and Prior/Concomitant Medications ........................................................................ 48 
9.3.5. Analysis of Efficacy ........................................................................................................................... 49 
9.3.6. Analysis of Safety .............................................................................................................................. 50 
9.4. 
Procedure for Accounting for Missing, Unused, or Spurious Data .............................................. 50 
9.5. Procedure for Reporting Deviations From the Statistical Plan ........................................................... 50 
10. 
DIRECT ACCESS TO SOURCE DATA AND DOCUMENTS ............................................................ 50 
11. 
QUALITY CONTROL AND QUALITY ASSURANCE........................................................................ 50 
12. 
ETHICAL CONSIDERATIONS AND GCP COMPLIANCE .............................................................. 51 
12.1. GCP Compliance.................................................................................................................................... 51 
12.2. Institutional Review Board ................................................................................................................... 51 
12.3. Protocol Deviations/Violations .............................................................................................................. 51 
12.4. Informed Consent Requirements ......................................................................................................... 51 
13. 
DATA HANDLING AND RECORD KEEPING .................................................................................... 52 
13.1. Data Entry .............................................................................................................................................. 52 
13.2. Data Quality Control and Reporting ................................................................................................... 52 
13.3. Archiving of Data ................................................................................................................................... 52 
13.4. Records Retention .................................................................................................................................. 52 
13.5. Amendments to the Protocol ................................................................................................................. 53 
14. 
REFERENCES ........................................................................................................................................... 54 
APPENDIX 1: IRIS COLOR CHART .............................................................................................................. 55 
APPENDIX 2: VISUAL ACUITY CHARTS .................................................................................................... 56 
APPENDIX 3: ADRENERGIC AND CHOLINERGIC DRUGS ................................................................... 57 
APPENDIX 4: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING IMAGES FROM CCLRU58 
APPENDIX 5: TREATMENT GROUPS .......................................................................................................... 59 
 
 
 
 


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2. INTRODUCTION 
2.1. Investigational Products 
The combination of Nyxol (0.75% Phentolamine Ophthalmic Solution [POS]) and Low-Dose 
(0.4%) Pilocarpine Ophthalmic Solution (LDP) is to be studied for the indication of “temporary 
treatment of presbyopia.” Specifically, Nyxol would be dosed once daily at or near bedtime and 
LDP dosed in the morning. Nyxol + LDP dosing can potentially reduce pupil diameter (PD) size 
to ≤ 2.0 mm, which has the potential to improve near visual acuity (VA) due to a “pinhole” 
effect. In this trial, Nyxol and Placebo are referred to as Treatment 1, and LDP and No Treatment 
are referred to as Treatment 2. 
Treatment 1 (Masked) 
The test product is Nyxol® Eye Drops – 0.75% Phentolamine Ophthalmic Solution (Nyxol), a 
non-selective alpha-1 and alpha-2 adrenergic receptor antagonist that inhibits contraction of the 
iris dilator muscle, resulting in a smaller pupil size. Note that the concentration of 0.75% refers 
to phentolamine free base and is the same—and used in place of—1% phentolamine mesylate 
drug substance, which was how Nyxol had been described in all prior studies.  
Placebo control is Nyxol vehicle alone. 
One drop of Nyxol or Placebo will be administered in each eye daily at or near bedtime 
(approximately 8PM-10PM) starting the night of Visit 1 (Screening/Baseline) or at the appropriate 
evening in order for Treatment 1 to be taken daily for 3 to 4 consecutive days immediately prior to 
Visit 2.  
Subjects will be given enough Nyxol or Placebo for 4 doses. 
Treatment 2 (Unmasked) 
Low-Dose (0.4%) Pilocarpine Ophthalmic Solution (LDP) is a direct-acting cholinergic, 
parasympathomimetic agent which acts through direct stimulation of muscarinic receptors and 
smooth muscle such as the iris and secretory glands. Pilocarpine reduces pupil size through 
stimulating contraction of the iris sphincter muscle. 
For those who receive LDP, one drop of LDP will be administered in each eye by Site Staff at 
Time 0 minutes during Visit 2. 
For those who receive No Treatment, no drop of LDP will be administered by Site Staff at Time 
0 minutes during Visit 2. 
2.2. Findings From Nonclinical and Clinical Studies 
Nyxol 
Detailed findings from nonclinical and clinical studies and potential risk are provided in the 
Investigators’ Brochure (IB) (2020).  
Nyxol has been assessed in 7 Investigator-initiated and sponsored Phase 1 and Phase 2 clinical 
trials. Across all trials, 168 of 232 adult subjects were exposed to at least one dose of 
Phentolamine Mesylate Ophthalmic Solution. The data presented below are from Nyxol alone. 
In prior clinical trials, Nyxol has demonstrated a consistent ability to decrease PD by 
approximately 20% (~1-1.5 mm) in both mesopic and photopic conditions. Nyxol has been 
found to be efficacious for more than 24 hours with an onset of action of approximately 30 to 60 
minutes. Key PD data are summarized below (Table 1). 


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Table 1: Efficacy of 1% Nyxol in Reducing Pupil Diameter in Mesopic Conditions in Phase 
2 Trials 
Study 
Group 
Mesopic Conditions 
Pre-Treatment 
(Baseline) Pupil 
Diameter 
Post-Treatment 
Pupil Diameter Change (%) 
p-value 
compared 
to baseline 
p-value 
compared 
to placebo 
NYX-SNV 
(1) 
Placebo (N = 16) 
6.6 mm 
6.4 mm 
-0.2 mm (-3%) 
p = 0.08 
p < 0.0001 
1.0% Nyxol (N = 32) 
6.5 mm 
5.2 mm 
-1.3 mm (-20%) 
p < 0.0001 
NYX-01a2 
(2) 
Placebo (N = 38) 
6.25 mm 
6.31 mm 
0.07 mm (+1%) 
p = 0.6 
p < 0.0001 
1.0% Nyxol (N = 40) 
6.17 mm 
5.31 mm 
-0.86 mm (-14%) 
p < 0.0001 
NYXG-201 
(3) 
Placebo (N = 20) 
4.57 mm 
4.52 mm 
-0.05 mm (-1%) 
p = 0.6178 
p < 0.0001 
1.0% Nyxol (N = 19) 
4.69 mm 
3.70 mm 
-1.00 mm (-21%) 
p < 0.0001 
 
Nyxol was observed to be well tolerated at single doses up to and including 1.0% daily in each 
eye. This includes 59 subjects who received multiple doses of up to 1% Nyxol for at least 
14 days. Safety of the subjects in these trials was evaluated by adverse event (AE) monitoring, 
physical examinations, and vital sign assessments. Across all trials, no healthy volunteers or 
subjects reported a treatment-emergent serious adverse event (SAE). No deaths occurred in any 
of the trials. No clinically meaningful changes were observed in physical examinations or vital 
signs, including blood pressure (BP) and heart rate (HR). AEs reported were mild to moderate in 
intensity with the most common being transient conjunctival hyperemia and ocular irritation; 
however, Nyxol dosing at or near bedtime was observed to mitigate or minimize these side 
effects during the daytime. 
Phase 2 Trial in a Glaucomatous and Presbyopic Population (ORION-1, NYXG-201): 
ORION-1 (NYXG-201) was a double-masked, randomized, placebo-controlled, multi-center trial 
of 1% Nyxol compared with placebo ophthalmic solution for 14 days in subjects with open-angle 
glaucoma or ocular hypertension. After screening was performed based on inclusion and 
exclusion criteria, a total of 39 elderly subjects (median age of 63) were randomized into the trial 
(Nyxol arm, n = 19; placebo arm, n = 20). These subjects were either treatment-naïve or were 
previously taking intraocular pressure (IOP)-lowering medication and were washed out for 30 
days prior to dosing. Subjects took their study medication (Nyxol or placebo) in both eyes (OU) 
between 8PM and 10PM every evening for 14 days. Assessments were made on Day 1, Day 8, 
Day 15, and Day 16. The primary efficacy endpoint was change from baseline in mean diurnal 
IOP at Day 15. Mean diurnal IOP is the mean of the IOP measurements at 3 timepoints (8AM, 
10AM, 4PM). Secondary efficacy endpoints included change in PD, change in distance-corrected 
near visual acuity (DCNVA), and change in best-corrected distance visual acuity (BCDVA), as 
well as additional IOP analyses. Safety assessments included measurements of conjunctival 
hyperemia (using the Cornea and Contact Lens Research Unit [CCLRU] grading 4-point scale 
[0-3]), AEs, HR, BP, concomitant medications, and pregnancy. 
In summary, Nyxol had statistically significant reductions in PD under photopic and mesopic 
conditions that were sustained for 36 hours post-dosing (Table 1). A statistically significant 
number of subjects treated with Nyxol compared to placebo demonstrated ≥ 1 line of 
improvement in DCNVA (63.2% with Nyxol vs 20.0% with placebo), with a trend for a 2-line 
improvement at all timepoints. There was no statistical difference in conjunctival hyperemia 


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compared to placebo. In summary, daily evening dosing of Nyxol was found to be well tolerated 
with no daytime conjunctival redness and demonstrated improvement in DCNVA with sustained 
PD reduction in a glaucomatous and presbyopic population. Smaller pupil size can have 
beneficial effects in improving near VA in presbyopic subjects, which is relevant to the VEGA-1 
trial. 
Regarding safety, Nyxol 1% was well tolerated and there were no major ocular or systemic 
safety issues. An evening dose regimen minimized eye redness during the daytime while 
benefiting near VA in an elderly population. The incidence of treatment-emergent adverse events 
(TEAEs) was higher in the Nyxol arm compared with the placebo arm (31.6% versus 5.0%) but 
all TEAEs were mild in severity, with no serious TEAEs or TEAEs leading to withdrawal or 
study medication discontinuation. Most TEAEs were considered related to study medication, 
with the most frequent being conjunctival hyperemia and burning on instillation (each seen in 
16% of subjects treated with Nyxol). Although conjunctival redness scores increased in the 
Nyxol arm at Day 8, Day 15, and Day 16, the scores in the Nyxol arm at any post-baseline 
timepoint did not demonstrate a statistically significant difference from scores in the placebo 
arm. Mean systolic and diastolic BPs and HRs were relatively unchanged and remained within 
normal range throughout the duration of the trial and were similar between arms. Neither 
biomicroscopic nor ophthalmoscopic examination showed any clinically significant 
abnormalities at Screening or at Day 15. There was no worsening of distance VA, near VA, or 
IOP.  
Phase 2 Trial in Patients With Severe NVD (NYX-SNV): 
NYX-SNV was a double-masked, randomized, placebo-controlled, single-dose trial assessing the 
tolerability and effect of a single topical drop of 1.0% solution of phentolamine mesylate in 
Tears Naturale II in each eye or Tears Naturale II (placebo) on PD, contrast sensitivity (CS), VA, 
and wavefront aberrometry. A total of 24 patients (median age of 39) with severe night vision 
(SNV) complaints were randomly assigned 2:1 to treatment groups (active treatment, n = 16; 
placebo control, n = 8). Each group was treated with one drop of test article in each eye. The 
primary endpoint was a statistically significant improvement in the mean change in monocular 
CS scores under mesopic conditions at each of 5 spatial frequencies. Key secondary endpoints 
included measurements of low-contrast visual acuity (LCVA) under mesopic and photopic 
conditions, change in PD, and percent of subjects with an improvement in CS (at multiple 
frequencies), which were recorded at baseline (prior to treatment administration) and 
approximately 2 hours after administration. 
 
In summary, in NYX-SNV, 1% Nyxol demonstrated statistically significant reductions in PD and 
improvement in LCVA under photopic and mesopic lighting conditions, as well as individual CS 
frequency improvements. Treatment with 1% Nyxol further exhibited a statistically significant 
reduction in aberration errors (errors that affect light transmission in specific PD sizes). 
Additionally, mean PD decreased an average of 1.3 mm (p < 0.0001), or ~20%, for phentolamine 
mesylate–treated patients, whereas mean PD of placebo-treated patients did not significantly 
change between pre-treatment and post-treatment. The difference in mean PD change between 
treatment groups was also statistically significant (1.1 mm; p < 0.0001) (Table 1). 
 
No SAEs or other AEs were reported during the trial. Overall, study treatment appeared to be 
well tolerated. No meaningful differences in mean HR or mean systolic and diastolic BP between 
treatment groups were observed. Treatment with phentolamine mesylate caused a statistically 
significant elevation in mean change from baseline in conjunctival hyperemia between the 2 
treatment groups (+38.6 mm versus +12.1 mm for placebo; p < 0.0004; range, 0 mm [no redness] 


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– 100 mm [maximal redness]). The mean change in IOP of phentolamine mesylate–treated eyes 
from screening to 2 to 3 hours post-treatment (-1.8 mmHg) was statistically significant (p < 
0.0004). 
 
It is important to note that similar results were found in NYX-01a2, which was a 15-day, double-
masked, randomized, placebo-controlled trial in patients with severe dim light vision 
disturbances (DLD). The NYX-01a2 trial demonstrated a dose response favoring 1% Nyxol. 
Further, statistically significant reductions in PD, trends in improvement in LCVA under bright 
and dim lighting conditions were shown. Durability of effect on PD was observed 24 hours later 
for Nyxol with daily morning doses. 
Pilocarpine 
A detailed report of pilocarpine, its product label, properties, and potential risk are provided in 
the IB (2020). 
Pilocarpine hydrochloride has not been studied in prior clinical trials by Ocuphire. However, 
there is extensive literature on its ability, at various concentrations, to reduce PD. These pupil-
constricting effects are counteracted by a dose-dependent side effect profile, notably browache 
and stinging in the eye upon topical application of the drug.  
In an effort to better characterize an efficacious dose of pilocarpine that would have minimal side 
effects, Ocuphire conducted an expansive review of the medical literature concerning 
experiments of various doses of pilocarpine and their effects on PD and side effect profile. 
Leavitt et al. found that normal pupils constrict to dilute concentrations of 0.25% or 0.125% 
pilocarpine (1.7 and 0.6 mm, respectively) but constrict insignificantly at concentrations of 
0.0313% or 0.0625% (4). These decreases in PD were found to occur within 15 minutes of 
dosing and peak pupil size reduction occurred at 30 to 60 minutes (4). On the other hand, 2% 
pilocarpine, although efficacious in decreasing PD by upwards of 3 mm, caused significant side 
effects including worsened visual field and acuity (5). 
When considering the effects on PD and the potential side effects of higher doses of pilocarpine, 
Ocuphire found that a range of 0.3% to 0.6% pilocarpine may provide moderate efficacy with 
potentially limited side effects. Moreover, based on these data, Ocuphire hypothesizes that the 
combination of 0.4% pilocarpine, when added to phentolamine mesylate, can have an additive or 
synergistic effect to produce a long duration of pupil reduction in an optimal range of PD for 
treating presbyopia without causing undue adverse side effects due to eye redness, eye stinging, 
or browache. 
2.3. Design Justification 
Presbyopia is an age-related condition with onset most common in people over 40 years old (6). 
Patients experience blurred near vision, difficulty seeing in dim light, and eye strain. In young 
healthy eyes, lenses are able to focus light from objects at different distances by a process called 
accommodation. During accommodation, muscles surrounding the lens contract, causing the lens 
to change shape, thus increasing the focusing power of the eye. This allows for dynamic, clear 
vision at both near and far distances. With increasing age, the lens becomes stiffer, which limits 
the eye’s ability to adjust its focus for reading or for other tasks that require clear vision at near 
distances.  
Presbyopia has a significantly negative impact on quality of life (7), interfering with daily 
activities such as reading, use of computers or hand-held devices, and seeing the dashboard of a 


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car. It is estimated that 100 million Americans have presbyopia and this number is expected to 
grow as the population above the age of 40 increases (8). 
Limitations of Existing Treatments for Presbyopia 
Despite ongoing research, there are currently no noninvasive, pharmacological treatments for 
presbyopia that have been approved by the US Food and Drug Administration (FDA). The 
available treatments for presbyopia include reading glasses, bifocals, gradients, bifocal contact 
lenses, and multifocal intraocular lenses (IOLs). Reading glasses can be inconvenient and must 
be taken off and put on frequently throughout the day to see objects at far and near distances, 
respectively. Many patients express frustration with losing or forgetting their reading glasses. 
Moreover, some patients find glasses unflattering, and contact lenses for presbyopia have their 
own set of drawbacks regarding eye strain and limitation of use. 
A small portion of patients elect surgical intervention, including laser treatment to achieve 
monovision and insertion of KAMRA Inlays, a plastic implant into the cornea of the non-
dominant eye to increase its depth of field. The risks of such interventions are those associated 
with all ocular surgeries, such as a potential decrease in CS and the creation or worsening of 
DLD.  
Nyxol Opportunity in Presbyopia 
PD management for the pharmacological treatment of presbyopia is a promising strategy and an 
intense area of focus by several other pharmaceutical companies. Nyxol alone has shown in 
multiple Phase 2 trials the ability to reduce PD size by 15% to 20% and improve near VA by 1 
line for at least 24 hours after a single application. Other pharmacological and device approaches 
have consistently demonstrated that reducing pupil size to a diameter of 1.6 to 2 mm range 
(“pinhole”) will lead to significant improvement in presbyopia symptoms by increasing depth of 
focus (9-11). The creation of a pinhole-sized pupil can allow the eye to focus on near objects 
with less dependence on reading adds or lenses. To achieve the pinhole, Ocuphire plans to 
evaluate the efficacy of a kit combination of Nyxol (dosed in the evening offering a 20% 
reduction in PD) and LDP (dosed in the daytime with an expected additional 20% reduction in 
PD).  
2.4. Route of Administration, Dosage Regimen, and Treatment Period 
As the intended route of administration for Nyxol, Placebo, and pilocarpine is topical 
ophthalmic, this is the route to be used in this study.  
The dose for one drop of Nyxol selected for this study, 0.75%, was based upon: 1) preclinical 
safety studies, 2) the results of the previous ophthalmic clinical studies described above and in 
the IB, and 3) clinical studies conducted with varying doses of drugs in the same class (12). 
Note that 0.75% POS, which expresses the phentolamine mesylate concentration as the free base, 
is the new nomenclature being used in place of 1% Phentolamine Mesylate Ophthalmic Solution, 
which was how Nyxol had been described in prior studies, including ORION-1. 
The dose for one drop of pilocarpine selected for this study is 0.4%. 
All treatments will be administered OU. 
2.5. Compliance 
This study will be conducted in compliance with the protocol and in accordance with Good 
Clinical Practice (GCP), the ethical principles set forth in the Declaration of Helsinki, and with 
the US Code of Federal Regulations (CFR). 


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2.6. Study Population 
The study population for this trial will be comprised of males and females between 40 to 64 
years of age with presbyopia. A sample size of approximately 152 subjects will be randomized 
into 1 of 4 treatment arms with the expectation that approximately 140 subjects will be evaluable 
for efficacy.  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Subjects will be randomized 4:3:3:4 into the above groups. 
After randomization, masked Treatment 1 study medications (Nyxol or Placebo) will be 
dispensed. Treatment 1 study medication will be taken by the subject at or near bedtime 
(approximately 8PM-10PM) starting the night of Visit 1 (Screening/Baseline) or at the 
appropriate evening in order for Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2. 
Treatment 2 (LDP or No Treatment) is unmasked and will be administered at Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments. 
Randomization will be stratified 
 (Appendix 1). The subjects will be 
recruited from approximately 20 investigational sites.  
 
