Clinical trial NCT04638660
Conditions: Dim Light Vision Disturbances
Keywords: Nyxol, Night Vision Disturbances, Glare, Halos, Starbursts
Sponsor: Ocuphire Pharma, Inc.
Start date: 2020-12-30; Completion date: 2022-05-19
Enrollment: 144 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objectives of this study are:

* To evaluate the efficacy of Nyxol to improve mesopic low contrast visual acuity (mLCVA) in subjects with Dim Light Vision Disturbances (DLD)
* To evaluate efficacy of Nyxol to improve visual performance
* To evaluate the safety of Nyxol

Detailed description:
Placebo-controlled, double-masked, multiple-dose, Phase 3 study in approximately 160 randomized subjects with DLD (approximately 136 that are evaluable for efficacy), evaluating safety and efficacy of Nyxol in subjects with DLD following administration of Nyxol once daily (QD) at or near bedtime (at 8PM to 10PM) in both eyes (OU) for 14 days.

Following the successful completion of screening, each subject will be stratified by iris color (light/dark irides) and will then be randomized to treatment (masked) 1:1, Nyxol or placebo (vehicle).

Treatment (Nyxol or placebo) will be administered in both eyes (OU) by the subjects at or near bedtime each day.

At the first visit subjects will be screened for study eligibility.

Treatment visits will occur 2 times: Day 8 (+1 day)/Visit 2 and Day 15 (+1 day)/Visit 3. mLCVA evaluations shall be performed on each of these days.

A follow-up visit (Visit 4) phone call will occur 1 to 3 days after Visit 3.

At select sites OPD Scan measurements will be made using wavefront abhermettry.

Arms: arm 1: One drop in both eyes at or near bedtime (8PM to 10PM) arm 2: One drop in both eyes at or near bedtime (8PM to 10PM)
Interventions: intervention 1: Phentolamine Ophthalmic Solution 0.75% intervention 2: Phentolamine Ophthalmic Solution Vehicle (Placebo)
Intervention descriptions: intervention 1: 0.75% phentolamine ophthalmic solution (Nyxol), a non-selective alpha-1 and alpha-2 adrenergic antagonist intervention 2: Topical sterile ophthalmic solution

Protocol documents:
Prot_000.pdf: 
 
CONFIDENTIAL 
 
Clinical Study Phase 3 Protocol 
OPI-NYXDLD-301 
LYNX-1 
 
RANDOMIZED, PLACEBO-CONTROLLED, DOUBLE-MASKED STUDY OF THE 
SAFETY AND EFFICACY OF NYXOL (0.75% PHENTOLAMINE OPHTHALMIC 
SOLUTION) IN SUBJECTS WITH DIM LIGHT VISION DISTURBANCES 
 
 
Ocuphire Pharma, Inc. 
37000 Grand River Avenue, Suite 120 
Farmington Hills, MI 48335 
 
 
 
 
 
Original: 
October 28, 2020 
Amendment 1: 
 
November 6, 2020 
Amendment 2: 
March 25, 2021 
 
 
 
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 
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SUMMARY OF CHANGES FOR AMENDMENT 2 
Bold underlined text shows additions; strikethrough text shows deletions. For changes that 
affect multiple sections of the protocol, the change is listed once at the first instance below, 
and each subsequent protocol section incorporating that change is also listed at that point. 
Administrative and minor editing changes not affecting the content or conduct of the protocol 
have been made; these are not listed. None of the changes made affect the risks to the subjects 
in this study. 
Section: Location 
Description of Change 
Rationale for Change 
Synopsis: Inclusion 
Criteria, p.13 
5.1  Subject Inclusion 
Criteria, p.41 
7.
 under mesopic conditions (prior to
illumination) in at least one eye. This test may be
repeated once following an additional 5 minutes of
adaptation to the mesopic light conditions if the
initial results do not meet this criterion.
Global change to 
modify mesopic PD 
eligibility criterion 
from
 
mm and to clarify that 
one repeated screening 
of PD under mesopic 
conditions is 
acceptable during the 
Screening/Baseline 
visit 
Synopsis: Inclusion 
Criteria, p.13 
5.1  Subject Inclusion 
Criteria, p.41 
8. ≤ 230 (20/100 Snellen or worse) Early Treatment
Diabetic Retinopathy Study (ETDRS) letters (20/63
Snellen or worse) in mLCVA score in at least one
eye using the Precision Vision Illuminator Cabinet
with 5% translucent contrast chart and a mesopic filter
at 4 m.
Global change to 
modify mLCVA 
eligibility criterion 
from 20 letters (20/100 
Snellen or worse) to 30 
letters (20/63 Snellen 
or worse) 
4.4.3  Study 
Medication 
Administration, p.37 
The first dose of study medication will be taken at 
8PM to 10PM on the evening of the Baseline Visit 
(Day 1). 
 
 
 
Global change to 
modify and clarify the 
contact lens 
discontinuation 
requirements for 
contact lens wearers 
Synopsis: Exclusion 
Criteria, p.14 
5.2  Subject Exclusion 
Criteria, p.42 
12. Unwilling or unable to discontinue use of contact
lenses at screening until study completion, except for
keratoconus subjects who may wear contacts up to 24 
hours prior to on their scheduled visits 
 
 
 
 (for 
both types of lenses) before on Day 8 and Day 15. 
7.2.1  Visit 1 
(Screening/Baseline)/ 
Day 1, p.47 
 
 
 
 
7.2.1  Visit 1 
(Screening/Baseline)/ 
Day 1, p.48 
The first dose of study medication will be taken at 
8PM to 10PM on the evening of the Baseline Visit 
(Day 
 
 
 


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7.2.2  Visit 2 
(Treatment)/Day 8 ± 1 
day), p.49 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
7.2.3  Visit 3 
(Treatment)/Day 15 ± 
1 day), p.50 
If
 
 
 
Synopsis: Treatment 
Visits, p.17 
Treatment visits will occur 2 times: Day 8 (+±1 
day)/Visit 2 and Day 15 (+±1 day)/Visit 3. 
Global change to 
remove the restriction 
that the Day 8 and Day 
15 clinic visits have to 
be performed in the 
8AM to 10AM time 
window and to open 
the visit window to ± 1 
day 
Synopsis: Study 
Medications, Dose and 
Mode of 
Administration, p.21 
Nyxol® Eye Drops (Phentolamine Ophthalmic 
Solution): 
One drop of Nyxol QD at or near bedtime (8PM to 
10PM) OU for 14 days (+±1 day) for subjects 
randomized to active treatment. 
Placebo (Nyxol vehicle): 
One drop of placebo QD at or near bedtime (at 8PM 
to 10PM) OU for 14 days (+±1 day) for subjects 
randomized to placebo. 
Synopsis: Duration of 
Subject Participation 
and Study, p.21 
The total length of subject participation is 
approximately 3 weeks with 3 clinic visits and one 
telephone call follow-up summarized below: 
• 
Visit 1 (Screening/Baseline)/Day 1 
• 
Visit 2 (Treatment)/Day 8 (+±1 day) at 
approximately 8AM to 10AM (+2 hours) 
• 
Visit 3 (Treatment)/Day 15 (+±1 day) at 
approximately 8AM to 10AM (+2 hours) 
Table 2. Screening, 
Baseline, Treatment, 
and Follow-Up Visits 
and Procedures, p.34 
 
Screening/ 
Baseline 
[a] 
Treatment Treatment 
TC 
Follow-
Up 
Day 
1 
8 +± 1 
Day (8 ‒ 
10 AM  + 
2 hr) 
 
15 +± 1 
Day (8 ‒ 10 
AM  + 2 hr) 
 
1 to 3 
days after 
Visit 3 
 
4.5  Expected 
Duration of Subject 
Participation, p.38 
The total length of subject participation is 
approximately 3 weeks, with 3 clinic visits and one 
telephone call follow-up summarized below: 
• Visit 1 (Screening/Baseline)/Day 1 
• Visit 2 (Treatment)/Day 8 (+±1 day) 
• Visit 3 (Treatment)/Day 15 (+±1 day) 


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7.2.2  Visit 2 
(Treatment)/Day 8 ± 1 
day), p.49 
At Visit 2, subjects should be scheduled for this visit 
between approximately 8AM and 10AM (+2 hour). 
the medication box, complete with opened bottles and 
any unopened study medication, and diary will be 
returned to the study site where the site will account 
for opened and unopened study medication and the 
study medication box will be re-dispensed with the 
unopened medication. 
7.2.3  Visit 3 
(Treatment)/Day 15 ± 
1 day), p.50 
At Visit 3, subjects should be scheduled for this visit 
between approximately 8AM and 10AM (+2 hour). 
the subject will bring their used dropper bottles with 
any unused medications and diary with them for 
purposes of drug accountability. The last day of study 
treatment is the day before Visit 3; no further study 
medication will be dispensed at Visit 3. 
7.2.1  Visit 1 
(Screening/Baseline)/ 
Day 1. p.47 
Prior to further screening, the subject must be asked to 
complete the subject questionnaire. 
 
 
 
 
 
, Then subjects shall be assessed in each 
eye for PD and for mLCVA before and during 
illumination of the contralateral eye with a 
 
”) (
 
To clarify that subjects 
need their refraction 
confirmed at the 
Screening/Baseline 
visit. 
7.2.1  Visit 1 
(Screening/Baseline)/ 
Day 1, p.48 
If the subject meets all eligibility criteria, they will 
be randomized to a treatment group. If a subject 
fails the screen, they can return for a rescreening 
(signing a new informed consent and assigning a 
new subject number) at the investigator’s 
discretion. 
To clarify that 
rescreening of subjects 
who initially fail 
screening is 
acceptable. 
 