3. OBJECTIVES AND PURPOSE 
The VEGA-1 study is a randomized, placebo-controlled, double-masked study of the safety and 
efficacy of Nyxol (0.75% POS) with 0.4% LDP in subjects with presbyopia. 
The objectives of this study are: 
Primary objective 
• To evaluate the efficacy of Nyxol + LDP to improve DCNVA compared to Placebo alone 
in subjects with presbyopia 
 
Secondary objectives 
• To evaluate the efficacy of Nyxol + LDP to improve DCNVA compared to Placebo alone 
in subjects with presbyopia 
• To evaluate the efficacy of Nyxol + LDP to prevent < 1 line of loss of BCDVA 
• To evaluate the efficacy of Nyxol + LDP to improve DCNVA compared to Nyxol alone 
or LDP alone   
• To evaluate the ability of Nyxol + LDP to produce the “pinhole” pupil size 
(approximately 1.6-2 mm) 
• To evaluate the efficacy of Nyxol + LDP to improve BCIVA and BCDVA 
• To evaluate the effect of iris color on the efficacy of Nyxol + LDP  
• To evaluate the ocular and systemic safety of Nyxol + LDP and each component 
individually 


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The Sponsor intends to use this Phase 2 study to evaluate Nyxol + LDP for the chronic indication 
of “temporary treatment of presbyopia.” 
 
4. STUDY DESIGN 
This is a placebo-controlled, double-masked, multiple-dose, Phase 2 study in approximately 152 
randomized subjects with presbyopia (approximately 140 evaluable for efficacy), evaluated for 
safety and efficacy following administration of masked Nyxol or Placebo (Treatment 1) at or 
near bedtime for 3 to 4 days, with administration of LDP or No Treatment (Treatment 2) at the 
next visit (Visit 2) in the morning. Measurements will be made at multiple visits and timepoints, 
and analysis will consist of comparison across 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Subjects will be randomized 4:3:3:4 into the above groups. 
After randomization, masked Treatment 1 study medications (Nyxol or Placebo) will be 
dispensed. Treatment 1 study medication will be taken by the subject at or near bedtime 
(approximately 8PM-10PM) starting the night of Visit 1 (Screening/Baseline) or at the 
appropriate evening in order for Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2. 
Treatment 2 (LDP or No Treatment) is unmasked and will be administered at Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments. 
Treatment 1 (Nyxol or Placebo) will be administered to both eyes (OU) by the subject.  
Treatment 2 (LDP or No Treatment) will be administered OU by the Site Staff. 
The study eye is defined as the eye with worse Baseline DCNVA. In the case where both eyes 
have the same Baseline DCNVA, the study eye will be the right eye (OD). The non-study eye 
will be the fellow eye. The study eye and fellow eye will both be evaluated at all assessments. 
4.1.Primary and Secondary Endpoints 
Efficacy: 
The primary efficacy endpoint is the percent of subjects with ≥ 15 letters of improvement in 
photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP compared to Placebo alone. 
The improvement in binocular DCNVA for each subject is relative to the subject’s own Baseline 
value (Visit 1). 
Secondary efficacy endpoints (for the study eye, the fellow eye, and binocular) will include: 
• 
 
 
 
  
 
 
 


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• 
 
 
 
  
 
 
 
 
  
Measurements: 
• DCNVA Reading/Near will be measured under photopic and mesopic conditions by a 
high-contrast Near Visual Acuity Chart in the 
 
 (Appendix 2) 
• BCDVA will be measured under photopic conditions by a high-contrast Standard Early 
Treatment 
 
  
• BCIVA will be measured under photopic conditions by a high-contrast Near Visual 
Acuity Chart in the 
 at 
 
 
• PD will be measured with a 
 (mm)  
All of the efficacy endpoints will be analyzed overall and by 
 at all timepoints. 
Subjects will also be analyzed by study eye, fellow eye, and binocular. 
In photopic lighting conditions, 
 
 
 In mesopic conditions, 
 
 
. Ambient 
lighting 
. The 
 
. Subjects will be allowed to acclimate to these lighting conditions (with 
the eyes open 
 to the set of PD and VA 
measurements). Subjects will sit in the exam chair facing directly at the illuminated chart during 
the acclimation period and for all assessments.  
 for the scheduled remaining safety assessments (e.g., conjunctival 
hyperemia, AEs, subject questionnaire, etc.). The subject will be in the same room for all 
assessments, and every effort will be made to have the same person perform the measurements at 
all visits. 
For VA, measurements will be made in letters and will be converted to LogMAR and lines, as 
appropriate. 
All of the efficacy endpoints will also be analyzed by Modified Intention-to-Treat (mITT) and 
Per Protocol (PP) populations. 
 
Safety: 
The primary safety measures are conjunctival hyperemia, subjective ocular tolerability (Visit 2), 
and AEs.  


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Analysis for conjunctival hyperemia includes change from Baseline (Visit 1) in the conjunctival 
hyperemia grading scale (CCLRU images) at each visit, for study eye and fellow eye. Any 
increase of 2 units in conjunctival hyperemia between consecutive visits is considered an AE. 
Other safety measures include IOP, biomicroscopy, ophthalmoscopy, and systemic safety as 
measured by HR and BP. Urine pregnancy tests for females of childbearing potential will be 
conducted. 
Measurements: 
• Conjunctival hyperemia will be assessed visually with a grading scale (0-3) using 
images from the CCLRU  
• Subjective ocular tolerability will be measured on a 4-point scale (0-3) 
• IOP will be measured with the Tono-Pen 
4.2.Description and Schedule of Visits and Procedures 
Study procedures are shown in detail in Table 2. 
 
 


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Table 2: Visit Schedule 
 
Day [b] 
Time (hr) [c] 
Informed consent 
Subject Number assigned 
Medical/Ophthalmic history 
Demographics  
Prior/Concomitant medications 
Urine pregnancy test [d] 
Heart rate/Blood pressure 
 
 
 
 
 
 
  
 
 
  
 
 
Adverse events 
Randomization assigned [a] 
Treatment 1: Nyxol or Placebo [k] 
Treatment 2: 0.4% LDP or No Treatment [l] 
Subjective ocular tolerability [m] 
Abbreviations: BCDVA, best-corrected distance visual acuity; BCIVA, best-corrected intermediate visual acuity; CCLRU, Cornea and Contact Lens 
Research Unit; DCNVA, distance-corrected near visual acuity; LDP, low-dose pilocarpine; N/A, not applicable; VA, visual acuity. 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
  
 
. 
 
 


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4.3. Measures Taken to Minimize/Avoid Bias 
This is a placebo-controlled, double-masked, randomized, 4-arm Phase 2 study.  
4.4.Study Medications 
Study Medication Identification 
The POS treatment medication is phentolamine mesylate; its chemical name is 3-[N-(4,5-
dihydro-1H-imidazol-2-ylmethyl)-4-methylanilino]phenol;methanesulfonic acid. It is a clean 
white to off-white, odorless crystalline powder with a molecular weight of 377.46 g/mol. 
Solutions of phentolamine mesylate are acid to litmus. It is freely soluble in water and in ethanol, 
and slightly soluble in chloroform. It melts at about 178ºC. 
The properties (Table 3) of phentolamine mesylate are described below. 
Table 3: 
Drug Substance and Drug Product Identifiers 
Established name 
Phentolamine mesylate – parent phentolamine 
CAS registry number 
65-28-1 – parent 50-60-2 
Chemical class 
An alpha-adrenergic receptor antagonist, it is a 
member of the following classes: imidazoles, of 
phenols, is a tertiary amino compound and a 
substituted aniline. 
Chemical name 
3-[N-(4,5-dihydro-1H-imidazol-2-ylmethyl)-4-
methylanilino]phenol;methanesulfonic acid 
Molecular formula 
C18H23N3O4S– parent C17H19N3O 
Molecular weight (g/mol) 
377.140– parent 281.352 
Drug name/formulation 
Nyxol / aqueous isotonic solution  
Concentration active 
Mesylate salt 1% – parent 0.75% 
Manufacturer drug substance 
 
Manufacturer drug product, Placebo 
 
Storage requirements 
 
 
 
  
Stored at the site, drug will be placed in a secured 
location (locked) with no access for unauthorized 
personnel. 
 


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Nyxol (0.75% Phentolamine Ophthalmic Solution) is a clear, colorless to slightly brown, sterile, 
non-preserved, isotonic, buffered aqueous solution containing 1% phentolamine mesylate 
(equivalent to 0.75% phentolamine free base), mannitol, and sodium acetate. Placebo for Nyxol 
is a clear, colorless, sterile, non-preserved, isotonic, buffered aqueous solution containing 
mannitol and sodium acetate. 
 
 The product is preservative 
free.  
For the proposed study, a second treatment medication (0.4% Pilocarpine Hydrochloride 
Ophthalmic Solution) will be used. Pilocarpine hydrochloride 1%, 2%, and 4% are approved by 
the FDA and available generically by multiple pharmaceutical manufacturers including Sandoz 
Inc, Somerset LLC, Akorn Inc, and Alcon Laboratories. For this study, a lower dose of 0.4% 
Pilocarpine Hydrochloride Ophthalmic Solution is planned.  
Pilocarpine hydrochloride is the hydrochloride salt of (+)-pilocarpine (parent compound), a 
medication used to treat increased pressure inside the eye and dry mouth. Its chemical name is 
(3S,4R)-3-ethyl-4-[(3-methylimidazol-4-yl)methyl]oxolan-2-one hydrochloride, and it has a 
molecular weight of 244.72 g/mol. Pilocarpine hydrochloride is a hygroscopic, odorless, bitter-
tasting white crystal or powder which is soluble in water and alcohol and virtually insoluble in 
most non-polar solvents. It melts at about 204.5°C. Its aqueous solubility in buffer at pH 7.4 is > 
36.7 μg/mL. The parent (+)-pilocarpine is a natural alkaloid extracted from plants of the genus 
Pilocarpus with cholinergic agonist activity. 
The properties (Table 4) of pilocarpine hydrochloride are described below. 
Table 4: 
Drug Substance and Drug Product Identifiers 
Established name 
Pilocarpine hydrochloride – parent (+)-pilocarpine 
CAS registry number 
54-71-7 – parent 92-13-7 
Chemical class 
A cholinergic parasympathomimetic agent, 
pilocarpine predominantly binds to muscarinic 
receptors, thereby inducing exocrine gland 
secretion and stimulating smooth muscle in the 
bronchi, urinary tract, biliary tract, and intestinal 
tract. When applied topically to the eye, this agent 
stimulates the sphincter pupillae to contract, 
resulting in miosis; stimulates the ciliary muscle 
to contract, resulting in spasm of accommodation; 
and may cause a transitory rise in IOP followed by 
a more persistent fall due to opening of the 
trabecular meshwork and an increase in the 
outflow of aqueous humor. 
Chemical name 
(3S,4R)-3-ethyl-4-[(3-methylimidazol-4-
yl)methyl]oxolan-2-one hydrochloride  
or  
(3S-cis)-2(3H)-Furanone, 3-ethyl-dihydro-4-[(1-
methyl-1H-imidazol-5-yl)methyl] mono-
hydrochloride 


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Molecular formula 
C11H17ClN2O2 – parent C11H16N2O2 
Molecular weight (g/mol) 
244.72 – parent 208.261 
Investigational product 
name/formulation 
PILOCARPINE HYDROCHLORIDE 0.4% 
ophthalmic solution 
Aqueous isotonic solution, compounded by Pine 
Pharmaceuticals LLC and released by Iuvo 
BioScience LLC  
Concentration active 
0.4% 
Drug used in compounding the 
investigational product 
PILOCARPINE HYDROCHLORIDE 4%- 
pilocarpine hydrochloride solution/ drops  
NDC 61314-0206-15 
Manufacturer drug product used for 
compounding 
Sandoz, Inc. 
Storage requirements 
Stored at room temperature (15°C to 25°C, 59°F 
to 77°F). 
Stored in a secured location (locked) with no 
access for unauthorized personnel.  
Pilocarpine hydrochloride 0.4% investigational drug is 
 
. In addition to the active drug, the 
formulation also comprises the following inactive excipients: benzalkonium chloride NF, 
hypromellose 2910 USP, sodium citrate USP, boric acid NF, sodium chloride USP and sterile 
water for injection. The pH is adjusted with sodium hydroxide NF (10%) and hydrochloric acid 
NF (37%) to achieve a target pH of 3.5 to 5.5. 
Pilocarpine hydrochloride is packaged in 15-mL LDPE dropper bottles for investigational use in 
the Investigator’s office. Pilocarpine Hydrochloride 0.4% Ophthalmic Solution is stored at room 
temperature (15°C to 25°C, 59°F to 77°F). 
4.4.1. Packaging and Labeling 
Nyxol and its placebo are packaged in unit-of-use 1-mL low-density polyethylene (LDPE) 
dropper bottles with a fill volume specification of 0.6 mL for single-dose investigational use. 
Each bottle is wrapped with an aluminum foil overwrap impermeable to water and oxygen. Each 
individual pouch is labeled with an investigational label showing the study protocol number and 
other relevant information, including a statement “Caution – New Drug – Limited by Federal 
(US) Law to Investigational Use.”  
LDP, one of the two options for Treatment 2, is provided in 15-mL LDPE dropper, multi-use 
bottles for investigational use in the Investigator’s office. It will be labeled with an 
investigational label showing the study protocol number and other relevant information, 
including a statement “Caution – New Drug – Limited by Federal (US) Law to Investigational 
Use.” 


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4.4.2. Storage of Study Medication and Dispensing 
Prior to dispensing, all investigational material must be stored in a secure location with strictly 
limited access documented by signature of authorized persons who may dispense investigational 
materials.  
Treatment 1 study medication (Nyxol and Placebo) should be shipped and 
 
 
Treatment 1 study 
medication must not be frozen and must be protected from light.  
Treatment 2 study medication (LDP) should be stored at room temperature (15°C to 25°C, 59°F 
to 77°F).  
All investigational study medication must be stored in a secure facility, with access limited to the 
Investigator and authorized staff. 
4.4.3. Study Medication Administration 
Treatment 1 study medication will be distributed by the Investigator or designee at Visit 1 after 
randomization has been assigned. The subject will instill one drop once daily at or near bedtime 
(approximately 8PM-10PM) OU for 3 to 4 days. 
Treatment 2 study medication will be administered by the designated, unmasked, Site Staff 
member, distinct from the Site Staff member recording measurements/assessments at Visit 2. 
4.4.4. Study Medication Accountability 
4.4.4.1. Receipt and Disposition of Study Medication 
The Investigator or designee (e.g., study coordinator or pharmacist) will maintain a full 
accountability record for both Treatment 1 and Treatment 2 study medications and will be 
responsible for recording the receipt, dispensing, and return of all supplies of the study 
medication using the inventories supplied by Ocuphire. The Investigator or designee will account 
for both Treatment 1 and Treatment 2 study medications. The monitor will review dispensing 
and study medication accountability records during site visits and at the completion of the study 
and note any discrepancies.  
4.4.4.2. Return of Study Medication 
When the study is completed or is terminated by Ocuphire, all study material including used and 
unused study medication bottles will be returned to Ocuphire (or its designee) or destroyed under 
the direction of same. All study medication accounting procedures must be completed before the 
study is considered completed. A final study medication disposition will be completed by the 
study coordinator. 
4.5. Expected Duration of Subject Participation 
The total length of subject participation is approximately 5 to 9 days (accommodating for 
weekends) with 2 clinic visits and one telephone call follow-up as summarized below: 
• Screening/Baseline Visit 1 (Day 1)  
• Treatment Visit 2 (Day 4 to 6)  
• Follow-up Call Visit 3 (Day 5 to 9)  
The execution of the entire study (first subject screen through last randomized subject 
completed) is expected to take approximately 6 to 9 months. 


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4.6.Randomization and Procedure for Breaking the Code 
At Visit 1, a randomization code for allocating subjects to Treatment 1 will be prepared by a 
masked biostatistician not connected with the study. Subjects will be then randomized 4:3:3:4 
into 1 of 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Randomization will be stratified 1:1 by iris color (light/dark irides). 
At the initiation of study-related procedures, every subject who is screened is assigned a subject 
number within numerical order within each site. Once a subject is qualified for the study, the 
subject is assigned a randomization number in the order provided by the biostatistician. 
The Treatment 1 study medications will be masked to Investigators, Site Staff, study subjects, 
the clinical research organization (CRO), and Ocuphire. Only in case of medical emergency or 
occurrence of SAEs will the randomization code be unmasked by the Medical Monitor and made 
available to the Investigator, Ocuphire, and/or other personnel involved in the monitoring or 
conduct of this study. 
Treatment 2 (LDP or No Treatment) is unmasked and will be administered on Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments.  
Randomization for both Treatments will occur at Visit 1. 
4.7.Collection of Data 
Study-specific data that have been outlined in the protocol will be entered into the clinical 
database by individual(s) designated by the Investigator. Data are verified electronically using a 
series of online programmed edit checks that have been created by the Clinical Data Manager 
and programmed by the Clinical Data Programmer or designee. Data discrepancies will be 
brought to the attention of the clinical team and investigated by the Study Monitor and Site Staff. 
Study Monitors will review and verify all data collected in the electronic Case Report Form 
(eCRF) against any applicable source documentation during remote review or scheduled 
monitoring visits. The Study Monitor will work closely with the Site Staff to address any 
discrepancies that have been found so that proper resolutions can be made and documented in the 
clinical database. An audit trail within the system will track all changes made to the data. 
4.8.Completed Subject 
A completed subject is defined as one who completes all planned procedures through the end of 
Visit 2. 
4.9.Non-completing Subject 
A non-completing subject is defined as one who exits the study by their own volition or at the 
discretion of the Investigator and/or the Medical Monitor prior to completing all of the study 
procedures required in this protocol. Any subject may decide to voluntarily withdraw from the 
study at any time without prejudice.  


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4.9.1. Study Medication Discontinuation 
The study medication may be discontinued for the following reasons: 
• Adverse Events: AEs include clinically significant laboratory abnormalities and 
intercurrent diseases reported by the subject or observed by the Investigator with 
documentation on the eCRF. 
• Death: If a subject dies, the AE(s) that caused the death should be documented on the 
eCRF and be noted as serious and fatal. 
• Disallowed concurrent medication: Any medication not allowed by the protocol would 
be a protocol violation. 
• Lack of efficacy: A subject may elect to discontinue participation in the study for a 
perceived lack of efficacy. 
• Investigator decision: A subject may be discontinued for reasons other than those 
bulleted previously if the Investigator thinks it is not in the best interest of the subject to 
continue. 
• Pregnancy:  A subject may be discontinued from study medication if pregnancy occurs 
while on study. 
• Other: If there is any other reason for subject discontinuation, such as pregnancy, this 
should be noted on the eCRF. 
The reason for premature study medication discontinuation should be entered onto the 
appropriate eCRF.  
4.9.2. Reasons for Withdrawal From Study 
• Subject withdraws consent. 
• Subject is lost to follow-up. 
• Subject withdraws for other reason. 
4.9.3. Entire Study Terminated 
The entire study may be terminated by Investigators or Ocuphire. Prompt, written notice of 
reasonable cause to the other party (Ocuphire or Investigators, respectively) is required. Prompt 
notice to the Institutional Review Board (IRB) and to regulatory authorities is also required. 
4.9.4. Actions After Discontinuation 
All subjects who discontinue study medication due to a report of an AE must be followed up and 
provided appropriate medical care until their signs and symptoms have remitted or stabilized or 
until medical assessments have returned to acceptable or pre-study limits. 
For any subject who chooses to withdraw consent or who is non-compliant, every possible effort 
should be made by the Investigator to assure the 6-hour measurements are assessed prior to 
discontinuation, in addition to a follow-up telephone call that includes assessments for AEs, 
concomitant medications, and subject-evaluated conjunctival hyperemia.  
4.10. Completed Study 
The study is completed when all randomized subjects have completed or discontinued the study, 
all eCRFs have been completed, and all eCRF data entered into the database. Final database lock 


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will occur after the last randomized subject completes last visit, all data have been entered, and 
all queries resolved.  
4.11.Procedure After the Completion of the Study 
When the study is completed, the CRO will provide Ocuphire and the Investigator with a brief 
(i.e., 1-3 pages) report, containing a description of the study, the number of subjects enrolled, the 
number of subjects completed, the number of subjects who dropped out and why, efficacy 
findings, and AEs. 
 