 


3/25/2021


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INVESTIGATOR’S AGREEMENT 
 
OPI-NYXDLD-301 
LYNX-1 
 
RANDOMIZED, PLACEBO-CONTROLLED, DOUBLE-MASKED STUDY OF 
THE SAFETY AND EFFICACY OF NYXOL (0.75% PHENTOLAMINE 
OPHTHALMIC SOLUTION) IN SUBJECTS WITH DIM LIGHT VISION 
DISTURBANCES 
 
 
Original Protocol:  October 28, 2020 
Amendment 1: 
November 6, 2020 
Amendment 2:  
March 25, 2021 
 
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 electronic records; 
electronic signatures (21 CFR 11, the protection of human subjects (21 CFR 50), financial 
disclosure by Clinical Investigators 921 CFR 54), 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 
Ann Grace 
 
 
Medical Monitor 
Benjamin Rubin, MD 
(
 
              
  
 
 
 
 


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ABBREVIATIONS AND TERMS 
Abbreviation 
Full term 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ARP 
All Randomized Population 
ARVO 
Association for Research in Vision and Ophthalmology 
BAT 
Brightness Acuity Tester 
BCDVA 
best-corrected distance visual acuity  
BP 
blood pressure 
°C 
degrees Celsius 
CCLRU 
Cornea and Contact Lens Research Unit 
CFR 
Code of Federal Regulations 
CHF 
congestive heart failure 
CI 
confidence interval 
CRA 
clinical research associate 
CRO 
clinical research organization 
CS 
contrast sensitivity 
°F 
degree Fahrenheit 
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 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HR 
heart rate 
IB 
Investigators’ Brochure 
ICH 
International Council for Harmonisation 
IND 
Investigational New Drug  
IOL 
intraocular lens 


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IOP 
intraocular pressure 
IRB 
Institutional Review Board 
ITT 
intention-to-treat 
IUD 
intrauterine device 
LASIK 
laser-assisted in situ keratomileusis 
LCVA 
low-contrast visual acuity 
LDPE 
low-density polyethylene 
logMAR 
logarithm of the Minimum Angle of Resolution 
LSM 
least squares mean 
max 
maximum 
MedDRA 
Medical Dictionary for Regulatory Activities 
mHCVA 
mesopic high-contrast visual acuity (BCDVA) 
mLCVA 
mesopic low-contrast visual acuity (BCDVA) 
mITT 
Modified intention-to-treat 
Nyxol® 
0.75% Phentolamine Ophthalmic Solution or 1% Phentolamine 
Mesylate Ophthalmic Solution 
OD 
oculus dexter (right eye) 
OR 
odds ratio 
OS 
oculus sinister (left eye) 
OTC 
over-the-counter 
OU 
oculus uterque (both eyes) 
PD 
pupil diameter 
pHCVA 
photopic high-contrast visual acuity (BCDVA) 
pLCVA 
photopic low-contrast visual acuity (BCDVA) 
PP 
Per Protocol 
PRK 
photorefractive keratectomy 
QD 
once daily 
RK 
radial keratectomy 
RMS 
root-mean square 
SAE 
serious adverse event 


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SOC 
system organ class 
SP 
Safety Population 
TEAE 
treatment-emergent adverse event 
US 
United States 
USP 
United States Pharmacopeia 
VA 
visual acuity 
 
 
 


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1 
STUDY SUMMARY 
Study Number 
OPI-NYXDLD-301 
Clinical Phase 
Phase 3 
Type of Study 
Randomized, placebo-controlled, double-masked, multiple-
dose study of the safety and efficacy of Nyxol (0.75% 
Phentolamine Ophthalmic Solution) in subjects with dim light 
vision disturbances (DLD). 
Name of 
Investigational 
Product 
Nyxol® Eye Drops – 0.75% Phentolamine Ophthalmic 
Solution 
Duration of Study  
Approximately 3 weeks, including screening, treatment, and 
follow-up. 
Rationale 
DLD, which includes night vision disturbances, affects patients 
under mesopic or scotopic conditions. 
 
 
 
 
 
 
 
When the pupil dilates in people with DLD, peripheral 
aberrations scatter light rays resulting in these visual 
aberrations, a loss of mesopic low-contrast visual acuity 
(mLCVA) and contrast sensitivity (CS). It is proposed that a 
moderate pharmacologic reduction of pupil size (20-25%) by 
the application of an alpha-1 antagonist has the potential to 
mitigate DLD in many affected individuals. 
Phentolamine is a non-selective alpha-1 and alpha-2 adrenergic 
antagonist acting on the adrenergic receptors and inhibiting 
contraction of the smooth muscle. When topically applied, 
phentolamine inhibits contraction of the iris dilator muscle, 
resulting in a smaller pupil size. 
Previous Phase 2 clinical trials showed that Nyxol significantly 
decreases pupil diameter (PD) and improves mLCVA in DLD 
subjects after single- and multi-dose (14 days) treatment. 
Nyxol was generally well-tolerated in the eye (most common 
complaint was transient mild-to-moderate ocular hyperemia 
and eye irritation) and there were no clinically meaningful 
systemic effects as measured by heart rate (HR) and blood 
pressure (BP).  


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Study Objectives 
The objectives of this study are: 
• To evaluate the efficacy of Nyxol to improve mLCVA 
in subjects with DLD 
• To evaluate the efficacy of Nyxol to improve visual 
performance  
• To evaluate the safety of Nyxol 
• To evaluate any additional benefits of Nyxol in 
treating DLD subjects 
The Sponsor intends to use this first Phase 3 registration study 
to evaluate Nyxol for the indication of “treatment of symptoms 
associated with dim light or night vision disturbances.” 
Design 
Placebo-controlled, double-masked, multiple-dose, Phase 3 
study in approximately 160 randomized subjects with DLD 
(approximately 136 that are evaluable for efficacy), evaluating 
safety and efficacy of Nyxol in subjects with DLD following 
administration of Nyxol once daily (QD) at or near bedtime (at 
8PM to 10PM) in both eyes (OU) for 14 days. 
Following the successful completion of screening, each subject 
will be stratified by iris color (light/dark irides) and will then 
be randomized to treatment (masked) 1:1, Nyxol or placebo 
(vehicle). 
Treatment (Nyxol or placebo) will be administered OU by the 
subjects at or near bedtime each day. 
 
 
 
 
 
 
 
 
The study eye and non-study eye, as well as 
binocular, will be evaluated at all appropriate assessments.  
Subject Population  
Approximately 160 randomized (approximately 136 that are 
evaluable for efficacy) subjects experiencing DLD 
Inclusion Criteria 
1. Males or females ≥ 18 years of age 
2. Subject-reported DLD (likely subjects with a history of 
multifocal IOLs, post–laser-assisted in situ keratomileusis 
[LASIK], corneal scars, and keratoconus) 


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3. Ability to comply with all protocol-mandated procedures 
independently and to attend all scheduled office visits 
4. Otherwise healthy and well-controlled subjects 
5. Able and willing to give written consent to participate in 
this study 
6. Able to self-administer study medication 
7. 
 
This test may be repeated 
once following an 
 of adaptation to the 
 if the initial results do not meet 
this criterion. 
8. ≤ 30 Early Treatment Diabetic Retinopathy Study 
(ETDRS) letters (20/63 Snellen or worse) in mLCVA score 
in at least one eye using the Precision Vision Illuminator 
Cabinet with 5% translucent contrast chart and a mesopic 
filter at 4 m. 
9. ≥ 10 ETDRS letters (≥ 2 lines) improvement in mLCVA in 
at least 
 
 
 
 
Exclusion Criteria 
Ophthalmic: 
1. Prior unresolved dry eye diagnosis, taking prescription 
drops for dry eye, or taking artificial tear drops routinely 
for dry eye  
2. Prior history of fluctuating vision 
3. Clinically significant ocular disease as deemed by the 
Investigator (e.g., untreated cataract, glaucoma, corneal 
edema, uveitis, severe keratoconjunctivitis sicca, retina 
degeneration, loss of visual field due to glaucoma or 
stroke, branch retinal vein occlusion, retina flare) that 
might interfere with the study  
4. Known hypersensitivity to any topical alpha-adrenoceptor 
antagonists 
5. Known allergy or contraindication to any component of 
the vehicle formulation 
6. History of cauterization of the punctum or punctal plug 
(silicone or collagen) insertion or removal 


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7. Ocular trauma, ocular surgery (e.g., IOLs) or laser 
procedure (e.g., LASIK, photorefractive keratectomy 
[PRK]) within 6 months prior to screening 
8. Use of any topical prescription or over-the-counter (OTC) 
ophthalmic medications of any kind (including artificial 
tear drops) 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.) 
9. Recent or current evidence of ocular infection or 
inflammation in either eye (such as current evidence of 
clinically significant blepharitis, conjunctivitis, or a 
history of herpes simplex or herpes zoster keratitis at 
screening in either eye). Subjects must be symptom free 
for at least 7 days. 
10. History of diabetic retinopathy, diabetic macular edema, or 
dry or wet macular degeneration 
11. 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, etc.) 
12. Unwilling or unable to discontinue use of contact lenses 
on their scheduled visits at least 1 hour for soft contact 
lenses and at least 8 hours for hard gas-permeable contact 
lenses before ophthalmic assessments at screening, and at 
least 8 hours (for both types of lenses) before Day 8 and 
Day 15. 
13. People with undiagnosed dry eye, at the determination of 
the Investigator. Dry eye evaluation should be based on 
one of the following dry eye tests: 
a. TearLabs osmolarity test (> 308 mOsm/L in 
either eye or an inter-eye difference ≥ 10 
mOsm/L) 
b. Corneal fluorescein staining (≥ grade 2 in the 
inferior or ≥ grade 1 in the central zone using 
the National Eye Institute scale) 
c. Tear break-up time of < 5 seconds 
Systemic: 
14. 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 


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blockage or congestive heart failure [CHF]; severe 
diabetes as defined below) 
a. Predisposition to severe hypoglycemia (2 or 
more serious hypoglycemic episodes requiring 
assistance within the past year) 
b. Any hospitalization or emergency room visit 
due to poor diabetic control within the past 6 
months 
c. Currently untreated diabetes mellitus or 
previously untreated subjects who initiated oral 
anti-diabetic medication or insulin within 3 
months prior to Day 1 
d. Any sign of diabetic retinopathy in either eye 
e. Uncontrolled diabetes mellitus defined by 
Hemoglobin A1c > 12% taken within the last 
3 months 
15. Clinically significant systemic disease (e.g., severe 
diabetes as previously defined, myasthenia gravis, cancer, 
hepatic, renal, endocrine, or cardiovascular disorders) that 
might interfere with the study 
16. 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  
17. Participation in any investigational study within 30 days 
prior to screening and during the conduct of the study 
18. 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), Visit 2 (Treatment), and 
Visit 3 (Treatment) examinations and must intend to not 
become pregnant during the study 
19. Resting HR outside the specified range (50-110 beats per 
minute) at Visit 1 (Screening/Baseline). HR may be 


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repeated only once if outside the specified range following 
at least a 5-minute rest period in the sitting position 
20. Hypertension with resting diastolic BP > 105 mmHg or 
systolic BP > 160 mmHg at Visit 1 (Screening/Baseline). 
BP may be repeated only once if outside the specified 
range following at least a 5-minute rest period in the 
sitting position 
Screening/Baseline 
Visit 
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, where the 
dose of study treatment is given. 
Once a subject arrives at the study center, a member of the 
study center 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 identification number is assigned. 
The start of screening includes an explanation of the study, a 
medical and ophthalmic history, demographics, and a review of 
prior/concomitant medications. During this time, eligibility for 
 shall be determined first. 
 