5. SUBJECT INCLUSION AND EXCLUSION CRITERIA 
5.1 
Subject Inclusion Criteria 
1. Males or females ≥ 40 and ≤ 64 years of age. 
2. Able to comply with all protocol-mandated procedures independently and to attend all 
scheduled office visits. 
3. Able and willing to give signed informed consent. 
4. Able to self-administer study medication throughout the study period. 
5. BCDVA of 0.0 LogMAR (20/20 Snellen equivalent) or better in each eye under photopic 
conditions. 
6. DCNVA of 0.4 LogMAR (20/50 Snellen equivalent) or worse under photopic conditions in 
each eye and binocularly. 
7. Subjects who depend on reading glasses or bifocals in which their binocular best-corrected 
near VA is 0.1 LogMAR (20/25 Snellen equivalent) or better. 
 
5.2 
Subject Exclusion Criteria 
Subjects excluded from the study will be individuals with the following characteristics: 
Ophthalmic (in either eye): 
1. Use of any topical prescription or over-the-counter (OTC) ophthalmic medications of any 
kind within 7 days of Screening until study completion, with the exception of lid scrubs with 
OTC products (e.g., OCuSOFT® lid scrub, SteriLid®, baby shampoo, etc.). 
2. Use of any OTC artificial tears (preserved or unpreserved) at least once per day within 7 days 
of Screening until study completion. 
3. Current use of any topical ophthalmic therapy for dry eye (e.g., Restasis, Xiidra, etc.). 
4. Tear break-up time of < 5 seconds or corneal fluorescein staining (≥ grade 2 in the inferior 
zone or ≥ grade 1 in the central zone using the National Eye Institute scale). 
5. Clinically significant ocular disease (e.g., cataract, glaucoma, corneal edema, uveitis, retinal 
degeneration, loss of visual field, any macular pathology) that might interfere with the study 
as deemed by the Investigator. 
6. Recent or current evidence of ocular infection or inflammation in either eye (such as current 
evidence of clinically significant blepharitis, conjunctivitis, keratitis, etc.). Subjects must be 
symptom free for at least 7 days. 


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7. Any history of herpes simplex or herpes zoster keratitis. 
8. History of diabetic retinopathy or diabetic macular edema. 
9. Known allergy, hypersensitivity, or contraindication to any component of the phentolamine, 
pilocarpine, or vehicle formulations.  
10. History of cauterization of the punctum or punctal plug (silicone or collagen) insertion or 
removal. 
11. Ocular trauma, ocular surgery (e.g., IOLs), ocular laser treatment within the 6 months prior 
to Screening. Any subject with multifocal IOLs are excluded. 
12. History of any traumatic (surgical or nonsurgical) or non-traumatic condition affecting the 
pupil or iris (e.g., irregularly shaped pupil, neurogenic pupil disorder, iris atrophy, iridotomy, 
iridectomy, iritis, etc.). 
13. Unwilling or unable to discontinue use of contact lenses at Screening until study completion. 
14. Conjunctival hyperemia ≥ grade 2 on the CCLRU 4-point scale. 
Systemic: 
15. Known hypersensitivity or contraindication to alpha- and/or beta-adrenoceptor antagonists 
(e.g., chronic obstructive pulmonary disease or bronchial asthma; abnormally low BP or HR; 
second- or third-degree heart blockage or congestive heart failure). 
16. Known hypersensitivity or contraindication to any systemic cholinergic 
parasympathomimetic agents. 
17. Clinically significant systemic disease (e.g., uncontrolled diabetes, myasthenia gravis, 
cancer, hepatic, renal, endocrine, or cardiovascular disorders) that might interfere with the 
study as deemed by the Investigator. 
18. Initiation of treatment with, or any changes to, the current dosage, drug, or regimen of any 
systemic adrenergic or cholinergic drugs within 7 days prior to Screening or during the study. 
19. Participation in any investigational study within 30 days prior to Screening.  
20. Females of childbearing potential who are pregnant, nursing, planning a pregnancy, or not 
using a medically acceptable form of birth control. Acceptable methods include the use of at 
least one of the following: intrauterine device (IUD), hormonal (oral, injection, patch, 
implant, ring), barrier with spermicide (condom, diaphragm), or abstinence. A female is 
considered to be of childbearing potential, unless she is 1 year postmenopausal or 3 months 
post-surgical sterilization. All females of childbearing potential including those with 
post-tubal ligation must have a negative urine pregnancy test result at Visit 1 
(Screening/Baseline).  
21. Resting HR outside the specified range of 50 to 110 beats per minute following at least a 
5-minute rest period in the sitting position at the Screening Visit 1. HR may be repeated only 
once if outside the specified range, following another 5-minute rest period in the sitting 
position. 
22. Hypertension with resting diastolic BP > 105 mmHg or systolic BP > 160 mmHg following 
at least a 5-minute rest period in the sitting position at the Screening Visit 1. BP may be 
repeated only once if outside the specified range, following another 5-minute rest period in 
the sitting position.  
 


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6. TREATMENT OF SUBJECTS 
A sample size of approximately 140 subjects (40 for Nyxol + LDP and Placebo alone arms and 
30 for each of the other 2 arms) who are evaluable for efficacy is needed for the study.  
Eligible subjects randomized into 1 of 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Subjects will be randomized 4:3:3:4 into the above groups. Randomization will be stratified by 
iris color (light/dark irides). 
It is assumed that there will be approximately 7% drop-out between Visit 1 and end of Visit 2. 
To account for this drop-out, a total of approximately 152 subjects will be randomized into the 
study.  
6.1.Treatment Adherence 
All subjects will be instructed on the importance of following the once-daily dosing regimen for 
Treatment 1. Beginning the day of Visit 1 (Screening/Baseline), subjects will instill one drop of 
Treatment 1 study medication in each eye at or near bedtime (approximately 8PM-10PM) and 
repeat this procedure daily up to 3 additional times, for a total of up to 4 doses.  
Effort should be made to schedule Visit 2 when the patient is onsite for Visit 1, and, if possible, 
Visit 2 should be scheduled within 3 or 4 days of Visit 1. If Visit 2 will be greater than 4 days 
after Visit 1, the subject should not start their medication until 3 or 4 days prior to Visit 2 and 
should take their medication for those consecutive days immediately prior to Visit 2. 
Subjects are to bring unused bottles into the study site on Visit 2. Adherence to Treatment 1 will 
be measured by counting the dropper bottles at the start of the study and those remaining at each 
study visit.  
Treatment 2 study medication will be administered by a designated Site Staff member, distinct 
from the Site Staff member recording measurements/assessments, at Visit 2. 
6.2.Concomitant Medications 
As noted in the exclusion criteria (Section 5.2), use of any topical prescription or OTC 
ophthalmic medications of any kind within 7 days of Screening, with the exception of lid scrubs 
with OTC products (e.g., OCuSOFT® lid scrub, SteriLid®, baby shampoo, etc.) is prohibited.  
Additionally, initiation of treatment with or any changes to the current dosage, drug, or regimen 
of any systemic adrenergic or cholinergic drugs (Appendix 3) within 7 days prior to Screening, 
or during the study, is prohibited. However, a subject can be treated with a systemic 
adrenoceptor antagonist, for example, as long as the particular agent and its dose and regimen 
had been consistent for the 7 days prior to Screening, and there was no reason to believe that 
alteration would be necessary at some point later during the study. 
If there is any question about whether a medication is acceptable, the Medical Monitor should 
be consulted before proceeding. 
Use of all medications should be documented on the appropriate eCRF. Investigators are 
encouraged to contact the Medical Monitor for any questions regarding allowed medications. 


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Judgment of continued study participation by the subject, and inclusion of this subject’s 
subsequent visits in the safety and efficacy analysis will be made by Ocuphire. 
All medications that the subject has taken within 30 days prior to the Screening Visit and during 
the study will be recorded in the eCRF. The name of the drug, dose, route of administration, 
duration of treatment, and indication will be recorded for each medication. For combination and 
products (e.g., Contac®, Cosopt®) and non-combination products, the generic name is desired. 
The use of routine ophthalmic diagnostic pharmaceutical agents (e.g., fluorescein and local 
anesthetic) will be allowed and should be documented. Any change in dosing parameters should 
also be recorded in the eCRF. 
7. ASSESSMENT OF EFFICACY 
7.1.Specification of the Efficacy Parameters 
The primary efficacy endpoint is the percent of subjects with ≥ 15 letters of improvement in 
photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP compared to Placebo alone. 
The improvement in binocular DCNVA for each subject is relative to the subject’s own Baseline 
value (Visit 1). 
Secondary efficacy endpoints, which will be assessed for the study eye, the fellow eye, and 
binocular can be found in Section 4.1.  
All of the efficacy endpoints will be analyzed overall and by light/dark irides at all timepoints.  
All of the efficacy endpoints will also be analyzed by mITT and PP populations. 
7.2.Assessing, Recording, and Analyzing of Efficacy Parameters 
VA and PD assessments will be measured at Visit 1 (Screening/Baseline) and Visit 2 
(Treatment), as described in Section 4.1. 
The photopic lighting 
 
 
 
. The mesopic lighting 
 
 
 
 
When near vision is being tested, 
 
 The luminance 
 
 
. Subjects will be allowed to acclimate to these lighting conditions (with 
the eyes open normally for a 
 to the set of PD, DCNVA, BCDVA, 
and BCIVA). Subjects will sit in the exam chair facing directly at the illuminated chart during 
the acclimation period and for all assessments.  
Room lights may be turned on for the scheduled remaining safety assessments (e.g., conjunctival 
hyperemia, AEs, subject questionnaire, etc.). The subject should be in the same room for all 
assessments, and every effort will be made to have the same person perform the measurements at 
all visits. 
7.2.1.Visit 1 (Screening/Baseline) – Day 1 
Individuals who are potential subjects are identified by the study center to schedule the 
Screening Visit. The Screening Visit shall occur the same day as the Baseline Visit.  


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Once a subject arrives at the study center, a member of the Site Staff will interview the 
individual as to their qualifications for participation in the study, and if the subject wishes to 
continue, the informed consent form is signed, and a subject number is assigned.  
The start of Screening includes an explanation of the study, a medical and ophthalmic history, 
and demographics. 
 
This shall be 
followed by a urine pregnancy test for females of childbearing potential (
 
), and HR/BP (
  
The subject will then undergo several VA measurements, including photopic BCDVA as well as 
mesopic and photopic DCNVA (
 In addition, subjects will undergo 
measurement of photopic best-corrected intermediate visual acuity (BCIVA) and mesopic 
BCDVA. 
The screening assessment will also include an ophthalmic examination that includes assessment 
of PD, biomicroscopy, dry eye examination with tear break-up time testing and corneal 
fluorescein staining (
), intraocular pressure (IOP) measurement (using a 
Tono-Pen), and direct or indirect ophthalmoscopy without dilation.  
In addition, conjunctival hyperemia will be assessed visually with a 4-point grading scale using 
images from the CCLRU (
).  
If all eligibility criteria are met, the subject will be randomized into the study.  
After randomization, masked Treatment 1 study medications (Nyxol or Placebo) will be 
dispensed.  
All treatments and assessments will be administered OU. 
Please see Table 2 for details on measurements expected at Visit 1. 
All treatments and assessments will be administered OU. 
7.2.2. Visit 2 (Treatment 2) – Day 4 
Visit 2 may occur 3 to 5 days after Visit 1. On the morning of Visit 2, subjects will undergo 
certain assessments immediately prior to receiving Treatment 2 at 0 minutes: 
• Urine pregnancy test for females of childbearing potential 
• HR/BP  
• Concomitant medications  
• 
 
 
 
 
Treatment 2 (LDP or No Treatment) is unmasked and will be administered on Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments.  
After instillation of Treatment 2 (LDP or No Treatment), subjects will undergo certain 
assessments at multiple timepoints after Time 0 (from 30 minutes to 6 hours): 
• PD 


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• DCNVA (mesopic and photopic), BCDVA (mesopic and photopic), BCIVA (photopic) – 
monocular and binocular 
• Conjunctival hyperemia 
• Adverse events (AEs) 
• Subjective ocular tolerability 
Biomicroscopy, IOP, and ophthalmoscopy will be performed at the 6-hour timepoint. 
There is a window of 
 for every timepoint to complete measurement of the primary 
endpoint of photopic DCNVA. Sites should start pupil diameter measurements approximately 5 
minutes prior to each timepoint in order to allow sufficient time to perform the primary endpoint of 
photopic DCNVA at the target timepoint. 
7.2.3.Visit 3 (Safety Follow-Up Call) – Day 5  
For Visit 3, which will occur 1 to 3 days after Visit 2, subjects will receive a safety follow-up 
call to review concomitant medications and AEs. 
7.2.4. Unscheduled Visits 
An unscheduled visit may be any visit to the Investigator other than the specific visits requested 
in the protocol as possibly required for the subject’s ophthalmic condition. The Investigator will 
perform all procedures necessary to evaluate the study participant at these visits and record any 
AEs in the eCRF. 
As noted in Section 4.9.4, every possible effort should be made by Investigators to assure that 
subjects who discontinue early from the study have a telephone follow-up that includes 
assessments of AEs, concomitant medications, and subject-evaluated conjunctival hyperemia. 
7.2.5. Visit Variation 
Visit 2 may occur 3 to 5 days after Visit 1. Visit 3 occurs 1 to 3 days (any time) after Visit 2.  
8. ASSESSMENT OF SAFETY 
8.1.Specification of Safety Parameters 
The primary safety measures are conjunctival hyperemia, subject-evaluated ocular tolerability, 
and AEs. Other safety measures include biomicroscopy, IOP (as measured with the Tono-Pen), 
and systemic safety (as measured by HR and BP). Urine pregnancy tests for females of 
childbearing potential will be conducted. 
The assessment of safety will be evaluated by: 
• Conjunctival hyperemia measured with CCLRU images using a 4-point scale (0-3) 
(Appendix 4). 
o None (0) = Normal. Appears white with a small number of conjunctival blood 
vessels easily observed 
o Mild (+1) = Prominent, pinkish-red color of both the bulbar and palpebral 
conjunctiva 
o Moderate (+2) = Bright, scarlet red color of the bulbar and palpebral conjunctiva 
o Severe (+3) = Beefy red with petechiae, dark red bulbar and palpebral conjunctiva 
with evidence of subconjunctival hemorrhage  


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• Subject-evaluated ocular tolerability measured on a 4-point scale. 
o 0 – No discomfort 
o 1 – Mild discomfort 
o 2 – Moderate discomfort 
o 3 – Severe discomfort 
• Biomicroscopy of the anterior segment includes evaluation of lids, lashes, cornea, 
conjunctiva, iris, lens, and anterior chamber. Fluorescein staining will be used for tear 
break-up time and/or corneal fluorescein staining assessments. 
• Ophthalmoscopy (direct or indirect) without PD to evaluate the vitreous and posterior 
pole. 
• IOP is measured with the Tono-Pen. 
• HR and BP (as per the site’s normal equipment and procedures). 
• AEs 
8.2. Assessing, Recording, and Analyzing Safety Parameters 
The timing for recording safety parameters may be found in Table 2. 
8.3.Adverse Events and Serious Adverse Events 
All AEs and SAEs that occur following consent and until the final study visit should be collected 
and recorded on the AE or SAE eCRF page. Only TEAEs will be summarized (Section 8.3.1). 
All AEs/adverse reactions occurring during the study (i.e., once the subject has signed the 
informed consent) must be documented, regardless of the assumption of causal relationship, on 
the respective eCRF. All TEAEs/treatment-emergent adverse reactions must be documented 
from the time the subject receives the study medication until the subject’s participation in the 
study has been completed. If a subject has ongoing AEs/adverse reactions at the time of study 
completion or discontinuation from the study, the ongoing AEs/adverse reactions must be 
followed up and provided appropriate medical care until the signs and symptoms have remitted 
or stabilized or until medical assessments have returned to acceptable or pre-study limits. 
Documentation of AEs/adverse reactions includes start date and end date, severity, relationship 
to study medications, action(s) taken, seriousness, and outcome.  
8.3.1. Adverse Event Definitions 
The following definitions of terms apply to this section: 
Adverse event. An AE is any untoward medical occurrence associated with the use of a study 
medication in humans, whether or not it is considered drug related. An AE can therefore be any 
unfavorable and unintended sign (including abnormal laboratory findings), symptom, or disease 
temporarily associated with the use of the study medication, whether or not related to the study 
medication. Study medication includes the investigational drug under evaluation and the 
comparator product or vehicle placebo that is given or administered during any phase of the 
study.  
Medical conditions/diseases present before starting the investigational treatment are only 
considered AEs if they worsen after starting the investigational treatment. Abnormal test results 


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constitute AEs only if they induce clinical signs or symptoms, are considered clinically 
significant, or require therapy. 
The occurrence of AEs should be sought by open-ended questioning of the subject at each visit 
during the study. At each clinic assessment/visit, study personnel should ask the following 
question: “Have you had any problems since your last assessment/visit?” AEs also may be 
detected when they are volunteered by the subject during or between visits or through study 
assessments. 
Life-threatening adverse event or life-threatening suspected adverse reaction. An AE or 
suspected adverse reaction is considered “life-threatening” if, in the view of either the 
Investigator or Ocuphire, its occurrence places the subject or subject at immediate risk of death. 
It does not include an AE or suspected adverse reaction that, had it occurred in a more severe 
form, might have caused death.  
Serious adverse event or serious suspected adverse reaction. An AE or suspected adverse 
reaction is considered ‘‘serious’’ if, in the view of either the Investigator or Ocuphire, it results 
in any of the following outcomes:  
• Death 
• Life-threatening AE 
• Inpatient hospitalization or prolongation of existing hospitalization 
• Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions  
• Congenital anomaly/birth defect  
• Other medically important serious event 
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.  
Treatment on an outpatient emergency basis that does not result in hospital admission, or a 
hospitalization that is elective or is a preplanned treatment for a pre-existing condition that is 
unrelated to the medication under study and has not worsened since the start of the study, is not 
considered an SAE. 
Suspected adverse reaction means any AE for which there is a reasonable possibility that the 
drug caused the AE. For the purposes of Investigational New Drug (IND) safety reporting, 
“reasonable possibility” means there is evidence to suggest a causal relationship between the 
drug and the AE. Suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any AE caused by a drug.  
Unexpected adverse event or unexpected suspected adverse reaction. An AE or suspected 
adverse reaction is considered “unexpected” if it is not listed in the IB or is not listed at the 
specificity or severity that has been observed. “Unexpected,” as used in this definition, also 
refers to AEs or suspected adverse reactions that are mentioned in the IB as occurring with a 
class of drugs or as anticipated from the pharmacological properties of the drug but are not 
specifically mentioned as occurring with the particular drug under investigation. 
The study medication relationship for each AE/adverse reaction should be determined by the 
Investigator using these explanations: 


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• Not related 
• Unlikely related 
• Possibly related 
• Probably related 
• Definitely related 
• Unknown 
Unless the relationship is considered to be “Not related” or “Unlikely related” and there is any 
valid reason, even if undetermined, for suspecting a possible cause-and-effect relationship 
between the study medication and the occurrence of the AE, then the AE should be considered 
“related.”  
If the relationship between the AE/SAE and the investigational product is determined by 
Ocuphire to be anything other than “Not related” or “Unlikely related,” the event will be 
considered to be related to the investigational product for the purposes of expedited regulatory 
reporting. 
Severity of an AE is defined as a qualitative assessment of the level of discomfort of an AE as is 
determined by the Investigator or reported to him/her by the subject. The assessment of severity 
is made irrespective of study medication relationship or seriousness of the event and should be 
evaluated according to the following scale: 
• 1 = Mild: Present, but not distressing, and no disruption of normal daily activity 
• 2 = Moderate: Discomfort sufficient to reduce or affect normal daily activity 
• 3 = Severe: Incapacitating, with inability to work or perform normal daily activity 
A change in severity for a reported AE will require an end date for the previous severity and a 
new start and end date for the new severity. For example, a change in severity may go from mild 
to severe or from severe to moderate. In either case the start or end times/dates should be 
recorded.  
The term “severe” is used to describe the intensity of an event/reaction; the event/reaction itself 
may be of relatively minor medical significance (such as a severe headache). This is not the same 
as a “serious” AE, which is based on a subject/event outcome or action criteria usually associated 
with events that pose a threat to the subject’s life or vital functions. “Seriousness” (NOT 
severity) serves as a guide for defining regulatory reporting obligations. 
Should a pregnancy occur, it must be reported and recorded on the pregnancy form and emailed 
to 
, as well as in the eCRF. Pregnancy in itself is not regarded as 
an AE unless there is a suspicion that an investigational product may have interfered with the 
effectiveness of a contraceptive medication. 
The outcome of all pregnancies (spontaneous miscarriage, elective termination, normal birth, or 
congenital abnormality) must be followed up and documented even if the subject was 
discontinued from the study. 
Action taken in response to an AE is coded as: 
• Dose increased: An indication that a medication schedule was modified by addition; 
either by changing the frequency, strength, or amount. 