) 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 #18), 
and HR/BP (Exclusion Criteria #19-20). 
 
 
 
 
 
 
 
 
 
 
 
 
 
. Investigators should frequently remind 
subjects to blink between visual acuity assessments. 
The study eye of the subject 
 
in order for the subject to not screen fail. 


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Before further screening criteria, the following assessments 
shall be performed to establish baseline values: 
• Mesopic high-contrast visual acuity (mHCVA), 
photopic low-contrast visual acuity (pLCVA), and 
mesopic high-contrast distance-corrected near visual 
acuity (DCNVA) 
• Using an 
 
 
 and corneal topography 
measurements under 
 
• Conjunctival hyperemia, assessed visually with a 
grading scale (0-3) using images from the Cornea and 
Contact Lens Research Unit (CCLRU; Investigators 
are cautioned to appropriately note all observations of 
conjunctival hyperemia on the biomicroscopy 
electronic Case Report Form at screening) 
• Slit lamp biomicroscopy 
The subject must also meet dry eye Exclusion Criterion #13 to 
be included in the study at the determination of the 
investigator. Specifically, subjects with undiagnosed dry eye 
may undergo a dry eye test. 
Final screening must include IOP measurement (using a Tono-
Pen) and ophthalmoscopy. 
With the subject passing all eligibility criteria, the Screening 
Visit becomes the Baseline Visit. The subject will then be 
assigned a randomization number and randomized into the 
study. 
See Table 2 for details on measurements expected at each visit.  
The first dose of study medication must be taken by the subject 
at 8PM to 10PM on the Baseline Visit (Day 1). 
All treatments will be administered OU. 
Treatment Visits 
Treatment visits will occur 2 times: Day 8 (±1 day)/Visit 2 and 
Day 15 (±1 day)/Visit 3. mLCVA evaluations shall be 
performed on each of these days. 
 
 
 Other assessments as defined in Table 2 shall be 
performed at these visits. 
Follow-up Visit  
A follow-up visit (Visit 4) phone call will occur 1 to 3 days 
after Visit 3. Concomitant medications and adverse events 
(AEs) will be collected. 


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Number of 
Investigational Sites 
Approximately 15 sites 
Estimated Total 
Sample Size 
Approximately 160 randomized subjects, with approximately 
136 subjects that are evaluable for efficacy 
Sample Size 
Justification 
A sample size of approximately 136 subjects (68 per treatment 
group) that are evaluable for efficacy (i.e. who have a Day 8 
mLCVA measurement) is needed for the study. 
 
 
 
 
 
 
 
 
 
All subjects will be randomized into the study in a 1:1 ratio to 
one of the treatment arms (Nyxol or placebo), with a 
 
It is assumed that there will be approximately 15% drop-out. 
To account for this drop-out, a total of approximately 160 
subjects will be randomized into the study. 
Primary Efficacy 
Endpoint 
Percent of subjects with ≥ 15 ETDRS letters (≥ 3 lines) of 
improvement in the study eye compared to baseline in 
monocular mLCVA at Day 8 for the mITT population. 
Secondary and 
Exploratory Efficacy 
Endpoints 
Secondary efficacy endpoints (for the study eye, the non-study 
eye, the best of either eye, and binocular) will include: 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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study eye, the best of either eye, and binocular) shall be 
collected and analyzed for the sites that have an OPD Scanner 
and will include but are not limited to: 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
, later 
 
 
 
 
 
 
 
 
 
 


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. 
 
 
 
 
 
Subject will be 
allowed to acclimate to these lighting conditions (with the eyes 
open normally for a minimum of 
 prior to the set of 
PD, BCDVA, DCNVA, and wavefront error measurements). 
Subject 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 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. 
All of the efficacy endpoints will be analyzed overall, and by 
 at all timepoints. The mLCVA endpoints will 
be analyzed by baseline PD 
 and higher-
order aberrations subpoint. 
All efficacy endpoints will be analyzed by Modified Intention 
to Treat (mITT) and Per Protocol (PP) populations. 
 Safety Endpoints 
The primary safety measures are conjunctival hyperemia and 
AEs.  
Analysis for conjunctival hyperemia includes change from 
baseline (Day 1) in the conjunctival hyperemia grading scale 
(CCLRU images) at each visit (Day 8 and Day 15), for study 
eye and non-study eye. Any increase of 2 units in conjunctival 
hyperemia between consecutive visits is considered an AE. 
Other safety measures include intraocular pressure (IOP), 
biomicroscopy, ophthalmoscopy, dry eye measurements, and 


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systemic safety as measured by HR and BP. Urine pregnancy 
tests for females of childbearing potential will be conducted. 
Subject questionnaire values will be summarized by 
treatment group and visits (Day 1, Day 8, and Day 15). 
Measurements: 
• Conjunctival hyperemia will be assessed visually with 
a grading scale (0-3) using images from the CCLRU 
• IOP will be measured with the Tono-Pen 
• BP and HR will be measured manually or with 
available digital devices 
• Subject questionnaire will be a brief symptom survey 
Study Medications, 
Dose and Mode of 
Administration 
Nyxol® Eye Drops (Phentolamine Ophthalmic Solution): 
One drop of Nyxol QD at or near bedtime (8PM to 10PM) OU 
for 14 days (±1 day) for subjects randomized to active 
treatment. 
Placebo (Nyxol vehicle): 
One drop of placebo QD at or near bedtime (at 8PM to 10PM) 
OU for 14 days (±1 day) for subjects randomized to placebo. 
Duration of Subject 
Participation 
and Study 
The total length of subject participation is approximately 3 
weeks with 3 clinic visits and one telephone call follow-up 
summarized below: 
• Visit 1 (Screening/Baseline)/Day 1 
• Visit 2 (Treatment)/Day 8 (±1 day) 
• Visit 3 (Treatment)/Day 15 (±1 day)  
• Follow-up telephone call 1 to 3 days after Visit 3 
The execution of the entire study (first subject screened 
through last randomized subject completed) is expected to be 
approximately 9 to 12 months. 
 


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TABLE OF CONTENTS 
INVESTIGATOR’S AGREEMENT ........................................................................... 6 
PROCEDURES IN CASE OF EMERGENCY ............................................................ 7 
ABBREVIATIONS AND TERMS .............................................................................. 8 
1 
STUDY SUMMARY ............................................................................... 11 
TABLE OF CONTENTS .......................................................................................... 22 
LIST OF TABLES .................................................................................................... 24 
2 
INTRODUCTION ................................................................................... 25 
2.1 
Investigational Products ..................................................................... 25 
2.2 
Findings From Nonclinical and Clinical Studies ................................. 25 
2.3 
Design Justification ............................................................................ 28 
2.4 
Route of Administration, Dosage Regimen, and Treatment Period ..... 29 
2.5 
Compliance ........................................................................................ 30 
2.6 
Study Population ................................................................................ 30 
3 
OBJECTIVES AND PURPOSE ............................................................... 31 
4 
STUDY DESIGN..................................................................................... 32 
4.1 
Primary and Secondary Endpoints ...................................................... 32 
4.2 
Description and Schedule of Visits and Procedures ............................ 34 
4.3 
Measures Taken to Minimize/Avoid Bias ........................................... 35 
4.4 
Study Medications .............................................................................. 35 
4.4.1 
Packaging and Labeling .......................................................... 36 
4.4.2 
Storage of Study Medication and Dispensing .......................... 36 
4.4.3 
Study Medication Administration ........................................... 37 
4.4.4 
Study Medication Accountability ............................................ 37 
4.4.4.1 
Receipt and Disposition of Study Medication ................. 37 
4.4.4.2 
Return of Study Medication ........................................... 37 
4.5 
Expected Duration of Subject Participation ........................................ 38 
4.6 
Randomization and Procedure for Breaking the Code ......................... 38 
4.7 
Collection of Data .............................................................................. 38 
4.8 
Completed Subject ............................................................................. 39 
4.9 
Non-completing Subject ..................................................................... 39 
4.9.1 
Study Medication Discontinuation .......................................... 39 
4.9.2 
Reasons for Withdrawal From Study ...................................... 39 
4.9.3 
Entire Study Terminated ......................................................... 40 
4.9.4 
Actions After Discontinuation................................................. 40 
4.10 
Completed Study ................................................................................ 40 
4.11 
Procedure After the Completion of the Study ..................................... 40 
5 
SUBJECT INCLUSION AND EXCLUSION CRITERIA ........................ 41 
5.1 
Subject Inclusion Criteria ................................................................... 41 