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• Dose not changed: An indication that a medication schedule was maintained. 
• Dose reduced: An indication that a medication schedule was modified by subtraction, 
either by changing the frequency, strength, or amount. 
• Dose interrupted: An indication that a medication schedule was modified by temporarily 
terminating a prescribed regimen of medication. 
• Drug withdrawn: An indication that a medication schedule was modified through 
termination of a prescribed regimen of medication. 
• Not applicable: Determination of a value is not relevant in the current context. 
• Unknown: Not known, not observed, not recorded, or refused. 
Additional other action taken: 
• Concomitant medication 
• Hospitalization 
Outcome of an AE is coded as: 
• Fatal: The termination of life as a result of an AE. 
• Not recovered/not resolved: One of the possible results of an AE outcome that indicates 
that the event has not improved or the subject has not recuperated. 
• Recovered/resolved: One of the possible results of an AE outcome that indicates that the 
event has improved or the subject has recuperated. 
• Recovered/resolved with sequelae: One of the possible results of an AE outcome where 
the subject recuperated but retained pathological conditions resulting from the prior 
disease or injury. 
• Recovering/resolving: One of the possible results of an AE outcome that indicates that 
the event is improving. 
• Unknown: Not known, not observed, not recorded, or refused. 
In previous clinical studies of Nyxol, the most frequently reported AE was conjunctival 
hyperemia. 
In the present study, any increase of 2 units in conjunctival hyperemia between consecutive 
timepoints is considered an AE. 
Investigators are cautioned to use the appropriate verbatim term on the AE form to describe this 
observation:  
• Redness related to instillation that is transient (i.e., is no longer present 2 hours after 
instillation) = “conjunctival erythema upon instillation.” 
• Redness that is NOT transient (i.e., is present 2 hours after instillation) = “conjunctival 
hyperemia.” 
Expedited reporting of serious and unexpected adverse events: All SAEs (related and 
unrelated) will be recorded following subject signature of the informed consent and until the 
Safety Follow-Up Call (Visit 3/Day 5). Any SAEs “suspected” to be related to the study 
medication and discovered by the Investigator at any time after the study must be reported. 


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Any SAE that occurs must be reported to the CRO within 24 hours of its occurrence or within 
24 hours of learning of its occurrence. Recurrent episodes, complications, or progression of the 
initial SAE must be reported to the CRO as follow-up to the original episode within 24 hours of 
the Investigator receiving the information. Information about all SAEs will be collected and 
recorded on the SAE eCRF. All pertinent medical records and information collected during the 
treatment and follow-up of the subject should be maintained at the site with a copy emailed 
to VEGA1_Safety@Oculoscr.com. The Investigator must assess the SAE relationship and 
complete the SAE eCRF. The CRO may request additional information. Follow-up information 
(e.g., discharge summary) will be retained in the subject’s chart and a copy will be emailed to 
VEGA1_Safety@Oculoscr.com. In addition, all SAEs should be recorded on the AE eCRF page 
with the serious question marked “Yes.” 
It is the Investigator’s responsibility to notify the approving IRB of any SAEs on a timely basis 
as instructed by Ocuphire following Ocuphire’s determination of causality. All subjects who 
experience an SAE should be followed clinically and undergo the appropriate diagnostic 
evaluations until stabilization or resolution of the event. Ocuphire will report all SAEs to the 
FDA on the appropriate schedule depending if the event is drug related or not drug related, 
expected, or unexpected (based on the available information in the IB). 
Any death occurring during the study and follow-up period must be reported as an SAE. For any 
death occurring through the end of the study, regardless of the degree of relationship to study 
medication, the SAE resulting in the death must be reported to the CRO. A death occurring after 
completion of the study including the Safety Follow-Up Call, that is not reasonably associated 
with study medication administration, does not require completion of the SAE form. 
8.3.2. Follow-Up of Subjects After Adverse Events 
If an AE/adverse reaction occurs, the Investigator will institute support and/or treatment as 
deemed appropriate. All SAEs ongoing at the time of the last visit or discontinuation from the 
study will be followed up until the AE/adverse reaction is resolved or stabilized, the subject is 
lost to follow-up, or there is other resolution to the event.  
 
9. STATISTICS 
9.1.Sample Size 
A sample size of approximately 140 subjects (40 for Nyxol + LDP and Placebo alone arms and 
30 for each of the other 2 arms) who are evaluable for efficacy is needed for the study. The 
primary treatment comparison will be Nyxol + LDP compared to Placebo alone. Using α = 0.05 
significance and a two-tailed test, 76 total subjects (38 subjects each in the Nyxol + LDP and 
Placebo alone arms) evaluable for efficacy will provide at 
 
 
 
  
Subjects will be randomized 4:3:3:4 to Nyxol + LDP, Nyxol + No Treatment 2 (Nyxol alone), 
Placebo + LDP, and Placebo + No Treatment 2 (Placebo alone), respectively. 
It is assumed that there will be approximately 7% drop-out between Visit 1 and end of Visit 2. 
To account for this drop-out, a total of approximately 152 subjects will be randomized into the 
study. 
A diagram outlining treatment arms can be found in Appendix 5. 


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9.2.Analysis Populations 
Modified Intention-to-Treat: The mITT population will include all randomized subjects who 
received at least one drop of Treatment 1 and were administered Treatment 2 (LDP or No 
Treatment). The mITT population will be used to analyze efficacy endpoints. 
Per Protocol Population: The PP population included all subjects in the mITT who received 
one drop of Treatment 1 the day prior to Visit 2 and were administered Treatment 2 at Visit 2, 
had binocular DCNVA and BCDVA in photopic conditions at Baseline (Visit 1) and at Visit 2 
time 1 hour, and had no major protocol deviations. The PP population will be used for the 
primary endpoint analysis and to analyze efficacy endpoints.  
All Randomized Population (ARP): The ARP will include all randomized subjects. This 
population is also known as the Intention-to-Treat (ITT) population. The ARP will be used in 
confirmatory efficacy analyses. 
Safety Population (SP): The SP will include all randomized subjects who have received at least 
one drop of study treatment (Treatment 1 or Treatment 2). The SP will be used to summarize 
safety variables. 
9.3.Statistical Methods  
9.3.1. General Considerations 
All continuous variables will be summarized by treatment and timepoint (as applicable) using 
descriptive statistics (n, mean, median, standard deviation, minimum, and maximum). All 
categorical variables will be summarized by treatment and timepoint (as applicable) using 
frequency counts and percentages.  
All study data will be listed by treatment, subject, and timepoint (as applicable). 
All statistical tests will be performed using a significance level of 5% (two-tailed). The p-values 
for the analysis of secondary efficacy endpoints will be considered descriptive. 
9.3.2. Demographic and Baseline Characteristics 
Demographic and baseline characteristics, such as age, race, and sex, will be summarized by 
treatment arm using the mITT population, PP population, the ARP, and the SP. These data will 
also be provided in by-subject listings. 
9.3.3. Subject Disposition 
Subject disposition, including randomization, and completion and withdrawal from the study will 
be summarized using the ARP. These data will also be provided in by-subject listings. 
9.3.4. Medical History and Prior/Concomitant Medications 
Medical history will be coded using the latest version of Medical Dictionary for Regulatory 
Activities (MedDRA, Version 23.1) and will be summarized by treatment arm using the mITT 
population.  
Prior medications (medications with an end date prior to the date of randomization) and 
concomitant medications (medications with a start or end date after the date of randomization) 
will be coded using World Health Organization Drug Dictionary (WHODrug, Sept 2020) and 
will both be summarized by treatment arm using the SP. 
Medical history and prior and concomitant medications will also be provided in by-subject 
listings.  


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9.3.5. Analysis of Efficacy 
Efficacy will be assessed using the mITT and PP populations with subjects included in the 
treatment arm in which they were randomized. For the analysis of the primary efficacy endpoint, 
appropriate imputation techniques will be performed for missing observations if applicable; 
details will be provided in the study Statistical Analysis Plan. The primary comparison of interest 
is Nyxol + LDP and Placebo alone for the PP population. Confirmatory analyses may be 
performed using the ARP, with imputation performed for missing data. For the analysis of the 
secondary efficacy endpoints, only observed case data will be used. If warranted, confirmatory 
analyses using the ARP with imputation for missing data will also be performed for the 
secondary efficacy endpoints. 
For all efficacy endpoints, Baseline values are defined as the screening assessment values on 
Visit 1. 
All efficacy data will be summarized by treatment group, study day, and timepoint (Baseline and 
Visit 2), as appropriate. 
The primary efficacy endpoint is the percent of subjects with ≥ 15 ETDRS letters of 
improvement in photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP compared to 
Placebo alone. The improvement in binocular DCNVA for each subject is relative to the 
subject’s own Baseline value (Visit 1). The primary efficacy endpoint will be analyzed using a 
logistic regression model with 
 
The percentage of subjects in each treatment arm meeting the criteria, the odds ratio (OR) with 
95% confidence interval (CI) and p-value will be provided. The analysis will be performed using 
the mITT and PP populations, with subjects included in their randomized treatment regardless of 
the treatment they actually received. A sensitivity analysis will be completed for the primary 
efficacy endpoint using the same model but excluding the 
. 
Secondary efficacy endpoints are indicated in Section 4.1.  
Each of the continuous secondary efficacy endpoints will be analyzed using analysis of 
covariance (ANCOVA) with change from Baseline as the dependent variable, treatment, and 
light/dark irides as factors, and the respective Baseline value included as the covariate. Each 
ANCOVA will be performed using the mITT and PP populations with subjects included in their 
randomized treatment regardless of the treatment they actually received. The output from each 
ANCOVA will include the least-squares mean (LSM) and standard error for all treatment groups, 
along with the placebo-corrected LSM for each treatment difference, its 95% CI, and associated 
p-value.  
All continuous secondary endpoints derived from VA assessments, such as change in DCNVA, 
BCIVA, and BCDVA, will be analyzed using ETDRS letters correctly read.  
For each of the secondary endpoints related to percent of subjects achieving certain criteria, the 
analysis will be performed using a logistic regression model with treatment, light/dark irides, and 
the respective Baseline as a covariate. For each analysis, the percentage of subjects in each 
treatment arm meeting the criteria, the OR with 95% CI and p-value will be provided. For these 
endpoints, the mITT and PP populations will be used with subjects included in their randomized 
treatment regardless of the treatment they actually received. 
In addition, each secondary efficacy endpoint will be analyzed by
using the 
same model indicated above but without irides as a factor, as appropriate. Other subgroups, such 
as age, sex, and race, may be analyzed as well.   


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9.3.6. Analysis of Safety 
Safety will be assessed using the SP with subjects included in the treatment arm they actually 
received, regardless of their randomized treatment. Observed case data will be used; no 
imputation will be performed for missing safety data. A statistical analysis of conjunctival 
hyperemia will be conducted as described below; otherwise, no statistical analysis of safety data 
will be performed. 
For HR and BP, Baseline is defined as the Screening value. HR and BP values and change from 
Baseline in the values will be summarized by treatment arm and timepoint (Visit 1 and Visit 2).  
Observed values and change from Baseline (Visit 1) in conjunctival hyperemia at each timepoint 
(Visit 1 at 0 and 1 hour, and Visit 2 at 0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 
and 6 hours), will be summarized for the study eye and fellow eye. Treatments will be compared 
using the same ANCOVA model proposed for the continuous secondary efficacy endpoints. 
For IOP, Baseline is defined as the Screening value. Observed values and change from Baseline 
in IOP at each timepoint (Visit 1 and Visit 2) will be summarized for the study eye and the 
fellow eye. 
Ocular tolerability values will be summarized by treatment arm at each timepoint (Visit 1 and 
Visit 2). 
Verbatim descriptions of AEs will be coded using MedDRA. Only TEAEs (those that occur after 
the first dose of study medication or increasing in severity after initiation of study medication) 
will be summarized. TEAEs and SAEs will be summarized by treatment group, by system organ 
class (SOC), severity, and relationship to study medication. Deaths, withdrawal from study 
medication due to AEs, and withdrawal from the study due to AEs will each by summarized by 
treatment group. Note that in MedDRA, ocular events are coded to the SOC of “special senses.” 
Thus, using SOC in the summaries will provide a separation of ocular and non-ocular AEs. 
All safety data will be provided in by-subject listings.  
9.4. Procedure for Accounting for Missing, Unused, or Spurious Data 
For the summarization and analysis of efficacy data, the focus will be on observed case data 
only. As appropriate, confirmatory efficacy analyses will be performed using imputation for 
missing data; details of the imputation, if performed, will be included in the study Statistical 
Analysis Plan. For the summarization of safety data, observed case data only will be used. 
9.5.Procedure for Reporting Deviations From the Statistical Plan 
Any deviations from the statistical plan will be described and a justification given in the final 
Clinical Study Report.  
 
10. DIRECT ACCESS TO SOURCE DATA AND DOCUMENTS 
The Investigator will permit study-related monitoring visits, audits, IRB review, and regulatory 
inspection(s) by providing direct access to source data and documents. 
 
11. QUALITY CONTROL AND QUALITY ASSURANCE 
The progress of the study will be monitored by onsite, written, and telephone communications 
between personnel at the Investigator’s site and the Medical Monitor. Should the COVID-19 
pandemic restrict monitors from traveling to a site, remote review will be conducted to the extent 
possible, while still ensuring the study is monitored appropriately per applicable regulations and 


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guidelines. The Investigator will allow Ocuphire, the Study Monitor, and the Medical Monitor to 
inspect all eCRFs, subject records (source documents), signed consent forms, records of study 
medication receipt, storage, preparation, and disposition, and regulatory files related to this 
study.  
 
12. ETHICAL CONSIDERATIONS AND GCP COMPLIANCE  
12.1.GCP Compliance 
The proposed study is subject to all applicable governmental rules and regulations concerning 
the conduct of clinical trials on human subjects. This includes, but is not necessarily limited to, 
the approval of IRBs, the Helsinki Declaration, US FDA law, International Council for 
Harmonisation (ICH) GCP guidelines, obtaining prospective informed consent, monitoring of the 
conduct of the study and the completeness of the eCRFs by Ocuphire or its designee(s), and 
appropriate record retention by the Investigator.  
12.2.Institutional Review Board  
This protocol, materials used to recruit subjects, and materials used to document consent must be 
approved by the IRB prior to initiation of the study. Written IRB approval must adequately 
identify the protocol and informed consent. In addition to approving the protocol, the IRB must 
also approve the subject information and consent form, as well as any advertising tools that will 
be used for the study. Copies of all approved materials, all correspondence with the IRB and 
written approval from the IRB must be made available to Ocuphire, prior to the start of subject 
enrollment into the study. 
12.3.Protocol Deviations/Violations 
The Investigator should not deviate from the requirements of this protocol without prior written 
approval of the Medical Monitor or Sponsor, except in the event of a medical emergency. 
A reportable protocol deviation is defined as nonadherence to the protocol that involves 
inclusion/exclusion criteria, affects subject safety, rights or welfare, or has the potential to affect 
the integrity of the data. Examples of major protocol deviations include study enrollment by 
ineligible subject, loss of key data such as equipment malfunction (e.g., pupillometer), and/or use 
of a prohibited medication during the study.  
All protocol deviations will be reported by entering the event in the appropriate eCRF page. 
Protocol deviations should be reported to the IRB in accordance with IRB-specific guidelines. If 
there is any question as to whether the deviation is reportable, Ocuphire or designee and the IRB 
should be contacted. 
All changes to the protocol will be made by the Sponsor or designee as an approved amendment 
to the protocol, submitted to the FDA, and approved by the IRB prior to implementation.  
Changes implemented without prior approval will be considered protocol violations. 
12.4. Informed Consent Requirements 
Written informed consent will be obtained from each subject. A copy of the signed and dated 
consent document will be given to each subject. The original signed and dated informed consent 
document must be maintained in the study files at the Investigator’s site.  
The Investigator is responsible for ensuring that no subject is subject to any study-related 
examination or activity before that subject has given informed consent. The subject must give 


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written consent after the receipt of detailed information. The verbal explanation will cover all the 
elements specified in the written information provided for the subject. 
It should be emphasized that the subject is at liberty to withdraw consent to participate at any 
time, without penalty or loss of benefits to which the subject is otherwise entitled. Subjects who 
refuse to give or who withdraw written informed consent may not be included or continued in 
this study, but this will not impact on their subsequent care.  
The Investigator will inform the subject of the aims, methods, anticipated benefits, and potential 
hazards of the study, including any discomfort it may entail. The subject must be given every 
opportunity to clarify any points he/she does not understand and, if necessary, ask for more 
information. At the end of the interview, the subject may be given time to reflect if this is 
required, or if the subject requests more time. Subjects will be required to sign and date the 
informed consent form.  
A copy of the signed and dated consent document will be given to each subject. The original 
signed and dated informed consent document must be maintained in the study files at the 
Investigator’s site. Signed informed consent must be obtained prior to the conductance of any 
study procedures. 
 
13. DATA HANDLING AND RECORD KEEPING 
All procedures for the handling and analysis of data will be conducted using good computing 
practices meeting ICH and US FDA guidelines for the handling and analysis of data for clinical 
trials. 
13.1.Data Entry 
Study-specific data that have been outlined in the protocol will be entered into the clinical 
database by individual(s) designated by the Investigator.  
13.2.Data Quality Control and Reporting 
Data are verified electronically using a series of programmed edit checks that have been created 
by the Clinical Data Manager and programmed by the Clinical Data Programmer or designee. 
Data discrepancies will be brought to the attention of the clinical team and investigated by the 
clinical research associate (CRA) and Site Staff. CRAs will review and verify all data collected 
in the eCRF against source documentation during scheduled monitoring visits. The CRA will 
work closely with the Site Staff to address any discrepancies that have been found so that proper 
resolutions can be made and documented in the clinical database. An audit trail within the system 
will track all changes made to the data. 
13.3.Archiving of Data 
Archived versions of the database will be saved by Ocuphire consistent with ICH GCP 
guidelines, complying with whichever of the requirements is longer. Ocuphire will notify the 
Investigator when documents should be returned. 
13.4. Records Retention 
The Investigator’s site and clinical laboratory will retain all records related to the study in 
compliance with ICH GCP guidelines. 


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13.5.Amendments to the Protocol 
Modifications of the signed protocol are only possible by approved protocol amendments and 
with the agreement of all responsible persons. The procedure for approval of a protocol 
amendment is identical to that for approval of the protocol. The IRB must be informed of all 
protocol amendments and should be asked for its opinion as to whether a full re-evaluation of the 
ethical aspects of the study is necessary by the committee. This should be fully documented. 
The Investigator must not implement any deviation from or change to the protocol, without 
discussion with, and agreement by Ocuphire and prior review and documented 
approval/favorable opinion of the amendment from the relevant ethics committee, except where 
it is necessary to eliminate an immediate hazard to study subjects, or where the change(s) 
involves only logistical or administrative aspects of the study (e.g., change in monitor, change of 
telephone number). 
Protocol amendments will be submitted to the appropriate authority(ies) as required by the 
applicable regulatory requirement(s). 


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14. REFERENCES 
1. 
McDonald MB, Pitlick W, D.R. V, Meyer AR, E.D. D. Phentolamine Mesylate 
Treatment of Severe Night Vision Complaints. AAO Abstracts, PO433. 2010. 
2. 
Holladay JT, Meyer A, Pitlick B. Phentolamine Mesylate Ophthalmic Solution Once 
Daily Reduces Pupil Diameter and Improves Night Vision Disturbances. AAO Annual Meeting, 
PA025. 2018. 
3. 
Pepose JS, Pitlick B, Meyer A, Jaber R, Charizanis K, Slonim C, et al. Phentolamine 
mesylate ophthalmic solution provides long lasting pupil modulation and improves visual acuity. 
Investigative Ophthalmology & Visual Science. 2020;61(7):5100-. 
4. 
Leavitt JA, Wayman LL, Hodge DO, Brubaker RF. Pupillary response to four 
concentrations of pilocarpine in normal subjects: application to testing for Adie tonic pupil. Am J 
Ophthalmol. 2002;133(3):333-6. 
5. 
Edgar DF, Crabb DP, Rudnicka AR, Lawrenson JG, Guttridge NM, O'Brien CJ. Effects 
of dipivefrin and pilocarpine on pupil diameter, automated perimetry and LogMAR acuity. 
Graefes Arch Clin Exp Ophthalmol. 1999;237(2):117-24. 
6. 
AOA. American Optometric Association - Adult Vision: 41 to 60 Years of Age. 
7. 
McDonnell PJ, Lee P, Spritzer K, Lindblad AS, Hays RD. Associations of presbyopia 
with vision-targeted health-related quality of life. Arch Ophthalmol. 2003;121(11):1577-81. 
8. 
Fricke TR, Tahhan N, Resnikoff S, Papas E, Burnett A, Ho SM, et al. Global Prevalence 
of Presbyopia and Vision Impairment from Uncorrected Presbyopia: Systematic Review, Meta-
analysis, and Modelling. Ophthalmology. 2018;125(10):1492-9. 
9. 
Tucker J, Charman WN. The depth-of-focus of the human eye for Snellen letters. Am J 
Optom Physiol Opt. 1975;52(1):3-21. 
10. 
Dexl AK, Seyeddain O, Riha W, Hohensinn M, Hitzl W, Grabner G. Reading 
performance after implantation of a small-aperture corneal inlay for the surgical correction of 
presbyopia: Two-year follow-up. J Cataract Refract Surg. 2011;37(3):525-31. 
11. 
Park SY, Choi YJ, Jung JW, Choi M, Kim EK, Seo KY, et al. Clinical Efficacy of 
Pinhole Soft Contact Lenses for the Correction of Presbyopia. Semin Ophthalmol. 
2019;34(2):106-14. 
12. 
Hogan TS, McDaniel DD, Bartlett JD, Hart KK, Paggiarino DA. Dose-response study of 
dapiprazole HCl in the reversal of mydriasis induced by 2.5% phenylephrine. J Ocul Pharmacol 
Ther. 1997;13(4):297-302. 
 
 
 


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APPENDIX 1: IRIS COLOR CHART 
Study enrollment includes both light and dark-colored eyes. Examples of light irides (pictures a, 
b, c, d and e) and dark irides (pictures f, g and h) for the purposes of this study are detailed in the 
chart below.  
 
 
 
 
 
 
 
 
a = Light blue 
b = Dark blue 
c = Blue with peripupillary brown 
d = Uniform green 
e = Green with brown iris ring 
f = Central brown and peripheral green 
g = Brown with some peripheral green 
h = Brown 


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APPENDIX 2: VISUAL ACUITY CHARTS 
DCNVA will be measured using the Near Visual Acuity Chart (logarithmic Visual Acuity Chart 
2000, chart #1 and #2) in the Precision Vision Small 914 Illuminator Cabinet (light box) at 16 
inches (~40 cm) (letters recorded, later converted to LogMAR and number of lines). 
 
Near High-Contrast Chart 
 
            
 
 
BCDVA will be measured with a Standard ETDRS illuminated 100% high-contrast chart (on 
wall or stand) at 4 m. 
 
Distance High-Contrast Chart 
 
 
 
 
 
 
 


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APPENDIX 3: ADRENERGIC AND CHOLINERGIC DRUGS 
The following drugs are examples of drugs which cannot be used within 7 days prior to Screening or 
during the study unless the drug, dose, and regimen has been consistent for the 7 days prior to 
Screening. This list is not inclusive of all drugs in these classes. If there is any doubt, please 
consult with the Medical Monitor. 
Alpha-1-agonists 
Methyl 
norepinephrine 
Naphazoline 
Oxymetazoline 
Tetrahydrozoline 
Phenylephrine 
Xylometazoline 
 
Alpha-2-agonists 
Brimonidine 
Clonidine 
Guanfacine 
Guanabenz 
Guanoxabenz 
Guanethidine 
Xylazine 
Tizanidine 
Methyldopa 
Non-selective alpha-
antagonists 
Phenoxybenzamine 
Tolazoline 
Labetalol 
Carvedilol 
 
Alpha-1-antagonists  
Alfuzosin 
Prazosin 
Doxazosin 
Tamsulosin 
Terazosin 
 
Alpha-2-antagonists 
Atipamezole 
Idazoxan 
Yohimbine  
 
 
Acetylcholine receptor 
agonists 
Pilocarpine (M3 
receptors) 
 
Acetylcholine receptor 
antagonists 
Scopolamine 
Dicycloverine 
Tolterodine 
Oxybutynin 
Ipratropium 
Mamba Toxin 
(MT7) 
Pirenzepine 
Telenzepine 
 
Antivertigo 
Meclizine  
Scopolamine 
Gastrointestinal 
Atropine  
Belladonna 
 
Parkinsonism 
Amantadine 
Benztropine 
Biperiden 
Trihexyphenidyl 
 
 


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APPENDIX 4: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING IMAGES 
FROM CCLRU 
 
 
None (0) 
Mild (+1) 
Moderate (+2) 
Severe (+3) 
Normal. Appears 
white with a small 
number of 
conjunctival blood 
vessel easily 
observed 
Prominent, pinkish-
red color of both the 
bulbar and palpebral 
conjunctiva 
Bright, scarlet red 
color of the bulbar 
and palpebral 
conjunctiva 
Beefy red with 
petechiae, dark red 
bulbar and palpebral 
conjunctiva with 
evidence of 
subconjunctival 
hemorrhage  
 
 
  
 
 
 


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APPENDIX 5: TREATMENT GROUPS 
 

SAP_001.pdf: 
  
 
CONFIDENTIAL 
 
 
 
 
Ocuphire Pharma, Inc. 
 
STATISTICAL ANALYSIS PLAN 
 
 
 
Protocol Title: 
Randomized, Placebo-Controlled, Double-Masked Study of the 
Safety and Efficacy of Nyxol (0.75% Phentolamine Ophthalmic 
Solution) with Low-Dose (0.4%) Pilocarpine Eye Drops in 
Subjects with Presbyopia 
Study Number: 
OPI-NYXP-201 (VEGA-1) 
Phase: 
Phase 2 
Sponsor: 
Ocuphire Pharma, Inc. 
37000 Grand River Avenue, Suite 120 
Farmington Hills, MI 48335 USA 
 
Author: 
Summit Analytical, LLC 
8354 Northfield Blvd. 
Bldg. G Suite 3700 
Denver, CO 80238 
SAP Date: 
2021-06-10 
Status: 
FINAL V1.0 
 
 
 


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1. 
TABLE OF CONTENTS 
1. 
TABLE OF CONTENTS ............................................................................................................................... 2 
2. 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS................................................................... 5 
3. 
INTRODUCTION ......................................................................................................................................... 7 
3.1. 
Preface .............................................................................................................................................. 7 
3.2. 
Purpose of Analyses .......................................................................................................................... 7 
3.3. 
Summary of Statistical Analysis Changes to the Protocol ................................................................ 7 
4. 
STUDY OBJECTIVES AND ENDPOINTS ................................................................................................. 8 
4.1. 
Study Objectives ............................................................................................................................... 8 
4.2. 
Study Endpoints ................................................................................................................................ 8 
4.2.1. 
Primary Endpoints ........................................................................................................... 8 
4.2.2. 
Secondary Endpoints ....................................................................................................... 9 
5. 
STUDY METHODS .................................................................................................................................... 12 
5.1. 
General Study Design and Plan....................................................................................................... 12 
5.2. 
Inclusion – Exclusion Criteria and General Study Population ........................................................ 14 
5.3. 
Randomization and Masking........................................................................................................... 14 
5.4. 
Analysis Variables .......................................................................................................................... 15 
6. 
SAMPLE SIZE ............................................................................................................................................ 17 
7. 
GENERAL CONSIDERATIONS ............................................................................................................... 18 
7.1. 
Analysis Populations ....................................................................................................................... 18 
7.1.1. 
Modified Intention-to-Treat (mITT) ............................................................................. 18 
7.1.2. 
Per Protocol Population (PP) ......................................................................................... 18 
7.1.3. 
All Randomized Population (ARP) ............................................................................... 18 
7.1.4. 
Safety Population (SP) .................................................................................................. 18 
7.2. 
Covariates and Subgroups ............................................................................................................... 18 
7.2.1. 
Planned Covariates ........................................................................................................ 18 
7.2.2. 
Planned Subgroups ........................................................................................................ 18 
7.3. 
Management of Analysis Data ........................................................................................................ 19 
7.3.1. 
Data Handling ............................................................................................................... 19 
7.3.2. 
Missing Data ................................................................................................................. 19 
7.3.2.1. 
Handling of Missing Date Values ............................................................ 19 
7.3.2.2. 
Missing Baseline Data ............................................................................. 20 
7.3.2.3. 
Imputation Methods ................................................................................. 20 
7.3.3. 
Handling of Early Termination Visit Information ......................................................... 21 
7.3.4. 
Pooling of Investigational Sites ..................................................................................... 21 
7.3.5. 
Coding Conventions for Events and Medications ......................................................... 22 
7.3.6. 
Analysis Software ......................................................................................................... 22 
7.3.7. 
Study Data ..................................................................................................................... 22 
7.4. 
Planned Study Analyses .................................................................................................................. 23 
7.4.1. 
Statistical Summaries: Descriptive and Inferential ....................................................... 23 
7.4.2. 
Interim Analyses and Data Monitoring ......................................................................... 23 
7.4.3. 
Final Analysis and Publication of Study Results .......................................................... 23 
7.5. 
Multiple Testing Procedures ........................................................................................................... 23 
7.6. 
Baseline Values ............................................................................................................................... 23 
8. 
SUMMARY OF STUDY DATA ................................................................................................................ 24 
8.1. 
Subject Disposition ......................................................................................................................... 24 


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8.2. 
Protocol Deviations ......................................................................................................................... 24 
8.3. 
Demographics and Baseline Characteristics ................................................................................... 24 
8.4. 
Medical History .............................................................................................................................. 25 
8.5. 
Prior and Concurrent Medications .................................................................................................. 25 
8.6. 
Treatment Administration ............................................................................................................... 25 
9. 
EFFICACY ANALYSES ............................................................................................................................ 26 
9.1. 
Clinical Efficacy ............................................................................................................................. 26 
9.1.1. 
Primary Efficacy Analysis ............................................................................................ 26 
9.1.2. 
Secondary Efficacy Analyses ........................................................................................ 27 
10. SAFETY ANALYSES................................................................................................................................. 29 
10.1. 
Adverse Events ............................................................................................................................... 29 
10.2. 
Deaths, Serious Adverse Events and Other Significant Adverse Events ........................................ 30 
10.2.1. 
Deaths............................................................................................................................ 30 
10.2.2. 
Serious Adverse Events ................................................................................................. 30 
10.2.3. 
Adverse Events Leading to Withdrawal from the Study ............................................... 30 
10.2.4. 
Adverse Events Leading to Discontinuation of Study Medication ............................... 30 
10.3. 
Conjunctival Hyperemia ................................................................................................................. 30 
10.4. 
Subjective Ocular Tolerability ........................................................................................................ 30 
10.5. 
Vital Signs ....................................................................................................................................... 31 
10.6. 
IOP .................................................................................................................................................. 31 
10.7. 
Biomicroscopy and Ophthalmoscopy ............................................................................................. 31 
10.8. 
Other Safety Measures .................................................................................................................... 31 
11. REFERENCES ............................................................................................................................................ 32 
12. APPENDICES ............................................................................................................................................. 33 
12.1. 
List of Planned Tables ..................................................................................................................... 33 
12.2. 
List of Planned Listings .................................................................................................................. 39 
 
 


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LIST OF TABLES 
 
Table 1 
Visit Schedule ............................................................................................................... 13 
Table 2 
Snellen Equivalents to VA Assessment Letters Read ................................................... 27 
 
 
LIST OF FIGURES 
 
Figure 1 
Randomization schema by investigational treatment and irides 
type ................................................................................................................................ 15 
Figure 2 
SDTM, ADaM, and TFL Development and Validation ................................................ 22 
 
 
 


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2. 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Only abbreviations and terms relevant to the SAP are repeated herein. The reader is referred 
to the protocol for the complete and comprehensive list of abbreviations and definitions of 
terms for the study. 
Abbreviation/Term 
Definition 
ADaM 
Analysis Data Model 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ARP 
All Randomized Population 
ATC 
Anatomical Therapeutic Chemical  
BCDVA 
best-corrected distance visual acuity  
BP 
blood pressure 
°C 
degrees Celsius 
CCLRU 
Cornea and Contact Lens Research Unit 
CDISC 
Clinical Data Interchange Standards Consortium  
CFR 
Code of Federal Regulations 
CI 
confidence interval 
CSR 
clinical study report 
DCIVA 
distance-corrected intermediate visual acuity 
DCNVA 
distance-corrected near visual acuity 
DLD 
dim light vision disturbances (also referred to as night vision 
disturbances or NVD) 
eCRF 
electronic Case Report Form 
EDC 
electronic data capture 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
°F 
degrees Fahrenheit 
FDA 
Food and Drug Administration 
HR 
heart rate 
IOP 
intraocular pressure 
IUD 
intrauterine device 
LCVA 
low-contrast visual acuity 
LDP 
Low-Dose (0.4%) Pilocarpine Ophthalmic Solution 
LDPE 
low-density polyethylene 
LSM 
least-squares mean 
MCMC 
Markov Chain Monte Carlo 


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Abbreviation/Term 
Definition 
MedDRA 
Medical Dictionary for Regulatory Activities 
mITT 
Modified Intention-to-Treat 
Nyxol 
0.75% Phentolamine Ophthalmic Solution or 1% Phentolamine 
Mesylate Ophthalmic Solution  
Nyxol + LDP 
Nyxol dosed in the evening with LDP dosed during the day 
OD 
oculus dexter (right eye) 
OR 
odds ratio 
OU 
oculus uterque (both eyes) 
PD 
pupil diameter 
POS 
Phentolamine Ophthalmic Solution 
PP 
Per Protocol 
PT 
preferred term 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SDTM 
study data tabulation model 
SE 
standard error 
SOC 
system organ class 
SP 
Safety Population 
TEAE 
treatment-emergent adverse event 
TFL 
tables, figures, and listings 
VA 
visual acuity 
WHO-DD 
World Health Organization Drug Dictionary 


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3. 
INTRODUCTION 
3.1. 
Preface 
This document presents a statistical analysis plan (SAP) for Ocuphire Pharma, Inc. Protocol 
OPI-NYXP-201 (VEGA-1) (Randomized, Placebo-Controlled, Double-Masked Study of the 
Safety and Efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) with Low-Dose 
(0.4%) Pilocarpine Eye Drops in Subjects with Presbyopia). 
Reference materials for this statistical plan include the protocol OPI-NYXP-201 
(30NOV2020) and Case Report Forms (CRFs; Version 01FEB2021).  
The SAP described hereafter is an a priori plan. The SAP will be finalized and approved 
prior to unmasking of any study data.     
For the reasons stated here, the conduct of the study in the field is considered to be 
independent of any study outcome that might materialize upon enactment of the currently 
proposed statistical plan.  
3.2. 
Purpose of Analyses 
The VEGA-1 study is a randomized, parallel arm, double-masked, placebo-controlled study 
of the safety and efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) with Low-
Dose (0.4%) Pilocarpine Ophthalmic Solution (LDP) in subjects with presbyopia. 
The Sponsor intends to use this Phase 2 study to evaluate Nyxol + LDP for the chronic 
indication of “temporary treatment of presbyopia.” 
Post-hoc exploratory analyses not identified in this SAP may be performed to further 
examine the study data. These analyses will be clearly identified, where appropriate, in the 
final clinical study report (CSR). Additional analyses not prospectively identified in this SAP 
may also be completed for publications, or regulatory or funding inquiries. These analyses, if 
performed, may not be reported in the CSR but will be fully detailed in the document 
containing the additional analyses. 
3.3. 
Summary of Statistical Analysis Changes to the Protocol 
The wording of the first secondary objective was changed from “< 1 line of loss” to “>1 line 
of loss”. An additional secondary endpoint for the percentage of subjects with distance-
corrected near visual acuity (DCNVA) ≤ 20/15, ≤ 20/20, ≤ 20/25, ≤ 20/32, ≤ 20/40, ≤ 20/50, 
≤ 20/63, ≤ 20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 was added to Section 4.2.2. The 
secondary endpoints originally specified as the percentage of subjects with improvement in 
best-corrected distance visual acuity (BCDVA) was changed to improvement or loss in 
BCDVA. The Snellen ranges for secondary endpoints with discrete ranges were changed to 
25/50 to 20/63, 20/80 to 20/100, and 20/125 to 20/160. Otherwise, the analyses described in 
this analysis plan are consistent with the analyses described in the study protocol. 