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5.2 
Subject Exclusion Criteria .................................................................. 41 
6 
TREATMENT OF SUBJECTS ................................................................ 44 
6.1 
Treatment Adherence ......................................................................... 44 
6.2 
Concomitant Medications ................................................................... 44 
7 
ASSESSMENT OF EFFICACY .............................................................. 46 
7.1 
Specification of the Efficacy Parameters ............................................ 46 
7.2 
Assessing, Recording, and Analyzing Efficacy Parameters ................. 46 
7.2.1 
Visit 1 (Screening/Baseline)/Day 1 ......................................... 46 
7.2.2 
Visit 2 (Treatment)/Day 8 ....................................................... 49 
7.2.3 
Visit 3 (Treatment)/Day 15 ..................................................... 49 
7.2.4 
Follow-Up Phone Call Visit 4 ................................................. 50 
7.2.5 
Unscheduled Visits ................................................................. 51 
7.2.6 
Visit Variation ........................................................................ 51 
8 
ASSESSMENT OF SAFETY .................................................................. 52 
8.1 
Specification of Safety Parameters ..................................................... 52 
8.2 
Assessing, Recording, and Analyzing Safety Parameters .................... 52 
8.3 
Adverse Events and Serious Adverse Events ...................................... 52 
8.3.1 
Adverse Event Definitions ...................................................... 53 
8.3.2 
Follow-Up of Subjects After Adverse Events .......................... 57 
9 
STATISTICS ........................................................................................... 58 
9.1 
Sample Size ....................................................................................... 58 
9.2 
Analysis Populations .......................................................................... 58 
9.3 
Statistical Methods ............................................................................. 58 
9.3.1 
General Considerations ........................................................... 58 
9.3.2 
Demographic and Baseline Characteristics .............................. 59 
9.3.3 
Subject Disposition ................................................................. 59 
9.3.4 
Medical History and Prior/Concomitant Medications .............. 59 
9.3.5 
Analysis of Efficacy ............................................................... 59 
9.3.6 
Analysis of Safety ................................................................... 60 
9.4 
Procedure for Accounting for Missing, Unused, or Spurious Data ...... 61 
9.5 
Procedure for Reporting Deviations From the Statistical Plan ............. 61 
10 
DIRECT ACCESS TO SOURCE DATA AND DOCUMENTS ............... 62 
11 
QUALITY CONTROL AND QUALITY ASSURANCE ......................... 63 
12 
ETHICAL CONSIDERATIONS AND GCP COMPLIANCE .................. 64 
12.1 
GCP Compliance ................................................................................ 64 
12.2 
Institutional Review Board ................................................................. 64 
12.3 
Protocol Deviations/Violations ........................................................... 64 
12.4 
Informed Consent and Assent Requirements ...................................... 64 
13 
DATA HANDLING AND RECORD KEEPING ..................................... 66 


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13.1 
Data Entry .......................................................................................... 66 
13.2 
Data Quality Control and Reporting ................................................... 66 
13.3 
Archiving of Data ............................................................................... 66 
13.4 
Records Retention .............................................................................. 66 
13.5 
Amendments to the Protocol............................................................... 66 
14 
BIBLIOGRAPHY .................................................................................... 68 
APPENDIX 1: IRIS COLOR CHART ................................................................. 69 
APPENDIX 2: VISUAL ACUITY CHARTS ...................................................... 70 
APPENDIX 3: SUBJECT QUESTIONNAIRE .................................................... 72 
APPENDIX 4: ADRENERGIC AND CHOLINERGIC DRUGS ......................... 75 
APPENDIX 5: SCREENING PROCESS FOR INCLUSION/EXCLUSION 
CRITERIA ......................................................................................... 76 
APPENDIX 6: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING 
IMAGES FROM CCLRU .................................................................. 77 
 
LIST OF TABLES 
Table 1 
Efficacy of 1% Nyxol in Reducing Pupil Diameter Under Mesopic 
Conditions in Phase 2 Trials ......................................................... 25 
Table 2 
Screening, Baseline, Treatment, and Follow-Up Visits and 
Procedures .................................................................................... 34 
 


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2 
INTRODUCTION 
2.1 
Investigational Products 
The test product is Nyxol® Eye Drops – 0.75% Phentolamine Ophthalmic Solution 
(Nyxol), a non-selective alpha-1 and alpha-2 adrenergic antagonist. 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. 
2.2 
Findings From Nonclinical and Clinical Studies 
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 patients were exposed to at least one dose 
of Phentolamine Mesylate Ophthalmic Solution. 
In prior clinical trials, Nyxol has demonstrated a consistent ability to decrease pupil by 
approximately 20% (~1–1.5 mm) under both mesopic and photopic conditions. Key pupil 
diameter (PD) data are summarized below (Table 1). 
Table 1 
Efficacy of 1% Nyxol in Reducing Pupil Diameter Under 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-SNV1 
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-01a22 
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 
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 
*Two eyes per subject were included to calculate the endpoint 
 
Nyxol was observed to be well-tolerated at single doses up to and including 1.0% daily in 
each eye. This includes 59 patients who received multiple doses of up to 1% Nyxol for at 


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least 14 days. Safety of the patients in these trials was evaluated by adverse event (AE) 
monitoring, physical examinations, and vital sign assessments. Across all trials, no healthy 
volunteers or patients 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 in 
the evening was observed to mitigate or minimize these side effects during the daytime. 
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), visual acuity (VA), and wavefront aberrometry. A total of 
24 patients (median age of 39) with severe night vision complaints were randomly 
assigned 2:1 to treatment groups (active treatment, n = 16; placebo control, n = 8). Patients 
had to demonstrate at least a 2-line improvement in low-contrast visual acuity (LCVA) in 
dim light during illumination of the contralateral eye at screening. 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 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. 
The planned United States (US) Food and Drug Administration (FDA) primary endpoint 
was percent of subjects with 15 Early Treatment Diabetic Retinopathy Study (ETDRS) 
letters (3 lines) of improvement in mesopic LCVA (mLCVA) at a single timepoint. In this 
trial, even with a small sample size, there was a positive trend of 15-letter (3-line or 
greater) improvement in mLCVA (19% Nyxol vs 0% for placebo, p = 0.16) and photopic 
low-contrast visual acuity (pLCVA) (19% Nyxol vs 0% for placebo, p = 0.16). 
Additionally, greater fractions of Nyxol-treated eyes registered a 1-line (5-letter or greater) 
improvement in mLCVA (69% vs 31% for placebo, p = 0.029) and pLCVA (63% vs 13% 
for placebo, p = 0.017), as well as a 2-line (10-letter or greater) improvement in mLCVA 
(34% vs 6% for placebo, p < 0.03) and pLCVA (28% vs 0% for placebo, p < 0.02). 
Other distance VA measurements were made, including mesopic distance high-contrast 
visual acuity (mHCVA) and photopic distance high-contrast visual acuity (pHCVA). 
Greater fractions of Nyxol-treated eyes registered a 2-line (10-letter or greater) statistically 
significant improvement in mHCVA (25% vs 0% for placebo, p < 0.03), with a notable but 
not statistically significant trend in pHCVA (19% vs 0% for placebo, p = NS). Differences 
in mean change in VA between treatments were also seen. There were statistically 
significant improvements with 1% Nyxol from pre-treatment across all mean VA 
measurements (p < 0.0001). Further, mean mLCVA showed statistically significant 
improvement for both treatment groups 2 to 3 hours post-treatment, with the mean 


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magnitude of improvement for phentolamine mesylate–treated patients being over twice 
that of placebo-treated patients (8.0 vs 3.1 letters, respectively; p = 0.035). 
Additionally, mean PD decreased at a statistically significant amount of 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 change between treatment groups was also 
statistically significant (1.1 mm, p < 0.0001) (Table 1). In a post-hoc analysis that helped 
inform the Phase 3 trial design, there was an average of ~1.5 mm PD reduction in patients 
with baselines above 6 mm, compared to ~1 mm reduction in patients with baselines 
below 6 mm. Measurements were taken 2 to 3 hours after dosing.  
Total wavefront root-mean square (RMS) error is the summation of all aberrations 
measured with a wavefront device (VISX-CustomVue Aberrometer), delineated in µm 
(microns), RMS error for short. Higher-order RMS error is the summation of higher-order 
aberrations, including trefoil, coma, and spherical aberrations that because of their 
complex nature cannot be corrected with regular corrective lenses. Reduction in higher 
“errors” would be consistent with improvements in dim light vision disturbances (DLD). 
In a post-hoc analysis with the purposes to help inform future trials and commercial 
efforts, the difference in change between Nyxol and placebo treatment arms for both total 
RMS (0.42 µm, p = 0.0004) and higher-order RMS (0.17 μm, p < 0.0001) were 
statistically significant, with Nyxol-treated eyes showing improvement with a larger 
reduction in error. 
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). 
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 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. 
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 
patients with open-angle glaucoma or ocular hypertension. After screening was performed 
based on inclusion and exclusion criteria, a total of 39 elderly patients (median age of 63) 
were randomized into the trial (Nyxol arm, n = 19; placebo arm, n = 20). These patients 
were either treatment-naïve or were previously taking intraocular pressure (IOP)-lowering 
medication that was washed out for 30 days prior to dosing. Patients took their study 
medication (Nyxol or placebo) in both eyes (OU) between 8PM to 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 in mean diurnal IOP at Day 15 from baseline. Mean diurnal 


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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 
redness (using the Cornea and Contact Lens Research Unit [CCLRU] grading 4-point scale 
[0-3]), AEs, HR, BP, concomitant medications, and pregnancy. Highlights of this trial 
were presented at the 2020 annual meeting of the Association for Research in Vision and 
Ophthalmology (ARVO). 
In this study, Nyxol had statistically significant PD reductions under photopic and mesopic 
conditions that were sustained for 36 hours post-dosing. A statistically significant number 
of patients with Nyxol compared to placebo demonstrated ≥ 1 line of improvement in 
DCNVA, with a trend for 2- and 3-line improvements at all time points. There was no 
statistical difference in conjunctival hyperemia 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 symptoms of patients with DLD, which is relevant to the LYNX-1 trial. 
2.3 
Design Justification 
DLD, which includes night vision disturbances, affects patients under mesopic or scotopic 
conditions. DLD involves photic phenomena, such as glare, halos, and starbursts, which 
can be a result of a combination of ocular aberrations and light scatter, as well as the effect 
of multiple images simultaneously superimposed onto the retina, as in the case of 
multifocal intraocular lenses (IOLs; Rosen 2005). 
These visual disturbances can be debilitating to a variety of everyday activities, especially 
driving. The light emitted by traffic lights and other cars scatters and obscures most of the 
visual field, making driving in dim light conditions hazardous. Glare, in particular, can be 
dangerous while driving. 
Several surveys suggest that DLD are a clinically relevant concern. 
• More than half (53%) of vision-corrected Americans admit they feel uncomfortable 
when driving in the dark (Fejer and Girgis, 1992). 
• Up to 3% of patients with refractive procedures are unable to function visually at 
night (Pop and Payette, 2004; Fan-Paul, 2002; Holladay, 1999; Monestam and 
Lundqvist, 2006). 
• As many as 20% of people with multifocal IOLs complain of night vision 
disturbances (van Alphen, 1976). 
• In one study of 297 drivers given vision tests that correlate with accidents, 45% of 
the drivers who reported difficulty driving at night were unable to perform any of 
the tests with glare (Puell, 2004). 
 


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The effects of DLD can be mitigated by miosis, where the smaller pupil blocks the 
unfocused, peripheral aberrant rays of light, selectively allowing passage of the more 
centrally focused rays (Martinez, 1998). A miotic change in the pupil size can be achieved 
by modulating 1 of 2 or both opposing sets of muscles – the iris sphincter muscles 
controlled by the cholinergic nervous system and the iris dilator muscles controlled by the 
adrenergic nervous system (Yoshitomi, 1985; Steinhauer, 2004). An alpha-1 antagonist 
approach will act on the iris dilator muscle and produce a miotic effect (Yu, 2002). It is 
proposed that a moderate pharmacologic reduction of pupil size (20–25%) by the 
application of an alpha-1 antagonist has the potential to mitigate DLD in many affected 
individuals. 
The biggest challenge for the treatment of DLD is the lack of safe, tolerable, convenient, 
and effective treatments. Despite a large number of addressable patients with moderate-to-
severe DLD, there is no FDA-approved treatment on the market for DLD. Some 
commonly used tools such as tinted glasses are not effective, and in fact, may worsen 
patients’ vision at night. Off-label use of approved miotic agents, such as regular-strength 
pilocarpine, are unsuitable for the treatment of DLD because they reduce pupil size to a 
degree that may impede safe night vision and may cause loss of accommodation 
(Isopto prescribing information). 
Phentolamine is a non-selective alpha-1 and alpha-2 adrenergic antagonist acting on 
adrenergic receptors and is known to inhibit contraction of the iris dilator muscle, resulting 
in a smaller pupil size. 
Previous Phase 2 clinical trials showed that 1.0% Phentolamine Mesylate Ophthalmic 
Solution (0.75% Phentolamine Ophthalmic Solution) significantly decreases PD and 
improves mLCVA in DLD subjects after single- and multi-dose (14 days) treatment. 
Phentolamine Ophthalmic Solution was generally well-tolerated in the eye (the most 
common complaint was mild-to-moderate ocular hyperemia and eye irritation) and there 
were no clinically meaningful systemic effects as measured by HR and BP. 
2.4 
Route of Administration, Dosage Regimen, and Treatment Period 
As the intended route of administration for Nyxol is topical ocular, 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 (Hogan, 1997). 
Treatment (Nyxol or placebo) will be administered OU. Subjects will have one drop of 
treatment administered in each eye. 
Note that 0.75% Phentolamine Ophthalmic Solution, which expresses the phentolamine 
mesylate concentration in 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. 


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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. 
2.6 
Study Population 
A sample size of approximately 160 DLD subjects ≥ 18 years of age will be randomized in 
a 1:1 ratio to 1 of 2 masked treatment arms (Nyxol or placebo), with the expectation that 
approximately 136 subjects will be evaluable for efficacy. Randomization will be stratified 
by iris color (light/dark irides; Appendix 1). The subjects will be recruited from 
approximately 15 investigational sites. 


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3 
OBJECTIVES AND PURPOSE 
The LYNX-1 study is a randomized, placebo-controlled, double-masked, multiple-dose 
study of the safety and efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) in 
subjects with DLD. 
The objectives of this study are: 
• To evaluate the efficacy of Nyxol to improve mLCVA in subjects with DLD 
• To evaluate the efficacy of Nyxol to improve visual performance  
• To evaluate the safety of Nyxol 
• To evaluate any additional benefits of Nyxol in treating DLD subjects 
 
The Sponsor intends to use this first Phase 3 registration study to evaluate Nyxol for the 
indication of 
 
 


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4 
STUDY DESIGN 
4.1 
Primary and Secondary Endpoints 
Efficacy: 
The primary efficacy endpoint is the percent of subjects with ≥ 15 ETDRS letters 
(≥ 3 lines) of improvement in the study eye compared to baseline in monocular mLCVA at 
Day 8. 
The eye with the greatest mLCVA improvement 
 
 
 
 
 
 The study eye and 
non-study eye, as well as binocular, will both be evaluated at all appropriate assessments. 
All treatments will be administered OU. 
Secondary efficacy endpoints (for the study eye, the non-study eye, the best of either eye, 
and binocular) will include: 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Exploratory efficacy endpoints (for the study eye, the non-study eye, the best of either eye, 
and binocular) will be collected and analyzed for the sites that have an OPD Scanner and 
will include but are not limited to: 
• 
 
 
 
In addition, data captured by OPD Scan III per subject per applicable visit will be 
generated. These data include graphs, heatmaps, vision simulations, numerical data, point 
spread function, Zernike graphs, VA-ETDRS simulations, angle kappa, etc. Exploratory 
analyses may be completed using these data. 
Measurements (Appendix 2): 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety: 
The primary safety measures are conjunctival hyperemia and AEs. 
Analysis for conjunctival hyperemia includes change from baseline (Day 1) in the 
conjunctival hyperemia grading scale (CCLRU images) at each visit (Day 8 and Day 15), 
for study eye and non-study eye. Any increase of 2 units in conjunctival hyperemia 
between consecutive visits is considered an AE. 


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Other safety measures include IOP, biomicroscopy, ophthalmoscopy, dry eye 
measurements, and systemic safety as measured by HR and BP. Urine pregnancy tests for 
females of childbearing potential will be conducted. 
Subject questionnaire values will be summarized by treatment group and visits (Day 1, 
Day 8, and Day 15). 
Measurements: 
• Conjunctival hyperemia will be assessed visually with a grading scale (0-3) using 
images from the CCLRU.  
• IOP will be measured with the Tono-Pen. 
• BP and HR will be measured manually or with available digital devices. 
• Subject questionnaire will be a brief symptom survey (Appendix 3). 
 
4.2 
Description and Schedule of Visits and Procedures 
A sample size of approximately 160 DLD subjects ≥ 18 years of age will be randomized in 
a 1:1 ratio to 1 of 2 treatment arms (Nyxol or placebo), with the expectation that 
approximately 136 subjects will be evaluable for efficacy. Randomization will be stratified 
by iris color (light/dark irides). Study procedures are shown in detail in Table 2. 
Table 2 
Screening, Baseline, Treatment, and Follow-Up Visits and 
Procedures 
  
Day 
Visit 
Informed consent 
Subject ID number assigned 
Medical/ophthalmic history [b] 
Demographics 
Drug accountability and review 
of subject diary 
Concomitant medications [c] 
Urine pregnancy test [d] 
Heart rate/Blood pressure [e] 
 
 
 
 
 
 


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Screening/Baseline 
[a] 
Treatment 
Treatment 
TC Follow-
Up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse events 
X 
X 
X 
X 
Randomization number assigned 
X 
 
 
 
Medications dispensed/Re-
dispensed 
X 
X 
 
 
Diary dispensed/Re-dispensed 
X 
X 
 
 
Abbreviations: BAT, Brightness Acuity Tester; BCDVA, best-corrected distance visual acuity; CCLRU, 
Cornea and Contact Lens Research Unit; DCNVA, distance-corrected near visual acuity; mLCVA, mesopic 
low-contrast visual acuity 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
4.3 
Measures Taken to Minimize/Avoid Bias 
This is a placebo-controlled, double-masked, 1:1 randomized, 2-arm, Phase 3 study. 
4.4 
Study Medications 
 


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Study Medication Identification 
Established name 
Phentolamine mesylate – parent phentolamine 
CAS registry number 
65-28-1 – parent 50-60-2 
Chemical class 
An alpha-adrenergic 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 
377.140 – parent 281.352 
Drug name/formulation 
Nyxol / aqueous isotonic solution  
Concentration active 
1% – parent 0.75% 
Manufacturer drug substance 
 
Manufacturer drug product, placebo 
 
Storage requirements 
 
 
 
  
Stored in a secured location (locked) with no access 
for unauthorized personnel at site  
 
Formulation 
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), 
 
Placebo for Nyxol is a clear, colorless, sterile, non-preserved, isotonic, buffered aqueous 
solution containing 
. The pH of the study medications may be 
adjusted with 
 (United States Pharmacopeia [USP]) and/or 
 (USP) to 
 
4.4.1 
Packaging and Labeling 
The investigational products, active and placebo, are packaged in a 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 and 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”. 
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. 
 
 
 medication must not be frozen and must 


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be protected from light. 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 
Study medication will be dispensed by the Investigator or designee at the site on Visit 1 
(Screening/Baseline). 
The subject will instill one drop once daily (QD) at or near bedtime (8PM to 10PM) OU 
for 14 days (+ 1 day). The first dose of study medication will be taken at 8PM to 10PM on 
the evening of the Baseline Visit (Day 1). If the subject wears contact lenses, they must be 
removed prior to dosing and not replaced until the next morning at the earliest. 
4.4.4 
Study Medication Accountability 
 
Receipt and Disposition of Study Medication 
The Investigator or designee (e.g., study coordinator or pharmacist) shall maintain a full 
accountability record for the study medication and will be responsible for recording the 
receipt, dispensing, and return of all supplies of the study drug using the inventories 
supplied by the Sponsor. Each subject will receive sufficient study medication for the 
duration of the trial. A study medication box will be dispensed at Visit 1 
(Screening/Baseline; Day 1). As described in Section 7.2.1, the subject will empty any 
remaining contents from the unit-dose dropper bottle following each instillation. The 
subject should then empty the remaining contents and return the opened bottle to its foil 
pouch and place it in the medication box for return to the study site. All opened bottles as 
well as any unopened study medication will be returned at Visit 2 (Treatment; Day 8) and 
Visit 3 (Treatment; Day 15). 
At Visit 2, the box will be re-dispensed to the subject following study drug accountability 
and removal of opened study medication materials. Study medication accountability shall 
again be conducted at Visit 3, but the box will not be re-dispensed. The Investigator or 
designee must account for all received and returned study medication. The monitor will 
review dispensing and study medication accountability records during site visits and at the 
completion of the study and note any discrepancies. 
 
Return of Study Medication 
When the study is completed or is terminated by Ocuphire, all study materials, including 
used and unused study medication bottles, must 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. 