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4. 
STUDY OBJECTIVES AND ENDPOINTS 
Study objectives and endpoints defined in the protocol include safety and efficacy endpoints. 
Objectives and pre-specified endpoints are as follows: 
4.1. 
Study Objectives 
The objectives of this study are as follows: 
Primary objective 
• To evaluate the efficacy of Nyxol + LDP to improve distance-corrected near visual 
acuity (DCNVA) compared to Placebo alone in subjects with presbyopia 
 
Secondary objectives 
• To evaluate the efficacy of Nyxol + LDP to prevent >1 line of loss of best-corrected 
distance visual acuity (BCDVA) 
• To evaluate the efficacy of Nyxol + LDP to improve DCNVA compared to Nyxol 
alone or LDP alone   
• To evaluate the ability of Nyxol + LDP to produce the “pinhole” pupil size 
(approximately 1.6-2 mm) 
• To evaluate the efficacy of Nyxol + LDP to improve distance-corrected intermediate 
visual acuity (DCIVA) and BCDVA 
• To evaluate the effect of iris color on the efficacy of Nyxol + LDP  
• To evaluate the ocular and systemic safety of Nyxol + LDP and each component 
individually 
4.2. 
Study Endpoints 
4.2.1. 
Primary Endpoints 
Endpoints will be evaluated following administration of masked Nyxol or Placebo 
(Treatment 1) at or near bedtime for 3 to 4 days, with administration of LDP or No Treatment 
(Treatment 2) at the next visit (Visit 2) in the morning. Analysis will be completed for  4 
treatment arms: 
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP (LDP alone) 
• Placebo + No Treatment 2 (Placebo alone) 


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The primary efficacy endpoint is the percent of subjects with ≥ 15 letters of improvement in 
photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP compared to Placebo 
alone. The improvement in binocular DCNVA for each subject is relative to the subject’s 
own Baseline value (Visit 1). The analysis of the primary endpoint will be completed for the 
per-protocol (PP) population, as defined in Section 7.1.2 below. 
4.2.2. 
Secondary Endpoints 
Secondary endpoints for efficacy and safety assessments include the following: 
Efficacy: 
Secondary efficacy endpoints will be analyzed by study eye, fellow eye, and binocular unless 
otherwise indicated, and will include: 
• Percentage of subjects with improvement of ≥ 15 letters in DCNVA (photopic) at 1 
hour and with < 5 letters of loss in photopic binocular BCDVA from Baseline (Visit 
1) to Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours comparing 
Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone  
• Percentage of subjects with improvement of ≥ 5, ≥ 10, and ≥ 15 letters in DCNVA 
(photopic) from Baseline (Visit 1) to Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 
hours, and 6 hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and 
Placebo alone  
• Change in DCNVA (photopic) from Baseline (Visit 1) to Visit 2 at 30 minutes, 1 
hour, 2 hours, 3 hours, 4 hours, and 6 hours comparing Nyxol + LDP to Nyxol alone, 
Placebo + LDP, and Placebo alone   
• Percentage of subjects with improvement in DCNVA (mesopic) from Baseline (Visit 
1) to Visit 2 at 2 hours, 4 hours, and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing 
Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone   
• Change in DCNVA (mesopic) from Baseline (Visit 1) to Visit 2 at 2 hours, 4 hours, 
and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing Nyxol + LDP to Nyxol alone, 
Placebo + LDP, and Placebo alone   
• Percentage of subjects with improvement in DCIVA (photopic) from Baseline (Visit 
1) to Visit 2 at 1 hour, 3 hours, and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing 
Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone  
• Change in DCIVA (photopic) from Baseline (Visit 1) to Visit 2 at 1 hour, 3 hours, 
and 6 hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo 
alone   
• Percentage of subjects with improvement or loss in BCDVA (photopic) from 
Baseline (Visit 1) to Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 
hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing Nyxol + LDP to Nyxol alone, Placebo 
+ LDP, and Placebo alone   


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• Change in BCDVA (photopic) from Baseline (Visit 1) to Visit 2 at 30 minutes, 1 
hour, 2 hours, 3 hours, 4 hours, and 6 hours comparing Nyxol + LDP to Nyxol alone, 
Placebo + LDP, and Placebo alone   
• Percentage of subjects with improvement or loss in BCDVA (mesopic) from Baseline 
(Visit 1) to Visit 2 at 1 hour, 3 hours, and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters 
comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone   
• Change in BCDVA (mesopic) from Baseline (Visit 1) to Visit 2 at 1 hour, 3 hours, 
and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing Nyxol + LDP to Nyxol alone, 
Placebo + LDP, and Placebo alone   
• Change and percent change in Pupil Diameter (PD) (mesopic and photopic) from 
Baseline (Visit 1) to Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 
hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone   
• Percentage of subjects with a PD (mesopic and photopic) of < 2.4, < 2.2, < 2.0, < 1.8, 
< 1.6, and < 1.4 mm measured on Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 
hours, and 6 hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and 
Placebo alone  
• Percentage of subjects with a PD (mesopic and photopic) of 1.6 to < 2 mm, 2 to < 2.4 
mm, and 1.2 to < 1.6 mm measured at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 
and 6 hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo 
alone  
• Percentage of subjects with a ≥ 3-line improvement in photopic binocular DCNVA 
with a PD (photopic and mesopic) of 1.6 to < 2 mm at 30 minutes, 1 hour, 2 hours, 3 
hours, 4 hours, and 6 hours comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, 
and Placebo alone  
• Percentage of subjects with Baseline DCNVA (photopic and mesopic) ≤ 20/63, ≤ 
20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 with a ≥ 15-letter improvement in DCNVA 
on Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours comparing 
Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone  
• Percentage of subjects with Baseline DCNVA (photopic and mesopic) from 25/50 to 
20/63, 20/80 to 20/100, and 20/125 to 20/160 with a ≥ 15-letter improvement in 
DCNVA on Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours 
comparing Nyxol + LDP to Nyxol alone, Placebo + LDP, and Placebo alone 
• Percentage of subjects with improvement of ≥ 5, ≥ 10, and ≥ 15 letters in DCNVA 
(photopic) from Baseline (Visit 1) to Visit 2 at 0 minutes, comparing Nyxol to 
Placebo treated subjects  
• Change in DCNVA (photopic) from Baseline (Visit 1) to Visit 2 at 0 minutes, 
comparing Nyxol to Placebo treated subjects   
• Percentage of subjects with DCNVA (photopic and mesopic) ≤ 20/15, ≤ 20/20, ≤ 
20/25, ≤ 20/32, ≤ 20/40, ≤ 20/50, ≤ 20/63, ≤ 20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 


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Safety and Tolerability: 
• Conjunctival hyperemia 
• Subjective ocular tolerability (Visit 2) 
• Adverse events (AEs) 
• Vital signs (heart rate [HR] and blood pressure [BP]) 
• Intraocular pressure (IOP) 
• Biomicroscopy  
• Ophthalmoscopy 
• Urine pregnancy tests for females of childbearing potential 
 


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5. 
STUDY METHODS 
5.1. 
General Study Design and Plan 
This is a placebo-controlled, double-masked, randomized, 4-arm Phase 2 study in males and 
females between 40 to 64 years of age with presbyopia. A sample size of approximately 152 
subjects will be randomized into 1 of 4 treatment arms with the expectation that 
approximately 140 subjects will be evaluable for efficacy, where evaluable is defined as 
having received at least one drop of Treatment 1, administered Treatment 2 (LDP or No 
Treatment), and completed at least one photopic binocular DCNVA measurement after 
Treatment 2. 
Subjects are evaluated for safety and efficacy following administration of masked Nyxol or 
Placebo (Treatment 1) at or near bedtime for 3 to 4 days, with administration of LDP or No 
Treatment (Treatment 2) at the next visit (Visit 2) in the morning. Measurements are made at 
multiple visits and timepoints, and analysis compares 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP 
• Placebo + No Treatment 2 (Placebo alone) 
Subjects are randomized 4:3:3:4 into the above groups. 
After randomization, masked Treatment 1 study medications (Nyxol or Placebo) is 
dispensed. Treatment 1 study medication is taken by the subject at or near bedtime 
(approximately 8PM-10PM) starting the night of Visit 1 (Screening/Baseline) or at the 
appropriate evening in order for Treatment 1 to be taken daily for 3 to 4 consecutive days 
immediately prior to Visit 2. 
Treatment 2 (LDP or No Treatment) is unmasked and is administered at Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments. 
Treatment 1 (Nyxol or Placebo) is administered to both eyes (OU) by the subject.  
Treatment 2 (LDP or No Treatment) is administered OU by the Site Staff. 
The study eye is defined as the eye with worse Baseline photopic DCNVA. In the case where 
both eyes have the same Baseline photopic DCNVA, the study eye will be the right eye 
(OD). The non-study eye will be the fellow eye. The study eye and fellow eye will both be 
evaluated at all assessments. 
The schedule for assessments and timing of events is presented in Table 1. 
 
 


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Table 1 
Visit Schedule 
 
Visit 1 
Screening / 
Baseline [a] 
Visit 2 
Visit 3 
Safety  
Follow-Up 
Call 
Day [b] 
1 
4 (+2) 
8 – 10 AM (+2 hr) 
5 (+2) 
Time (hr) [c] 
N/A 
0* 
0.5 
1 
2 
3 
4 
6 
Any 
Informed consent 
X 
 
 
 
 
 
 
 
 
Subject Number assigned 
X 
 
 
 
 
 
 
 
 
Medical/Ophthalmic history 
X 
 
 
 
 
 
 
 
 
Demographics  
X 
 
 
 
 
 
 
 
 
Prior/Concomitant medications 
X 
X 
 
 
 
 
 
 
X 
Urine pregnancy test [d] 
X 
X 
 
 
 
 
 
 
 
Heart rate/Blood pressure 
X 
X 
 
 
 
 
 
X 
 
Pupil diameter (photopic) [e] 
X 
X 
X 
X 
X 
X 
X 
X 
 
DCNVA (photopic) [f] 
X 
X 
X 
X 
X 
X 
X 
X 
 
BCDVA (photopic) [f] 
X 
X 
X 
X 
X 
X 
X 
X 
 
DCIVA (photopic) [f] 
X 
X 
 
X 
 
X 
 
X 
 
Pupil diameter (mesopic) [e] 
X 
X 
X 
X 
X 
X 
X 
X 
 
DCNVA (mesopic) [f] 
X 
X 
 
 
X 
 
X 
X 
 
BCDVA (mesopic) [f]  
X 
X 
 
X 
 
X 
 
X 
 
Conjunctival hyperemia [g] 
X 
X 
X 
X 
X 
X 
X 
X 
 
Biomicroscopy 
X 
 
 
 
 
 
 
X 
 
Dry eye tests (tear break-up time and corneal
fluorescein staining) [h]  
X 
 
 
 
 
 
 
 
 
Intraocular pressure [i] 
X 
X 
 
 
 
 
 
X 
 
Ophthalmoscopy [j] 
X 
 
 
 
 
 
 
X 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Randomization assigned [a] 
X 
 
 
 
 
 
 
 
 
Treatment 1: Nyxol or Placebo [k] 
X 
 
 
 
 
 
 
 
 
Treatment 2: 0.4% LDP or No Treatment [l] 
 
X* 
 
 
 
 
 
 
 
Subjective ocular tolerability [m] 
 
X 
X 
X 
X 
X 
X 
X 
 
Abbreviations: BCDVA, best-corrected distance visual acuity; DCIVA, distance-corrected intermediate visual acuity; CCLRU, Cornea and 
Contact Lens Research Unit; DCNVA, distance-corrected near visual acuity; LDP, low-dose pilocarpine; N/A, not applicable; VA, visual acuity. 
[a] If the subject meets all the inclusion criteria and none of the exclusion criteria, this Screening Visit becomes the Baseline Visit. A 
randomization number is assigned after the subject is qualified. 
[b] An additional 2 days is permitted between Visit 1 and Visit 2, as well as between Visit 2 and Visit 3. Every effort should be made to bring 
the patient back within the 3 or 4 days. 
[c] There is a window of +/- 10 minutes for every timepoint to complete measurement of the primary endpoint of photopic DCNVA. Sites 
should start pupil diameter measurements approximately 5 minutes prior to each timepoint in order to allow sufficient time to perform the 
primary endpoint of photopic DCNVA at the target timepoint.  
[d] Urine pregnancy test is for females of childbearing potential. 
[e] Pupil diameter – NeurOptics VIP-300 pupillometer (mm) measured in mesopic and photopic lighting conditions. 
[f] VA performed on each eye and binocular. 
[g] Conjunctival hyperemia – grading scale (0-3) using images from CCLRU. 
[h] Dry eye tests – if either meets the exclusion criteria, subject will be excluded from study. 
[i] Intraocular pressure – Tono-Pen. 
[j] Direct or indirect ophthalmoscopy without dilation. Use of 90 D lens (indirect) is allowed. 
[k] One drop of Nyxol or Placebo (Nyxol vehicle) in each eye daily at bedtime (approximately 8PM-10PM) starting evening of Visit 1 until 
Treatment Visit 2. There will be at least 3 (up to 4) evenings of dosing with Nyxol or Placebo prior to Treatment Visit 2.  
*[l] One drop of 0.4% LDP in each eye. Time Zero (0 minutes) begins after Treatment 2 and all pupil diameter and acuity measurements 
will be done prior to LDP instillation. Assessment of hyperemia will be done after LDP instillation.  
[m] Subjective ocular tolerability – 4-point scale. Will be assessed at the beginning of each specified timepoint except for Time 0 minutes, 
where it will be assessed after instillation of Treatment 2. 
 


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5.2. 
Inclusion – Exclusion Criteria and General Study Population 
The study population will be approximately 152 subjects between 40 to 64 years of age with 
presbyopia, with approximately 140 evaluable subjects.  
Individuals who are potential subjects are identified by the study center to schedule the 
Screening Visit. The Screening Visit shall occur the same day as the Baseline Visit.  
Once a subject arrives at the study center, a member of the Site Staff will interview the 
individual as to their qualifications for participation in the study, and if the subject wishes to 
continue, the informed consent form is signed, and a subject number is assigned.  
The start of Screening includes an explanation of the study, a medical and ophthalmic 
history, and demographics. Eligibility for Inclusion Criteria #1 to 4 as well as Exclusion 
Criteria #1 to 3, 5 to 13, and 15 to 19 shall be determined and only eligible subjects will 
continue. This shall be followed by a urine pregnancy test for females of childbearing 
potential (Exclusion Criterion #20), and HR/BP (Exclusion Criteria #21-22).  
The subject will then undergo several VA measurements, including photopic BCDVA as 
well as mesopic and photopic DCNVA (Inclusion Criteria #5-7). In addition, subjects will 
undergo measurement of photopic DCIVA and mesopic BCDVA. 
The screening assessment will also include an ophthalmic examination that includes 
assessment of PD, biomicroscopy, dry eye examination with tear break-up time testing and 
corneal fluorescein staining (Exclusion Criterion #4), intraocular pressure (IOP) 
measurement (using a Tono-Pen), and direct or indirect ophthalmoscopy without dilation.  
In addition, conjunctival hyperemia will be assessed visually with a 4-point grading scale 
using images from the Cornea and Contact Lens Research Unit (CCLRU) (Exclusion 
Criterion #14).  
If all eligibility criteria are met, the subject will be randomized into the study.  
5.3. 
Randomization and Masking 
A randomization code for allocating subjects to treatment will be prepared by a masked 
biostatistician not connected with the study. At the initiation of study-related procedures, 
every subject who is screened is assigned a subject number within numerical order within 
each site. Once a subject is qualified for the study, the subject is assigned a randomization 
number in the order provided by the biostatistician. 
Subjects will be then randomized 4:3:3:4 into 1 of 4 treatment arms:  
• Nyxol + LDP 
• Nyxol + No Treatment 2 (Nyxol alone) 
• Placebo + LDP (LDP alone) 
• Placebo + No Treatment 2 (Placebo alone) 
Randomization will be stratified 1:1 by iris color (light/dark irides). 


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A randomization schema for evaluable subjects by investigational treatment (Treatment 1 
and Treatment 2) and irides type is described in Figure 1. 
Figure 1 
Randomization schema by investigational treatment and irides 
type 
 
The Treatment 1 study medications will be masked to Investigators, Site Staff, study 
subjects, Ocuphire, and all other study personnel involved in the conduct of the study. Only 
in case of medical emergency or occurrence of SAEs will the randomization code be 
unmasked by the Medical Monitor and made available to the Investigator, Ocuphire, and/or 
other personnel involved in the monitoring or conduct of this study. 
Treatment 2 (LDP or No Treatment) is unmasked and will be administered on Visit 2 by a 
designated, unmasked, Site Staff member, distinct from the Site Staff member recording 
measurements/assessments.  
Randomization for both Treatments will occur at Visit 1. 
5.4. 
Analysis Variables 
Variables to be summarized include demographics and baseline characteristics, medical 
(non-ocular) and ocular history, concomitant medications, and study drug accountability.  
Efficacy variables include: 
• DCNVA (i.e., Near VA) 
• BCDVA (i.e., Distance VA) 


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• DCIVA (i.e., Intermediate VA) 
• Pupil diameter (PD) 
Safety variables include: 
• Conjunctival hyperemia (eye redness) measured with a CCLRU card 4-point scale: 
o None (0) = Normal Appears white with a small number of conjunctival blood 
vessel easily observed. 
o Mild (+1) = Prominent, pinkish-red color of both the bulbar and palpebral 
conjunctiva. 
o Moderate (+2) = Bright, scarlet red color of the bulbar and palpebral 
conjunctiva. 
o Severe (+3) = Beefy red with petechiae, dark red bulbar and palpebral 
conjunctiva with evidence of subconjunctival hemorrhage.   
• Subjective ocular tolerability measured on a 4-point scale: 
o 0 – No discomfort 
o 1 – Mild discomfort 
o 2 – Moderate discomfort 
o 3 – Severe discomfort 
• AEs 
• Vital signs (HR and BP) 
• IOP 
• Biomicroscopy  
• Ophthalmoscopy 
• Urine pregnancy tests for females of childbearing potential 
 


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6. 
SAMPLE SIZE 
A sample size of approximately 140 subjects (40 for Nyxol + LDP and Placebo alone arms 
and 30 for each of the other 2 arms) who are evaluable for efficacy is needed for the study. 
The primary treatment comparison will be Nyxol + LDP compared to Placebo alone. Using α 
= 0.05 significance and a two-tailed test, 76 total subjects (38 subjects each in the Nyxol + 
LDP and Placebo alone arms) evaluable for efficacy will provide at least 80% power to 
detect a difference of 23% between the arms in percent of subjects with ≥ 15 letters of 
improvement in photopic binocular DCNVA at 1 hour. This calculation is based on a 5% 
Placebo effect and a 28% Nyxol + LDP treatment effect.  
Subjects will be randomized 4:3:3:4 to Nyxol + LDP, Nyxol + No Treatment 2 (Nyxol 
alone), Placebo + LDP, and Placebo + No Treatment 2 (Placebo alone), respectively. 
It is assumed that there will be approximately 7% drop-out between Visit 1 and end of Visit 
2. To account for this drop-out, a total of approximately 152 subjects will be randomized into 
the study. 


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7. 
GENERAL CONSIDERATIONS 
7.1. 
Analysis Populations 
The following analysis populations will be defined for this study.  
7.1.1. 
Modified Intention-to-Treat (mITT) 
The mITT population will include all randomized subjects who received at least one drop of 
Treatment 1 and were administered Treatment 2 (LDP or No Treatment). The mITT 
population will be used to analyze selected efficacy endpoints, with subjects included in their 
randomized treatment regardless of the treatment they actually received. 
7.1.2. 
Per Protocol Population (PP) 
The PP population included all subjects in the mITT population who received one drop of 
Treatment 1 the day prior to Visit 2 and were administered Treatment 2 at Visit 2, had 
binocular DCNVA and BCDVA in photopic conditions at Baseline (Visit 1) and at Visit 2 
time 1 hour, and had no major protocol deviations. The PP population will be used for the 
primary endpoint analysis and to analyze all efficacy endpoints, with subjects included in 
their randomized treatment regardless of the treatment they actually received. 
7.1.3. 
All Randomized Population (ARP) 
The ARP will include all randomized subjects. This population is also known as the 
Intention-to-Treat (ITT) population. The ARP will be used in confirmatory efficacy analyses, 
with subjects included in their randomized treatment regardless of the treatment they actually 
received. 
7.1.4. 
Safety Population (SP) 
The SP will include all randomized subjects who have received at least one drop of study 
treatment (Treatment 1 or Treatment 2). The SP will be used to summarize safety variables, 
using the actual treatment a subject received. 
7.2. 
Covariates and Subgroups 
7.2.1. 
Planned Covariates 
Planned covariates include baseline values for the given assessment.  
7.2.2. 
Planned Subgroups 
Subgroup analyses by irides type (light, dark) will be completed for efficacy endpoints.  
Other possible subgroups include age, sex, and race. 