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4.5 
Expected Duration of Subject Participation 
The total length of subject participation is approximately 3 weeks, with 3 clinic visits and 
one telephone call follow-up summarized below: 
• Visit 1 (Screening/Baseline)/Day 1 
• Visit 2 (Treatment)/Day 8 (±1 day) 
• Visit 3 (Treatment)/Day 15 (±1 day) 
• Follow-up telephone call 1 to 3 days after Visit 3 
 
The execution of the entire study (first subject screened through last randomized subject 
completed) is expected to be approximately 9 to 12 months. 
4.6 
Randomization and Procedure for Breaking the Code 
A randomization code for allocating subjects to treatment will be prepared by a masked 
biostatistician not connected with the study. Subjects will be stratified by iris color 
(light/dark irides). 
At the initiation of study-related procedures, every subject who is screened is assigned a 
subject identification number in numerical order within site. Once a subject is qualified 
for the study, the subject is assigned a randomization number in the order provided by the 
biostatistician. 
The study medications will be masked to Investigators, study 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. 
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 a targeted subset of 
data collected in the electronic Case Report Form (eCRF) against any applicable source 
documentation during remote review or scheduled monitoring visits. The specific data 
selected for source data verification will be detailed in Clinical Monitoring Plan. 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. 


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4.8 
Completed Subject 
A completed subject is defined as one who completes all planned procedures through the 
end of Visit 3. 
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. 
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 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. 
• Other: If there is any other reason for subject discontinuation 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. 
 


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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 Day 8 Visit 2 mLCVA and other 
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 the study, all eCRFs 
have been completed, and all eCRF data entered into the database. Final database lock will 
occur after the last randomized subject completes last visit, all data have been entered, and 
all queries have been 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. 


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5 
SUBJECT INCLUSION AND EXCLUSION CRITERIA 
5.1 
Subject Inclusion Criteria 
1. Males or females ≥ 18 years of age 
2. Subject-reported DLD (likely subjects with a history of multifocal IOLs, post–laser-
assisted in situ keratomileusis [LASIK], corneal scars, and keratoconus) 
3. Ability to comply with all protocol-mandated procedures independently and to attend 
all scheduled office visits 
4. Otherwise healthy and well-controlled subjects 
5. Able and willing to give written consent to participate in this study 
6. Able to self-administer study medication 
7. 
 
 
 
8. ≤ 30 Early Treatment Diabetic Retinopathy Study (ETDRS) letters (20/63 Snellen or 
worse) in mLCVA score in at least one eye using the 
 
 
9. 
 
 
 
 
 
5.2 
Subject Exclusion Criteria 
Excluded from the study will be individuals with the following characteristics: 
Ophthalmic (in either eye): 
1. Prior unresolved dry eye diagnosis, taking prescription drops for dry eye, or taking 
artificial tear drops routinely for dry eye  
2. Prior history of fluctuating vision 
3. Clinically significant ocular disease as deemed by the Investigator (e.g., untreated 
cataract, glaucoma, corneal edema, uveitis, severe keratoconjunctivitis sicca, retina 
degeneration, loss of visual field due to glaucoma or stroke, branch retinal vein 
occlusion, retina flare) that might interfere with the study  
4. Known hypersensitivity to any topical alpha-adrenoceptor antagonists 
5. Known allergy or contraindication to any component of the vehicle formulation 
6. History of cauterization of the punctum or punctal plug (silicone or collagen) insertion 
or removal 
7. Ocular trauma, ocular surgery (e.g., IOLs), or laser procedure (e.g., LASIK, 
photorefractive keratectomy [PRK]) within 6 months prior to screening 


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8. Use of any topical prescription or over-the-counter (OTC) ophthalmic medications of 
any kind (including artificial tear drops) 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.) 
9. Recent or current evidence of ocular infection or inflammation in either eye (such as 
current evidence of clinically significant blepharitis, conjunctivitis, or a history of 
herpes simplex or herpes zoster keratitis at screening in either eye). Subjects must be 
symptom free for at least 7 days. 
10. History of diabetic retinopathy, diabetic macular edema, or dry or wet macular 
degeneration 
11. 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, etc.) 
12. Unwilling or unable to discontinue use of contact lenses on their scheduled visits at 
least 1 hour for soft contact lenses and at least 8 hours for hard gas-permeable contact 
lenses before ophthalmic assessments at screening, and at least 8 hours (for both types 
of lenses) before Day 8 and Day 15 
13. People with undiagnosed dry eye, at the determination of the Investigator. Dry eye 
evaluation should be based on one of the following dry eye tests: 
a. TearLabs osmolarity test (> 308 mOsm/L in either eye or an inter-eye 
difference ≥ 10 mOsm/L) 
b. Corneal fluorescein staining (≥ grade 2 in the inferior or ≥ grade 1 in the central 
zone using the National Eye Institute scale) 
c. Tear break-up time of < 5 seconds 
 
Systemic: 
1. 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 [CHF]); severe diabetes as described below) 
a. Predisposition to severe hypoglycemia (2 or more serious hypoglycemic 
episodes requiring assistance within the past year) 
b. Any hospitalization or emergency room visit due to poor diabetic control 
within the past 6 months 
c. Currently untreated diabetes mellitus or previously untreated subjects who 
initiated oral anti-diabetic medication or insulin within 3 months prior to Day 1 
d. Any sign of diabetic retinopathy in either eye 
e. Uncontrolled diabetes mellitus defined by Hemoglobin A1c > 12% taken 
within the last 3 months 


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2. Clinically significant systemic disease (e.g., severe diabetes as previously defined, 
myasthenia gravis, cancer, hepatic, renal, endocrine, or cardiovascular disorders) that 
might interfere with the study 
3. 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 (Appendix 4) 
4. Participation in any investigational study within 30 days prior to screening and during 
the study 
5. 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), Visit 2 (Treatment), and Visit 3 (Treatment) 
examinations and must intend to not become pregnant during the study 
6. Resting HR outside the specified range (50–110 beats per minute) at Visit 1 
(Screening/Baseline). HR may be repeated only once if outside the specified range 
following at least a 5-minute rest period in the sitting position 
7. Hypertension with resting diastolic BP > 105 mmHg or systolic BP > 160 mmHg at 
Visit 1 (Screening/Baseline). BP may be repeated only once if outside the specified 
range following at least a 5-minute rest period in the sitting position 
 


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6 
TREATMENT OF SUBJECTS 
Approximately 160 DLD subjects ≥18 years of age will be randomized in a 1:1 ratio to 1 
of 2 treatment arms (Nyxol or placebo), with the expectation that approximately 
136 subjects will be evaluable for efficacy. Randomization will be stratified 
 
. There will be no treatment administered on Visit 4 (follow-up phone 
call). 
6.1 
Treatment Adherence 
All subjects must be instructed on the importance of following the once-daily dosing 
regimen and maintaining their dosing diaries. Beginning the day of Visit 1 
(Screening/Baseline; Day 1) subjects will instill one drop of study medication in the 
evening (at 8PM to 10PM) at or near bedtime OU. Subjects will repeat this instillation, 
using one bottle per day, 13 or 14 more times for a total of up to 15 doses over the course 
of the study. Subjects are to bring all opened bottles as well as any unopened study 
medication into the study site at Visit 2 and Visit 3. Treatment adherence will be measured 
by counting the dropper bottles at the start of the study and those remaining at each study 
visit. Subjects will also bring their diary to Visit 2 and Visit 3 and dosing will be reviewed 
by study staff. 
6.2 
Concomitant Medications 
As noted in the exclusion criteria (Section 5.1), 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. Many drugs, both prescription and OTC, contain active ingredients that can 
affect PD. This would include many eye drops, such as Visine®, that would be used to 
reduce redness, most cough or cold preparations, antihistamines and bronchodilators, most 
nose drops, most BP medications, many drugs used for migraines, and many other 
products. 
Additionally, initiation of treatment with or any changes to the current dosage, drug, or 
regimen of any systemic adrenergic or cholinergic drugs (Appendix 4) 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 shall be documented on the appropriate eCRF. Investigators are 
encouraged to contact the Medical Monitor for any questions regarding allowed 
medications. 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 taken by the subject 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 


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administration, duration of treatment, and indication will be recorded for each medication. 
For combination products (e.g., Contac®, Cosopt®), the brand name is required. For 
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. 


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7 
ASSESSMENT OF EFFICACY 
7.1 
Specification of the Efficacy Parameters 
The primary efficacy endpoint is the percent of subjects with ≥ 15 ETDRS letters 
(≥ 3 lines) of improvement in the study eye compared to baseline in monocular mLCVA at 
Day 8. 
 
 
 
 
 
The study eye and non-study eye, as well as binocular, will be evaluated at 
all appropriate assessments. Secondary efficacy endpoints can be found in Section 4.1. 
All of the efficacy endpoints will be analyzed overall, and by light/dark irides at all 
timepoints. The mLCVA endpoints will be analyzed by baseline PD 
 
) and 
 subpoint. 
7.2 
Assessing, Recording, and Analyzing Efficacy Parameters 
VA, PD, and wavefront error (total and higher order) will be measured at Visit 1 
(Screening/Baseline), Visit 2 (Treatment), and Visit 3 (Treatment) as described in 
Section 4.1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 to the set of PD, BCDVA, DCNVA, and wavefront error measurements). 
Subject 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 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. 
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 should occur the same day as the Baseline Visit, 
where the dose of study treatment is given. 


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Once a subject arrives at the study center, a member of the study center 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 identification 
number is assigned. 
If the subject wears soft contact lenses, they must be removed for at least 1 hour prior to 
ophthalmic assessments. If the subject wears hard gas-permeable contact lenses, they must 
be removed for at least 8 hours prior to testing. 
The start of screening includes an explanation of the study, a medical and ophthalmic 
history, demographics, and a review of prior/concomitant medications. During this time, 
eligibility for 
 must be determined first. Then, ophthalmic and 
non-ophthalmic criteria (
) shall be determined and only 
eligible subjects will continue. This shall be followed by a urine pregnancy test for females 
of childbearing potential 
 and HR/BP (
 
). 
Prior to further screening, the subject must be asked to complete the subject questionnaire. 
Subjects should then have their refraction confirmed using the High contrast distance chart 
as detailed in the Study Procedures Manual. Following a 
 
, subjects shall be assessed in each eye for PD 
and for mLCVA before and during illumination of the 
 
 (
 
 
 
 
 
 OD shall be the study 
eye. Investigators should frequently remind subjects to blink between visual acuity 
assessments. 
The study eye of the subject must meet
 in order for the 
subject to not screen fail. 
Before further screening criteria, the following assessments shall be performed to establish 
baseline values: 
• mHCVA, pLCVA, and mesopic high-contrast DCNVA 
• 
 
 corneal topography 
measurements under 
, and other data captured 
• Conjunctival hyperemia, assessed visually with a grading scale (0–3) using images 
from the CCLRU (Investigators are cautioned to appropriately note all observations 
of conjunctival hyperemia on the biomicroscopy eCRF at screening) 
• Slit lamp biomicroscopy 


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The subject must also meet dry eye Exclusion Criterion #13 to be included in the study at 
the determination of the investigator. Specifically, subjects with undiagnosed dry eye may 
undergo a dry eye test (one of the below): 
a. TearLabs osmolarity test (> 308 mOsm/L in either eye or an inter-eye 
difference ≥ 10 mOsm/L) 
b. Corneal fluorescein staining (≥ grade 2 in the inferior or ≥ grade 1 in the central 
zone using the National Eye Institute scale) 
c. Tear break-up time of < 5 seconds 
 
Final screening must include IOP measurement (using a Tono-Pen) and ophthalmoscopy. 
With the subject passing all eligibility criteria, the Screening Visit becomes the Baseline 
Visit. The subject will then be assigned a randomization number and randomized into the 
study. 
Through the screening process at Visit 1, all assessments will have been completed as 
defined in Table 2. A summary of the screening process is outlined in Appendix 5.  
If the subject meets all eligibility criteria, they will be randomized to a treatment group. If 
a subject fails the screen, they can return for a rescreening (signing a new informed 
consent and assigning a new subject number) at the investigator’s discretion. 
Site personnel will then demonstrate the proper instillation technique to the subject. The 
subject at home should be in a seated position and should tilt his or her head backward for 
administration of the study medication. The bottle of study medication should be held at an 
almost vertical position above the eye while the lower eyelid is pulled down gently, and 1 
drop is placed into the conjunctival cul-de-sac. The tip of the bottle should not touch the 
eye. After a drop is instilled in each eye, the subject should keep the eyes gently closed for 
approximately 30 seconds. After successful instillation of the drop in each eye, the subject 
should carefully empty any remaining contents as directed. 
The subject is given their medication dropper bottles in a kit and instructions regarding 
when to administer the eye drop (8PM to 10PM QD), and when to return to the clinic. All 
treatments will be administered OU. 
 
 
 
The first dose of study medication will be taken at 8PM to 10PM on the evening of the 
Baseline Visit (Day 1). If the subject wears contact lenses, they must be removed prior to 
dosing and not replaced until the next morning at the earliest. 
Each subsequent evening of dosing, the subject will administer 1 drop to each eye from a 
single new unit-dose bottle and close the eyes gently for 30 seconds, then empty the 


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remaining contents and store the opened bottle in the foil pouch and place it in the 
medication box for return to the study site at Visit 2 (Day 8). Each subsequent evening of 
dosing (approximately 24 hours between doses), the subject will follow the same 
procedures. 
The subject should be instructed to contact the Investigator should AEs of concern occur 
(e.g., shortness of breath, fainting, etc.). If the event is life-threatening, then the subject 
should go to the emergency room. 
7.2.2 
Visit 2 (Treatment)/Day 8 (± 1 day) 
If the subject wears contact lenses,
 prior 
to ophthalmic assessments. At Visit 2, the medication box, complete with opened bottles 
and any unopened study medication, and diary will be returned to the study site where the 
site will account for opened and unopened study medication and the study medication box 
will be re-dispensed with the unopened medication. 
The subjects will be asked if they had any problems with their eyes since the last visit, and 
if there have been any changes in their medical condition or concomitant medications 
since their last visit. Any adverse changes in the condition of the subject are recorded as 
an AE. 
In addition, at Visit 2 subjects shall have the following assessments, as defined in Table 2: 
•
Females of childbearing potential must take a urine pregnancy test.
•
Subject must have HR and BP assessments.
•
Subject shall be assessed using the subject questionnaire.
•
Pupil diameter
•
Subject shall be assessed for mLCVA, mHCVA, pLCVA and DCNVA (mesopic,
high-contrast conditions) as mentioned in Section 4.1.
•
OPD Scan measurements (for those sites with OPD Scan III), wavefront
aberrometry (total and higher-order RMS) and corneal topography measurements
under mesopic conditions, and other data captured
•
Eye redness (conjunctival hyperemia) will be assessed visually with a grading scale
(0-3) using images from the CCLRU.
•
Subject will have an IOP assessment.
7.2.3 
Visit 3 (Treatment)/Day 15 (± 1 day) 
During the second week of treatment, subjects will continue to administer 1 drop of study 
medication OU every night using a new bottle for each dose, then emptying the remaining 
contents of that bottle, return the opened bottle to its foil pouch and place it in the 
medication box for return to the study site at their next visit.  


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If the subject wears contact lenses, they must be removed for a minimum of 8 hours prior 
to ophthalmic assessments. At Visit 3, the subject will bring their used dropper bottles with 
any unused medications and diary with them for purposes of drug accountability. The last 
day of study treatment is the day before Visit 3; no further study medication will be 
dispensed at Visit 3. 
The subjects will be asked if they had any problems with their eyes from the last visit, and 
if there have been any changes in their medical condition or concomitant medications 
since their last visit. Any adverse changes in the condition of the subject are recorded as 
an AE. 
In addition, at Visit 3 subjects will have the following assessments, as defined in Table 2: 
•
Females of childbearing potential must take a urine pregnancy test.
•
Subject must have HR and BP assessments.
•
Subject shall be assessed using the subject questionnaire.
•
Pupil diameter
•
Subject shall be assessed for mLCVA, mHCVA, pLCVA, and DCNVA (mesopic,
high-contrast conditions) as mentioned in Section 4.1.
•
OPD Scan measurements (for those sites with OPD Scan III), wavefront
aberrometry (total and higher-order RMS) and corneal topography measurements
under mesopic conditions, and other data captured
•
Eye redness (conjunctival hyperemia) shall be assessed visually with a grading
scale (0-3) using images from the CCLRU.
•
Subject shall be assessed using biomicroscopy.
•
Subject must have an IOP assessment.
•
Subject shall be assessed using ophthalmoscopy.
7.2.4 
Follow-Up Phone Call Visit 4 
Up to 3 days after Visit 3, subjects will be called and asked if they had any problems with 
their eyes from the last visit, including whether their eyes are red, and if there have been 
any changes in their medical condition or concomitant medications since their last visit. 
Any changes in the condition of the subject are recorded as an AE. 
If an AE/adverse reaction is unresolved at the time of the last visit or if a subject reports a 
worsening of symptoms, every effort should be made to follow 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. 


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7.2.5 
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. 
7.2.6 
Visit Variation 
Visit 2 on Day 8 and Visit 3 on Day 15 may each be 1 day late. If the visit is late, the 
subject should be advised to take an additional dose from 1 of the 2 spare dropper bottles 
provided in the study medication box the night before the visit. The subject should then 
empty the remaining contents and return the opened bottle to its foil pouch and place it in 
the medication box for return to the study site at their next visit. The follow-up telephone 
call may occur up to 3 days after Visit 3. 


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8 
ASSESSMENT OF SAFETY 
8.1 
Specification of Safety Parameters 
The assessment of safety and tolerability is the secondary objective of this study. The 
assessment of safety will be evaluated by: 
• Conjunctival hyperemia measured with CCLRU images using a 4-point scale (0-3) 
(Appendix 6). 
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 
• Biomicroscopy of the anterior segment includes evaluation of cornea, conjunctiva, 
and anterior chamber. Fluorescein staining will be used. 
• IOP is measured with the Tono-Pen. 
• Ophthalmoscopy direct or indirect fundus exam without dilation includes optic 
nerve, macula, vessels, and periphery (note: Use of 90 D lens (indirect) is allowed). 
• HR and BP are measured as per the site’s normal equipment and procedures. 
• AEs 
• Brief subject questionnaire (Appendix 3) 
 
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 treatment-emergent adverse 
events/adverse reactions (TEAEs) will be summarized (Section 9.3.5). 
All AEs/adverse reactions occurring during the study (i.e., once the subject has signed the 
informed consent/assent) must be documented, regardless of the assumption of causal 
relationship, on the respective eCRF. All TEAEs/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 


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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 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 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 AE 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 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. 
SAE 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 


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• 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; or, if an IB is not required or available, is 
not consistent with the risk information described in the general investigational plan or 
elsewhere in the current application, as amended. “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: 
• 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”. 


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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. 
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. 
• 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 
 


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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 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 visits 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/assent and 
until the Follow-up Visit. Any SAEs “suspected” to be related to the study medication and 
discovered by the Investigator at any time after the study should be reported. 
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 


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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 form. 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 
 The 
Investigator must assess the SAE relationship and complete the SAE form. 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 
 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, 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 
Visits, 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. 


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9 
STATISTICS 
9.1 
Sample Size 
A sample size of approximately 136 subjects (68 per treatment group) that are evaluable 
for efficacy (i.e. who have a Day 8 mLCVA measurement) is needed for the study.  
 
 
 
 
 
 
 
All subjects will be randomized into the study in a 1:1 ratio to 1 of the 2 treatment arms 
(Nyxol or placebo), 
 
It is assumed that there will be approximately 15% drop-out. To account for this drop-out, 
a total of approximately 160 subjects will be randomized into the study. 
9.2 
Analysis Populations 
Modified Intention-to-Treat: The mITT will include all randomized subjects who 
received at least 1 dose of study medication. The mITT will be used for the primary 
endpoint analysis and to analyze efficacy endpoints. 
Per Protocol Population: The PP population includes all subjects in the mITT who 
received at least 5 doses of study medication during the first 7 days of dosing including the 
day prior to Day 8, have a Baseline and a Day 8 mLCVA measurement, and have no major 
protocol deviations. The PP population will be used 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 1 dose of study medication. 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). 


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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 group using the mITT, PP population, 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) and will be summarized by treatment group using the 
SP. 
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 and will 
both be summarized by treatment group using the SP. 
Medical history and prior and concomitant medications will also be provided in by-subject 
listings. 
9.3.5 
Analysis of Efficacy 
Efficacy will be assessed using the mITT and PP population 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. 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. 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 (Day 1). 
 