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7.3. 
Management of Analysis Data 
7.3.1. 
Data Handling 
Data from unscheduled visits will not be included in the analysis of efficacy or safety but will 
be listed.   
7.3.2. 
Missing Data 
The primary efficacy endpoint is the percentage of subjects with ≥ 15 letters of improvement 
from baseline in photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP 
compared to Placebo alone. For the analysis of the primary efficacy endpoint, imputation will 
be performed for missing efficacy data as specified in Section 7.3.2.3 for the analysis using 
the mITT, as well as the PP, if required. Confirmatory analyses may be performed using the 
ARP, also using imputation for missing data.   
Otherwise, there will be no substitutions made to accommodate missing data points for 
efficacy data. All data recorded on the CRF will be included in data listings that will 
accompany the CSR. 
Safety data will be imputed in limited situations. If the severity of an AE is missing, then the 
severity will remain missing. If relationship of the AE to study drug is missing, the 
relationship will remain missing. Missing or partial dates for AEs or concomitant 
medications will be imputed as described in Section 7.3.2.1. Otherwise, all summaries of 
safety endpoints will be completed using observed cases in the SP; no imputation will be 
completed. 
7.3.2.1. 
Handling of Missing Date Values 
Partial or Missing Dates 
The following conventions will be used to impute missing portions of dates for AEs and 
concomitant medications, if warranted. Note that the imputed values outlined here may not 
always provide the most conservative date. In those circumstances, the imputed value may be 
replaced by a date that will lead to a more conservative analysis. 
A. 
Start Dates 
1) 
If the year is unknown, then the date will not be imputed and will 
be assigned a missing value.  
2) 
If the month is unknown, then: 
i) If the year matches the first dose date year, then impute 
the month and day of the first dose date. 
ii) Otherwise, assign ‘January.’  
3) 
 If the day is unknown, then: 


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i) If the month and year match the first dose date month 
and year, then impute the day of the first dose date. 
ii) Otherwise, assign the first day of the month. 
B. 
Stop Dates 
1) 
If the year is unknown, then the date will not be imputed and will 
be assigned a missing value. 
2) 
If the month is unknown, then assign ‘December.’ 
3) 
If the day is unknown, then assign the last day of the month. 
 
7.3.2.2. 
Missing Baseline Data 
Every effort will be made to ensure that accurate baseline information on the subjects is 
collected. In the event that a subject is missing baseline information, the subject will be 
included in the SP for assessment of safety and excluded from the primary analyses. Each 
case of missing baseline data will be evaluated for potential inclusion in the exploratory 
endpoints. All baseline data will be observed cases, without imputation. 
7.3.2.3. 
Imputation Methods 
Imputation for efficacy data will only be performed for the primary efficacy endpoint using 
the mITT and ARP, as well as the PP, if required. If 5% or fewer data are missing at the Visit 
2, 1 hour time point in the Nyxol + LDP and Placebo alone treatment groups, an analysis 
with last observation carried forward (LOCF) for missing data will be applied within a 
treatment group. Neither the baseline (Visit 1) nor the Visit 2, 0 minutes time points will be 
used for imputation. If more than 5% of data are missing at the Visit 2, 1 hour time point in 
the Nyxol + LDP and Placebo alone treatment groups, multiple imputation will be employed 
to analyze incomplete data sets under the assumption that the mechanism responsible for the 
missing data is at worst characterized as missing at random (MAR).    
Multiple imputation is a simulation-based approach where missing values are replaced by 
multiple Bayesian draws from the conditional distribution of missing data given the observed 
data and covariates, creating multiple completed data sets. These completed datasets can then 
be analyzed using standard analysis methods. Rubin (1987) presented rules for how to 
combine the multiple sets of estimates to produce overall estimates, confidence intervals 
(CIs), and tests that adequately incorporate missing data uncertainty. 
Missing values for DCNVA will be imputed simultaneously based on an underlying joint 
normal distribution using a Markov Chain Monte Carlo (MCMC) method.   
The imputations will be done separately for each treatment group and will include the 
following variables in the imputation model: binocular photopic DCNVA at Visit 2 (30 
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours). No imputation will be applied to the 
Visit 2, 0 minutes time point. 
The number of imputations will be set to 500. The outcomes of interest (change from 
baseline) will be calculated from these imputed datasets. The treatment difference for each 


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imputed dataset will be evaluated using logistic regression. See Section 9.1 for details on 
these models. The estimates and standard errors (SEs) of the odds ratios based on the 500 
imputed datasets are then combined by applying Rubin’s rules for multiple imputed datasets. 
T-tests are also provided. SAS Proc MI, Proc LOGISTIC, and Proc MIANALYZE will be 
utilized for these analyses. The odds ratios with the corresponding 95% CI will also be 
presented.  
Example SAS code is provided below: 
PROC MI: 
 
 
 
proc mi data=original out=imputed seed=1223 nimpute=500; 
      
 
mcmc initial = EM; 
 
 
 
 
var VA_30M VA_1H VA_2H VA_3H VA_4H VA_6H; 
 
 
 
 
by trtname; 
ods output misspattern=msgpat varianceinfo=varinfo parameterestimates=param; 
 
 
 
run; 
 
PROC LOGISTIC: 
 
 
 
proc logistic data=imputed; 
         
 
by _imputation_; 
          
class trtname irides; 
          
model outcome = trtname irides base/cl rl firth; 
          
odds trtname; 
          
ods output odds=est_ds;  
         run; 
 
PROC MIANALYZE; 
 
 
 
proc mianalyze data=est_ds; 
    
 
 
modeleffects estimate; 
      
 
stderr stderr; 
      
 
ods select ParameterEstimates; 
 
   run; 
   
7.3.3. 
Handling of Early Termination Visit Information 
In the event that a subject is terminated early from this study on Visit 2, the early termination 
data for safety variables will be assigned to the closest scheduled time point on Visit 2. If the 
closest time point has valid data, the early termination data will be assigned to the next 
available time point. 
7.3.4. 
Pooling of Investigational Sites 
The data from all study centers will be pooled together for all planned analyses. 


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7.3.5. 
Coding Conventions for Events and Medications 
All AEs and medical history will be mapped to the Medical Dictionary for Regulatory 
Activities (MedDRA Version 23.1) system for reporting (preferred term and body system). 
Prior and concomitant medications will be coded using WHO-DD (World Health 
Organization Drug Dictionary) (Version September 2020, C Format). 
7.3.6. 
Analysis Software 
Data manipulation, tabulation of descriptive statistics, calculation of inferential statistics, and 
graphical representations will be performed primarily using SAS (release 9.4 or higher) for 
Windows. If the use of other software is warranted, the final CSR will detail what software 
was used and for what purposes. 
7.3.7. 
Study Data 
Study data identified in the schedule for time and events (Table 1) are collected, and source 
verified, on the electronic data capture (EDC) Datatrak One version 14.6.2.  
All study data will be formulated into regulatory-compliant data sets to provide transparency, 
traceability, and integrity of trial analysis results from the collection source. Observed study 
data will be mapped to the Clinical Data Interchange Standards Consortium (CDISC) Study 
Data Tabulation Model (SDTM) and serve as the source data from the trial. All study 
analyses will be completed using analysis data sets that are derived from the SDTM and 
follow the CDISC Analysis Data Model (ADaM) architecture.  
The methods for programming the CDISC SDTM and ADaM data sets are described in 
Figure 2. 
Figure 2 
SDTM, ADaM, and TFL Development and Validation 
Figure 3 
SDTM, ADaM, and TFL Development and Validation 
 
Where: 
1. Development, validation, and maintenance of SDTM domains 
2. Development and validation of ADaM data sets, with input source the appropriate 
SDTM domains. 
3. Development and validation of tables, figures, and listings (TFL), with input data 
source the SDTM domains and analysis specific ADaM data sets. 


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7.4. 
Planned Study Analyses 
7.4.1. 
Statistical Summaries: Descriptive and Inferential 
Categories for data presentation and analysis will consist of each treatment group: Nyxol + 
LDP, Nyxol + No Treatment 2 (Nyxol alone), Placebo + LDP, Placebo + No Treatment 2 
(Placebo alone).  
All statistical tests will be two-sided and a difference resulting in a p-value of less than or 
equal to 0.05 will be considered statistically significant. The p-values for the analysis of 
secondary efficacy endpoints and safety endpoints will be considered descriptive. All p-
values will be rounded to and displayed in four decimals. If a p-value less than 0.0001 
occurs, it will be shown in tables as <0.0001. 
Descriptive summaries of variables will be provided where appropriate. For continuous 
variables, the number of non-missing values (n), mean, standard deviation, median, 
minimum, and maximum will be tabulated by treatment group. For categorical variables, the 
counts and proportions of each value will be tabulated by treatment group. Expansion of 
descriptive table categories within each treatment may occur if such elaborations are thought 
to be useful. 
All study-related data collected will be presented in listings. Study-related data not subject to 
analysis according to this plan will not appear in any tables or graphs but will be included in 
the data listings. 
7.4.2. 
Interim Analyses and Data Monitoring 
No formal interim analysis or safety monitoring committee is planned for this study.  
7.4.3. 
Final Analysis and Publication of Study Results 
The final analysis will be completed after all subjects have completed the study.   
7.5. 
Multiple Testing Procedures 
There will be no adjustments for multiplicity and no formal multiple testing procedures are to 
be implemented with this analysis plan. 
7.6. 
Baseline Values 
Baseline values are the values obtained at Visit 1, prior to any treatment administration. If the 
Visit 1 value is missing, any value collected prior to treatment administration will be treated 
as the baseline. 
 


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8. 
SUMMARY OF STUDY DATA 
8.1. 
Subject Disposition 
A summary of the analysis sets includes the number and percentage of subjects by treatment 
group and overall for the following categories: subjects in the ARP, subjects in the SP, 
subjects in the mITT Population, and subjects in the PP Population. All percentages will be 
based on the number of subjects in the ARP.  
End of trial information will also be summarized in this table, including the number of 
subjects completing the study, the number of subjects who prematurely discontinued the 
study with reasons for withdrawal, the number of subjects completing the study medication 
dosing, and the number of subjects who prematurely discontinued the study medication with 
reasons for study medication discontinuation.   
A by-subject data listing of study completion information including the reason for premature 
study withdrawal, if applicable, will be presented. 
8.2. 
Protocol Deviations 
Major protocol deviations, as determined by a Sponsor blinded review of the data prior to 
database lock and unblinding of the study, may result in the removal of a subject’s data from 
the PP Population. The Sponsor or designee will be responsible for producing the final 
deviation file; this file will include a description of the protocol deviation and clearly identify 
whether this violation warrants exclusion from the PP Population. This file will be finalized 
prior to database lock. 
All protocol deviations will be presented in a by-subject data listing, with a flag to indicate if 
a deviation was considered major and/or exclusionary.  
8.3. 
Demographics and Baseline Characteristics 
Subject demographic data and baseline characteristics will be tabulated and summarized 
descriptively by treatment group and overall. The demographic data and baseline 
characteristics will be summarized for the mITT Population, PP Population, SP, and ARP. If 
the mITT population is equivalent to any of the other populations, then only the mITT 
version will be generated rather than repeating equivalent summaries.  
The demographics consist of age (year), sex, race, ethnicity, and study eye, iris color (light 
blue, dark blue, blue with peripupillary brown, uniform green, green with brown iris ring, 
central brown and peripheral green, brown with some peripheral green, or brown), and irides 
type (light or dark). A subject’s age in years is calculated using the date of the informed 
consent and date of birth. Age will be summarized using descriptive statistics. The number 
and percentage of subjects by sex, race, ethnicity, study eye, iris color, and irides type will 
also be reported. Percentages will be based on the total number of subjects in the study 
population presentation. 


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The following baseline characteristics will be summarized for study eye, fellow eye, and 
binocular (if applicable), using descriptive statistics: 
• BCDVA (photopic and mesopic) 
• DCNVA (photopic and mesopic) 
• DCIVA (photopic)  
• Pupil diameter (photopic and mesopic) 
• IOP 
All demographic and baseline information will be presented in by-subject listings. 
8.4. 
Medical History  
The number and percent of subjects with individual medical histories will be summarized for 
all subjects by treatment group and overall. Non-ocular and ocular medical history will be 
summarized separately.  
Medical history will be coded using the MedDRA Version 23.1 The number and percentage 
of subjects with any medical history will be summarized overall and for each system organ 
class (SOC) and preferred term (PT). Percentages will be calculated based on number of 
subjects in the SP.   
Subject medical history data including specific details will be presented in by-subject 
listings. 
8.5. 
Prior and Concurrent Medications 
The number and percentages of all concomitant medications will be summarized by 
treatment group, Anatomical Therapeutic Chemical (ATC) level 4, and PT. The total number 
of concomitant medications and the number and percentages of subjects with at least 1 
concomitant medication will be summarized by treatment group. All summaries will be 
performed using the SP.   
A concomitant medication is defined as any medication taken on or after the day of first 
exposure to study drug. 
Prior medications are defined as any medication that has a start and stop date prior to the day 
of first exposure to any study drug, collected from up to 30 days prior to Screening. The total 
number of prior medications and the number and percentages of subjects with at least 1 prior 
medication will be summarized by treatment group. 
8.6. 
Treatment Administration 
Treatment administration data for both Treatment 1 and Treatment 2 will be presented in by-
subject listings.  


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9. 
EFFICACY ANALYSES 
Unless otherwise noted, efficacy will be assessed using the mITT and PP populations, with 
subjects included in their randomized treatment regardless of the treatment they actually 
received. For the analysis of the primary efficacy endpoint, imputation will be performed for 
missing data as described in Section 7.3.2.3. Confirmatory analysis of the primary efficacy 
endpoint may be performed using the ARP, if different from the mITT, also using imputation 
for missing data. For the analysis of the secondary efficacy endpoints, only observed case 
data will be used.  
All efficacy assessment data, regardless of whether they are included in the analysis, will be 
presented in by-subject listings.  
9.1. 
Clinical Efficacy   
For all efficacy endpoints, Baseline is defined as Visit 1.  
All efficacy data will be summarized by treatment group for the Baseline assessment and for 
Visit 2 timepoints (0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours), as 
appropriate. 
9.1.1. 
Primary Efficacy Analysis 
The primary efficacy endpoint is the percent of subjects with ≥ 15 letters of improvement in 
photopic binocular DCNVA on Visit 2 at 1 hour with Nyxol + LDP compared to Placebo 
alone in the study eye. If the analysis using the PP Population shows a positive effect for 
Nyxol at the 0.05 level of significance, the primary endpoint will be considered met. The 
primary efficacy endpoint will be analyzed using a logistic regression model with treatment 
and light/dark irides as factors and the baseline DCNVA as a covariate. The percentage of 
subjects in each treatment group meeting the criteria, the odds ratio (OR) with 95% CI, and 
p-value will be provided. Example SAS code is as follows: 
proc logistic data=efficacydata;  
class trtname irides;  
model outcome = trtname irides base/cl rl firth; 
run; 
 
By including irides type as a factor in the primary efficacy analysis model, the model 
efficiency as well as a change in the treatment effect will be increased. Including this factor 
in the model will also make the results more generalizable to other studies in which the 
sample characteristics may differ from the current study [2].  
In addition, the primary efficacy endpoint will be analyzed by light/dark irides using the 
same model indicated above but without irides as a factor. For this subgroup analyses, 
observed case data only will be used; that is, missing values will not be imputed.   


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9.1.2. 
Secondary Efficacy Analyses  
Secondary efficacy endpoints are indicated in Section 4.2.2. Secondary efficacy endpoints 
will be analyzed by study eye, fellow eye, and binocular (if available) using the PP 
population, and the mITT population for selected endpoints (see the list of planned tables, 
Section 12.1, for details).  
All continuous secondary endpoints derived from VA assessments, such as change in 
DCNVA, DCIVA, and BCDVA, will be analyzed using Early Treatment Diabetic 
Retinopathy Study (ETDRS) letters correctly read. Any secondary endpoints using Snellen 
measurements will be converted from letters read as follows: 
Table 2 
Snellen Equivalents to VA Assessment Letters Read 
DCNVA/DCIVA letters 
Snellen 
BCDVA letters 
≥75 
20/15 
≥60 
70 - 74 
20/20 
55 - 59 
65 - 69 
20/25 
50 - 54 
60 - 64 
20/32 
45 - 49 
55 - 59 
20/40 
40 - 44 
50 - 54 
20/50 
35 - 39 
45 - 49 
20/63 
30 - 34 
40 - 44 
20/80 
25 - 29 
35 - 39 
20/100 
20 - 24 
30 – 34 
20/125 
15 - 19 
25 - 29 
20/160 
10 - 14 
<25 
20/200 
<10 
 
Each of the continuous secondary efficacy endpoints will be analyzed using analysis of 
covariance (ANCOVA), with observed value as the dependent variable, treatment, and 
light/dark irides as factors, and the respective baseline value included as the covariate. 
Another ANCOVA will be applied with change from baseline as the dependent variable 
using the same factors and covariates. 
The output from each ANCOVA will include the LSM and SE for all treatment groups, along 
with each LSM, the 95% CI and associated p-value for Nyxol + LDP vs. Nyxol alone, 
Placebo + LDP, and Placebo alone. Continuous pupil diameter endpoints will be assessed 
using the study eye, fellow eye, and best eye, which is defined as the eye with the greatest 
reduction in diameter. 
For each of the secondary endpoints related to percent of subjects achieving certain criteria, 
the analysis will be performed using a logistic regression model with treatment and light/dark 
irides as factors, and the respective baseline as a covariate. For each analysis, the percentage 
of subjects in each treatment group meeting the criteria will be presented, and the OR with 
95% CI and p-value will be provided for Nyxol + LDP vs. Nyxol alone, Placebo + LDP, and 
Placebo alone. Categorical pupil diameter endpoints will be assessed using the study eye, 
fellow eye, and either eye, which is defined as achieving a given criterion in either eye. 


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In addition, each secondary efficacy endpoint will be analyzed by light/dark irides using the 
same model indicated above but without irides as a factor.  


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10. 
SAFETY ANALYSES 
All safety analyses will be conducted using the SP unless specified otherwise below. All 
safety analyses will be completed using the actual treatment a subject received. Observed 
case data will be used; no imputation will be performed for missing safety data except for the 
limited situations described in Section 7.3.2. 
All safety data will be presented in by-subject listings. Unscheduled assessments will not be 
summarized but will be included in the listings.  
10.1. 
Adverse Events 
AEs will be coded using MedDRA, Version 23.1.   
Treatment-emergent adverse events (TEAEs) are defined as any AE that begins or worsens 
after initiation of the investigational product and through the subject’s last study visit (study 
completion or early termination). 
If the onset of an AE is on or after the date of first dose of study medication or is increasing 
in severity after first dose of study medication, then the AE will be considered treatment 
emergent. 
Only TEAEs will be summarized; all AEs (TEAE, non-TEAE) will be included in a by-
subject listing.  
The number and percent of subjects with any TEAEs will be summarized by SOC and PT by 
treatment group and overall. At each level of tabulation (e.g., at the PT level), subjects will 
be counted only once if they had more than one such event reported during the AE collection 
period.  
 
Note that in MedDRA, ocular events are coded to the SOC of “Eye Disorders”. Thus, using 
SOC in the summaries will provide a separation of ocular and non-ocular adverse events. 
The following summary tables will be presented for TEAE data:  
• 
Overall summary of TEAEs (completed for both the SP and mITT population) 
• 
Summary table of TEAEs by SOC and PT  
• 
Summary table of TEAEs by SOC, PT, and by greatest relationship level to study 
drug (not related, unlikely related, possibly related, probably related, definitely 
related, or unknown) 
• 
Summary table of TEAEs by SOC, PT, and maximum severity (mild, moderate, 
severe) 
• 
Summary table of serious TEAEs by SOC and PT  
• 
Summary table of TEAEs leading to withdrawal from the study by SOC and PT 
 


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• 
Summary table of TEAEs leading to study medication discontinuation by SOC and 
PT 
 
10.2. 
Deaths, Serious Adverse Events and Other Significant Adverse Events 
10.2.1. 
Deaths 
The AE listing will include all AEs, including deaths, regardless of causality; one of the 
columns in the listing will specify whether the AE was fatal.     
10.2.2. 
Serious Adverse Events 
The AE listing will include all AEs, including SAEs; one of the columns in the listing will 
specify whether the AE was an SAE.     
10.2.3. 
Adverse Events Leading to Withdrawal from the Study 
The AE listing will include all AEs, including AEs leading to withdrawal from the study; one 
of the columns in the listing will specify whether the AE led to withdrawal from the study.   
10.2.4. 
Adverse Events Leading to Discontinuation of Study Medication 
The AE listing will include all AEs, including AEs leading to discontinuation of study 
medication; one of the columns in the listing will specify whether the AE led to 
discontinuation of study medication.   
10.3. 
Conjunctival Hyperemia 
Results from the conjunctival hyperemia assessment, measured with a CCLRU card 4-point 
scale, will be summarized descriptively using counts and percentages for each treatment 
group at each time point (baseline, 0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 
and 6 hours) for the observed value and change from baseline. Separate summaries will be 
created for the study eye and the fellow eye. 
Additionally, conjunctival hyperemia will be summarized as a continuous variable. 
Treatments will be compared using the same ANCOVA model proposed for the continuous 
secondary efficacy endpoints. 
10.4. 
Subjective Ocular Tolerability 
Results from the subjective ocular tolerability assessment, measured on a 4-point scale, will 
be summarized descriptively using counts and percentages for each treatment group at each 
time point (0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours). 
Additionally, the categories “No Discomfort” and “Mild Discomfort” will be pooled into a 
single category and summarized descriptively, as will the categories “Moderate Discomfort” 
and “Severe Discomfort”.  


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Treatments will be compared for the two pooled categories using a Fisher’s exact test for 
Nyxol + LDP vs. Nyxol alone, Placebo + LDP, and Placebo alone. Separate summaries will 
be created for the study eye and the fellow eye.  
10.5. 
Vital Signs 
Descriptive statistics of observed values will be presented for vital sign data at each time 
point (baseline, 0 minutes, and 6 hours), including systolic BP (mmHg), diastolic BP 
(mmHg), and HR (bpm) by treatment group. Changes from baseline to each scheduled post-
baseline time point will be presented.  
10.6. 
IOP 
Observed values and change from baseline in IOP at 0 minutes and 6 hours will be 
summarized for the study eye and the fellow eye. Treatments will be compared using the 
same ANCOVA model proposed for the continuous secondary efficacy endpoints. 
10.7. 
Biomicroscopy and Ophthalmoscopy 
Results from biomicroscopic and ophthalmoscopic examinations at baseline and 6 hours will 
be summarized by treatment group for the study eye and the fellow eye. 
10.8. 
Other Safety Measures 
The efficacy endpoint “Percentage of subjects with improvement or loss in BCDVA 
(photopic) from Baseline (Visit 1) to Visit 2 at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 
and 6 hours of ≥ 5, ≥ 10, and ≥ 15 letters comparing Nyxol + LDP to Nyxol alone, Placebo + 
LDP, and Placebo” will be analyzed using the Safety Population for photopic BCDVA.  
Urine pregnancy tests for females of childbearing potential will be presented in by-subject 
listings. Dry eye tests (tear break-up time and cornea fluorescein staining), performed only at 
baseline, will also be listed. 
 
 
 


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11. 
REFERENCES 
[1] ICH E9 Expert Working Group. Statistical Principles for Clinical Trials: ICH 
Harmonized Tripartite Guideline, September 1998 
[2] Hauck WM, Anderson S, and Marcus SM, Should We Adjust for Covariates in Nonlinear 
Regression Analyses of Randomized Trials? Controlled Clin Trials 1998;19:249–256 
 
 


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12. 
APPENDICES 
12.1. 
List of Planned Tables 
The list of planned tables includes all of the main tables to be presented for the study. Cells 
that are highlighted in yellow are what we are considering for the top-line report.  
Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.1.1 
Subject Disposition  
X 
 
 
 
14.1.2.1/2/3/4 
Demographics and Baseline 
Characteristics 
X 
X 
X 
X 
14.1.3.1 
Non-ocular Medical History  
 
X 
 
 
14.1.3.2 
Ocular Medical History  
 
X 
 
 
14.1.4.1 
Prior Medications  
 
X 
 
 
14.1.4.2 
Concomitant Medications by Treatment 
Group 
 
X 
 
 
14.2.1.1/2/3 
Percent of Subjects with Improvement 
in Photopic DCNVA by Time Point 
X 
 
X 
X 
14.2.1.4/5 
Percent of Subjects with  Improvement 
in Photopic DCNVA by Time Point and 
Irides Type 
 
 
X 
X 
14.2.1.6/7 
Percent of Subjects with Improvement 
in Photopic DCNVA at 0 Minutes for 
Nyxol and Placebo Treated Subjects 
 
 
X 
X 
14.2.1.8/9 
Percent of Subjects with Improvement 
in Photopic DCNVA at 0 Minutes for 
Nyxol and Placebo Treated Subjects by 
Irides Type 
 
 
X 
X 
14.2.2.1/2 
Percent of Subjects with ≥ 15 Letters of 
Improvement in Photopic DCNVA and 
< 5 Letters of Loss in Photopic 
Binocular BCDVA by Time Point 
 
 
X 
X 
14.2.2.3/4 
Percent of Subjects with ≥ 15 Letters of 
Improvement in Photopic DCNVA and 
< 5 Letters of Loss in Photopic 
Binocular BCDVA by Time Point and 
Irides Type 
 
 
X 
X 
14.2.3.1/2 
Observed Values and Change from 
Baseline in Photopic DCNVA by Time 
Point 
 
 
X 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.2.3.3/4 
Observed Values and Change from 
Baseline in Photopic DCNVA by Time 
Point and Irides Type 
 
 
X 
X 
14.2.3.5/6 
Observed Values and Change from 
Baseline in Photopic DCNVA at 0 
Minutes for Nyxol and Placebo Treated 
Subjects 
 
 
X 
X 
14.2.3.7/8 
Observed Values and Change from 
Baseline in Photopic DCNVA at 0 
Minutes for Nyxol and Placebo Treated 
Subjects by Irides Type 
 
 
X 
X 
14.2.4.1/2 
Percent of Subjects with Improvement 
in Mesopic DCNVA by Time Point 
 
 
X 
X 
14.2.4.3/4 
Percent of Subjects with Improvement 
in Mesopic DCNVA by Time Point and 
Irides Type 
 
 
X 
X 
14.2.5.1/2 
Observed Values and Change from 
Baseline in Mesopic DCNVA by Time 
Point 
 
 
X 
X 
14.2.5.3/4 
Observed Values and Change from 
Baseline in Mesopic DCNVA by Time 
Point and Irides Type 
 
 
X 
X 
14.2.6.1/2 
Percent of Subjects with Improvement 
in Photopic DCIVA by Time Point 
 
 
X 
X 
14.2.6.3/4 
Percent of Subjects with Improvement 
in Photopic DCIVA by Time Point and 
Irides Type 
 
 
X 
X 
14.2.7.1 
Observed Values and Change from 
Baseline in Photopic DCIVA by Time 
Point 
 
 
 
X 
14.2.7.2 
Observed Values and Change from 
Baseline in Photopic DCIVA by Time 
Point and Irides Type 
 
 
 
X 
14.2.8.1/2 
Percent of Subjects with Improvement 
or Loss in Photopic BCDVA by Time 
Point 
 
X 
 
X 
14.2.8.3/4 
Percent of Subjects with Improvement 
or Loss in Photopic BCDVA by Time 
Point and Irides Type 
 
X 
 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.2.9.1 
Observed Values and Change from 
Baseline in Photopic BCDVA by Time 
Point 
 
 
 
X 
14.2.9.2 
Observed Values and Change from 
Baseline in Photopic BCDVA by Time 
Point and Irides Type 
 
 
 
X 
14.2.10.1 
Percent of Subjects with Improvement 
or Loss in Mesopic BCDVA by Time 
Point 
 
 
 
X 
14.2.10.2 
Percent of Subjects with Improvement 
or Loss in Mesopic BCDVA by Time 
Point and Irides Type 
 
 
 
X 
14.2.11.1 
Observed Values and Change from 
Baseline in Mesopic BCDVA by Time 
Point 
 
 
 
X 
14.2.11.2 
Observed Values and Change from 
Baseline in Mesopic BCDVA by Time 
Point and Irides Type 
 
 
 
X 
14.2.12.1 
Observed Values and Change from 
Baseline in Photopic Pupil Diameter by 
Time Point 
 
 
 
X 
14.2.12.2 
Observed Values and Change from 
Baseline in Photopic Pupil Diameter by 
Time Point and Irides Type 
 
 
 
X 
14.2.13.1/2 
Percent of Subjects with Photopic Pupil 
Diameter < 2.4, < 2.2, < 2.0, < 1.8, < 
1.6, and < 1.4 mm by Time Point 
 
 
X 
X 
14.2.13.3 
Percent of Subjects with Photopic Pupil 
Diameter < 2.4, < 2.2, < 2.0, < 1.8, < 
1.6, and < 1.4 mm by Time Point and 
Irides Type 
 
 
 
X 
14.2.14.1/2 
Percent of Subjects with Photopic Pupil 
Diameter 2 to < 2.4 mm, 1.6 to < 2 mm, 
and 1.2 to < 1.6 mm by Time Point 
 
 
X 
X 
14.2.14.3 
Percent of Subjects with Photopic Pupil 
Diameter 2 to < 2.4 mm, 1.6 to < 2 mm, 
and 1.2 to < 1.6 mm by Time Point and 
Irides Type 
 
 
 
X 
14.2.15.1/2 
Percent of Subjects with ≥ 15 Letter 
Improvement in Photopic Binocular 
 
 
X 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
DCNVA and Photopic Pupil Diameter 
1.6 to < 2 mm by Time Point 
14.2.15.3 
Percent of Subjects with ≥ 15 Letter 
Improvement in Photopic Binocular 
DCNVA and Photopic Pupil Diameter 
1.6 to < 2 mm by Time Point and Irides 
Type 
 
 
 
X 
14.2.16.1/2 
Observed Values and Change from 
Baseline in Mesopic Pupil Diameter by 
Time Point 
 
 
X 
X 
14.2.16.3 
Observed Values and Change from 
Baseline in Mesopic Pupil Diameter by 
Time Point and Irides Type 
 
 
 
X 
14.2.17.1/2 
Percent of Subjects with Mesopic Pupil 
Diameter < 2.4, < 2.2, < 2.0, < 1.8, < 
1.6, and < 1.4 mm by Time Point 
 
 
X 
X 
14.2.17.3 
Percent of Subjects with Mesopic Pupil 
Diameter < 2.4, < 2.2, < 2.0, < 1.8, < 
1.6, and < 1.4 mm by Time Point and 
Irides Type 
 
 
 
X 
14.2.18.1/2 
Percent of Subjects with Mesopic Pupil 
Diameter 2 to < 2.4 mm, 1.6 to < 2 mm, 
and 1.2 to < 1.6 mm by Time Point 
 
 
X 
X 
14.2.18.3 
Percent of Subjects with Mesopic Pupil 
Diameter 2 to < 2.4 mm, 1.6 to < 2 mm, 
and 1.2 to < 1.6 mm by Time Point and 
Irides Type 
 
 
 
X 
14.2.19.1/2 
Percent of Subjects with ≥ 15 Letter 
Improvement in Photopic Binocular 
DCNVA and Mesopic Pupil Diameter 
1.6 to < 2 mm by Time Point 
 
 
X 
X 
14.2.19.3 
Percent of Subjects with ≥ 15 Letter 
Improvement in Photopic Binocular 
DCNVA and Mesopic Pupil Diameter 
1.6 to < 2 mm by Time Point and Irides 
Type 
 
 
 
X 
14.2.20.1/2 
Percent of Subjects with Photopic 
Baseline DCNVA ≤ 20/63, ≤ 20/80, 
≤ 20/100, ≤ 20/125, and ≤ 20/160 With 
≥ 15-letter Improvement in Photopic 
DCNVA by Time Point 
 
 
X 
X 
14.2.20.3 
Percent of Subjects with Photopic 
Baseline DCNVA ≤ 20/63, ≤ 20/80, 
 
 
 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
≤ 20/100, ≤ 20/125, and ≤ 20/160 With 
≥ 15-letter Improvement in Photopic 
DCNVA by Time Point and Irides Type 
14.2.21.1/2 
Percent of Subjects with Photopic 
Baseline DCNVA from 20/50 to 20/63, 
20/80 to 20/100, and 20/125 to 20/160 
With ≥ 15-letter Improvement in 
Photopic DCNVA by Time Point 
 
 
X 
X 
14.2.21.3 
Percent of Subjects with Photopic 
Baseline DCNVA from 20/50 to 20/63, 
20/80 to 20/100, and 20/125 to 20/160 
With ≥ 15-letter Improvement in 
Photopic DCNVA by Time Point and 
Irides Type 
 
 
 
X 
14.2.22.1/2 
Percent of Subjects with Mesopic 
Baseline DCNVA ≤ 20/63, ≤ 20/80, 
≤ 20/100, ≤ 20/125, and ≤ 20/160 With 
≥ 15-letter Improvement in Mesopic 
DCNVA by Time Point 
 
 
X 
X 
14.2.22.3 
Percent of Subjects with Mesopic 
Baseline DCNVA ≤ 20/63, ≤ 20/80, 
≤ 20/100, ≤ 20/125, and ≤ 20/160 With 
≥ 15-letter Improvement in Mesopic 
DCNVA by Time Point and Irides Type 
 
 
 
X 
14.2.23.1/2 
Percent of Subjects with Mesopic 
Baseline DCNVA from 20/50 to 20/63, 
20/80 to 20/100, and 20/125 to 
20/160With ≥ 15-letter Improvement in 
Mesopic DCNVA by Time Point 
 
 
X 
X 
14.2.23.3 
Percent of Subjects with Mesopic 
Baseline DCNVA from 20/50 to 20/63, 
20/80 to 20/100, and 20/125 to 20/160 
With ≥ 15-letter Improvement in 
Mesopic DCNVA by Time Point and 
Irides Type 
 
 
 
X 
14.2.24.1 
Percent of Subjects with Photopic 
DCNVA ≤ 20/15, ≤ 20/20, ≤ 20/25, ≤ 
20/32, ≤ 20/40, ≤ 20/50, ≤ 20/63, ≤ 
20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 
by Time Point 
 
 
 
X 
14.2.24.2 
Percent of Subjects with Photopic 
DCNVA ≤ 20/15, ≤ 20/20, ≤ 20/25, ≤ 
20/32, ≤ 20/40, ≤ 20/50, ≤ 20/63, ≤ 
 
 
 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 
by Time Point and Irides Type 
14.2.25.1 
Percent of Subjects with Mesopic 
DCNVA ≤ 20/15, ≤ 20/20, ≤ 20/25, ≤ 
20/32, ≤ 20/40, ≤ 20/50, ≤ 20/63, ≤ 
20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 
by Time Point 
 
 
 
X 
14.2.25.2 
Percent of Subjects with Mesopic 
DCNVA ≤ 20/15, ≤ 20/20, ≤ 20/25, ≤ 
20/32, ≤ 20/40, ≤ 20/50, ≤ 20/63, ≤ 
20/80, ≤ 20/100, ≤ 20/125, and ≤ 20/160 
by Time Point and Irides Type 
 
 
 
X 
14.3.1.1.1/2 
Overall Summary of Treatment 
Emergent Adverse Events (TEAE) 
 
X 
X 
 
14.3.1.2 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class and 
Preferred Term 
 
X 
 
 
14.3.1.3 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class, 
Preferred Term, and Severity 
 
X 
 
 
14.3.1.4 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class, 
Preferred Term, and Relationship to 
Study Treatment 
 
X 
 
 
14.3.1.5 
Serious Treatment-Emergent Adverse 
Events (TEAE) by System Organ Class 
and Preferred Term 
 
X 
 
 
14.3.1.6 
Treatment-Emergent Adverse Events 
(TEAE) Leading to Withdrawal From 
the Study by System Organ Class and 
Preferred Term 
 
X 
 
 
14.3.1.7 
Treatment-Emergent Adverse Events 
(TEAE) Leading to Study Medication 
Discontinuation by System Organ Class 
and Preferred Term 
 
X 
 
 
14.3.3.1 
Categorical Summary of Conjunctival 
Hyperemia by Time Point 
 
X 
 
 
14.3.3.2 
Summary of Mean Conjunctival 
Hyperemia by Time Point 
 
X 
 
 
14.3.4 
Subjective Ocular Tolerability  
 
X 
 
 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.3.5 
Vital Signs by Time Point 
 
X 
 
 
14.3.6 
Intraocular Pressure (IOP) 
 
X 
 
 
14.3.7 
Biomicroscopy 
 
X 
 
 
14.3.8 
Ophthalmoscopy 
 
X 
 
 
 
12.2. 
List of Planned Listings 
Listing 
Description of Listing 
16.2.1.1 
Subject Disposition 
16.2.1.2 
Eligibility and Randomization 
16.2.2 
Treatment Assignment and Analysis Populations 
16.2.3 
Protocol Deviations 
16.2.4.1 
Demographics 
16.2.4.2 
Best Corrected Visual Acuity 
16.2.4.3 
General Medical History (Non-ocular) 
16.2.4.4 
Ocular Medical History 
16.2.4.5 
Prior Medications 
16.2.4.6 
Concomitant Medications 
16.2.5.1 
Study Drug Administration 
16.2.5.2 
LDP Administration 
16.2.6.1 
Distance Corrected Near Visual Acuity (DCNVA) 
16.2.6.2 
Best Corrected Distance Visual Acuity (BCDVA) 
16.2.6.3 
Distance Corrected Intermediate Visual Acuity (DCIVA) 
16.2.6.4 
Pupil Diameter 
16.2.7 
Adverse Events 
16.2.8 
Conjunctival Hyperemia 
16.2.9 
Subjective Ocular Tolerability 
16.2.10 
Biomicroscopy Examination 
16.2.11 
Ophthalmoscopic Examination 
16.2.12 
Vital Signs 
16.2.13 
Intraocular Pressure (IOP) Tonometry 
16.2.14 
Dry Eye Tests 
16.2.15 
Urine Pregnancy Test