 
 
 
 


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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 the respective baseline value included as the covariate. 
Each ANCOVA will be performed using the mITT and PP population, 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 both treatment groups, along with the placebo-corrected LSM, its 95% CI and 
associated p-value. 
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 group meeting the criteria, the OR with 95% CI 
and p-value will be provided. For these endpoints, the mITT and PP population 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
 using 
the same model and sensitivity analysis indicated above, as appropriate. Other subgroups, 
such as age, sex, and race, may be analyzed as well. 
9.3.6 
Analysis of Safety 
Safety will be assessed using the SP with subjects included in the treatment group 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 value is defined as the screening value. HR and BP values and 
change from baseline in the values will be summarized by treatment group and timepoint 
(Day 1, Day 8, and Day 15). 
Observed values and change from baseline (Day 1) in conjunctival hyperemia at each visit 
(Day 8 and Day 15), will be summarized for the study eye, the non-study eye, and the best 
of either eye. Treatments will be compared using the same ANCOVA model proposed for 
the continuous secondary efficacy endpoints. 
For IOP, baseline is defined as the baseline value of Visit 1 (Day 1). Observed values and 
change from baseline in IOP at Day 8 and Day 15 will be summarized for the study eye, 
the non-study eye, and the best of either eye. 


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Subject questionnaire values will be summarized by treatment group and timepoint (Day 1, 
Day 8, and Day 15). 
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. 


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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. 


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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 guidelines. The Investigator will allow 
Ocuphire, the Study Monitor, and the Medical Monitor to inspect all eCRFs, subject 
records (source documents), signed consent/assent forms, records of study medication 
receipt, storage, preparation, and disposition, and regulatory files related to this study. 


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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/assent must be approved by the IRB prior to initiation of the study. Written IRB 
approval must adequately identify the protocol and informed consent/assent. In addition to 
approving the protocol, the IRB must also approve the Subject Information and 
Consent/Assent/Parental 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 and Assent Requirements 
Written informed consent will be obtained from each adult subject. A copy of the signed 
and dated consent/assent document will be given to each subject or parent guardian. The 


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original signed and dated informed consent/assent 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/assent. The subject 
must give written consent/assent 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/assent to 
participate at any time, without penalty or loss of benefits to which the subject is otherwise 
entitled. Subjects who refuse to give, or withdraw, written informed consent/assent may 
not be included or continued in this study, but this will not impact 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 and/or legal 
guardian will be required to sign and date the informed consent form. 
A copy of the signed and dated consent/assent document will be given to each subject. The 
original signed and dated informed consent/assent document must be maintained in the 
study files at the Investigator’s site. Signed informed consent/assent must be obtained prior 
to the conductance of any study procedures. 


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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 a targeted subset of data collected in the eCRF against source documentation during 
remote review or scheduled monitoring visits. The specific data selected for source data 
verification will be detailed in Clinical Monitoring Plan. 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. 
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). 


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Protocol amendments will be submitted to the appropriate authority(ies) as required by the 
applicable regulatory requirement(s). 
 


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14 
BIBLIOGRAPHY 
1. Fan-Paul NI, Li J, Miller JS, Florakis GJ. Night vision disturbances after corneal refractive 
surgery. Surv Ophthalmol. 2002;47(6):533-46. 
2. Fejer TP, Girgis R. Night myopia: implications for the young driver. Can J Ophthalmol. 
1992;27(4):172-6. 
3. 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. 
4. Holladay JT, Dudeja DR, Chang J. Functional vision and corneal changes after laser in situ 
keratomileusis determined by contrast sensitivity, glare testing, and corneal topography. J 
Cataract Refract Surg. 1999;25(5):663-9. 
5. Isopto® Carpine (pilocarpine hydrochloride ophthalmic solution) 1%, 2% and 4% 
[prescribing information]. Initial U.S. Approval: 1974. 2010. 
6. Martinez CE, Applegate RA, Klyce SD, McDonald MB, Medina JP, Howland HC. Effect of 
pupillary dilation on corneal optical aberrations after photorefractive keratectomy. Arch 
Ophthalmol. 1998;116(8):1053-62. 
7. Monestam E, Lundqvist B. Long-time results and associations between subjective visual 
difficulties with car driving and objective visual function 5 years after cataract surgery. J 
Cataract Refract Surg. 2006;32(1):50-5. 
8. Pop M, Payette Y. Risk factors for night vision complaints after LASIK for myopia. 
Ophthalmology. 2004;111(1):3-10. 
9. Puell MC, Palomo C, Sanchez-Ramos C, Villena C. Mesopic contrast sensitivity in the 
presence or absence of glare in a large driver population. Graefes Arch Clin Exp Ophthalmol. 
2004;242(9):755-61. 
10. Rosen, Emanuel S. FRCSE Night vision disturbance, Journal of Cataract & Refractive 
Surgery. 2005;31(2):247-9. 
11. Steinhauer SR, Siegle GJ, Condray R, Pless M. Sympathetic and parasympathetic innervation 
of pupillary dilation during sustained processing. Int J Psychophysiol. 2004;52(1):77-86. 
12. van Alphen GW. The adrenergic receptors of the intraocular muscles of the human eye. 
Invest Ophthalmol. 1976;15(6):502-5. 
13. Yoshitomi T, Ito Y, Inomata H. Adrenergic excitatory and cholinergic inhibitory innervations 
in the human iris dilator. Exp Eye Res. 1985;40(3):453-9. 
14. Yu Y, Koss MC. alpha(1A)-adrenoceptors mediate sympathetically evoked pupillary dilation 
in rats. J Pharmacol Exp Ther. 2002;300(2):521-5. 


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APPENDIX 1: IRIS COLOR CHART 
Study enrollment includes both light and dark-colored eyes. Examples of 
 
) and 
 for the purposes of this study are detailed in the 
chart below. 
 
 
 


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APPENDIX 2: VISUAL ACUITY CHARTS 
BCDVA (distance) will be measured with a Standard 
 
 
 
 
Distance High-Contrast Chart 
 
 
 
 
 
 
 
 
 
 
 
 


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Distance Low-Contrast Chart 
 
DCNVA will be measured under 
 
 
 (letters recorded, later converted 
to logMAR and number of lines). 
 
Near High-Contrast Chart 
 


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APPENDIX 3: SUBJECT QUESTIONNAIRE 
Please use the pictures below to answer the questions in the following table. 
 
Representative Images of Dim Light Disturbances 
 
1. Circle the number below to describe the severity of your symptoms at night  
 
2. In terms of overall severity, how 
 
 
3. Please circle all of the statements below that apply to your level of avoiding driving at night 
due to dim light vision disturbances? 
 
 
 


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D. I only drive at night when I know the exact routes 
E. Occasionally I miss a social outing because it would require me to drive at night 
F. I have no problems when I drive at night 
 
4. For the following statements, circle the number to indicate to what extent does dim light 
vision disturbance impact your day-to-day life. 
 
5. I am satisfied with my night vision at the moment. 
 
6. My night vision has improved. 
 


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7. My night vision is 100% back to normal. 
8. Please indicate the
 
) 


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APPENDIX 4: 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 5: SCREENING PROCESS FOR INCLUSION/EXCLUSION CRITERIA 
 


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APPENDIX 6: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING IMAGES 
FROM CCLRU 

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) in Subjects with Dim Light Vision Disturbances 
Study Number: 
OPI-NYXDLD-301 (LYNX-1) 
Phase: 
Phase 3 
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: 
2022-03-28 
Status: 
FINAL V1.0 
1


A D S T- 0 7. 0 1
0 1J UL 2 0 1 1
P a g e 1
A D S T- 0 7
St atistic al A n al ysis Pl a n A p pr o v al F or m
S p o ns or:
Oc u p hir e P h ar m a, I nc .
Pr ot oc ol:
O PI -N Y X DL D -3 0 1
Pr ot oc ol Titl e:
R a n d o miz e d, Pl ac e b o -C o ntr oll e d, D o u bl e -M ask e d St u d y of t h e S af ety a n d Effic ac y of 
Nyx ol ( 0. 7 5 % P h e nt ol a mi n e O p ht h al mic S ol uti o n) i n S u bj ects wit h Di m Li g ht Visi o n 
Dist ur b a nc es
S A P V ersi o n:
Fi n al V 1 . 0
S A P D at e:
2 8 M A R 2 0 2 2
T h e st atistic al a n al ysis pl a n h as b e e n r e vi e w e d a n d a p pr o v e d.
S p o ns or :
Oc u p hir e P h ar m a, I nc .
_ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ 
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Mi n a S o oc h, M B A
D at e 
C E O, C o- F o u n d er, a n d C h air
_ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ 
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Mitc h ell Bri g ell, P h D
D at e 
H e a d of Cli nic al D ev el o p m e nt a n d Str at e gy
A ut h or :
S u m mit A n alytic al
_ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ 
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
B e n St ei n, P h D
D at e
Sr. Bi ost atistici a n
D o c u Si g n E n v el o p e I D: 7 A F A F 3 C 5- 4 B A A- 4 2 F A- 9 8 6 C- 6 7 C 7 3 7 9 5 0 8 0 3 
---
S U M MI T
A N A
L Y T
I  C A L 
(
D o c u Si g n e d b y : 
I 
 
 
 
 
 
 
  
 
 
(
D o c u Si g n e d b y; 
I 
 
 
 
 
 
 
(
D o c u Si g n e d b y : 
I 
 
 
 
 
 
 
 
2 8- M ar- 2 0 2 2 
2 9- M ar- 2 0 2 2 
2 8- M ar- 2 0 2 2 
2


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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 and Exploratory Endpoints ............................................................................ 8 
5. 
STUDY METHODS .................................................................................................................................... 11 
5.1. 
General Study Design and Plan ....................................................................................................... 11 
5.2. 
Inclusion – Exclusion Criteria and General Study Population ........................................................ 13 
5.3. 
Randomization and Blinding ........................................................................................................... 13 
5.4. 
Analysis Variables .......................................................................................................................... 13 
6. 
SAMPLE SIZE ............................................................................................................................................ 15 
7. 
GENERAL CONSIDERATIONS ............................................................................................................... 16 
7.1. 
Analysis Populations ....................................................................................................................... 16 
7.1.1. 
Modified Intention-to-Treat (mITT) ............................................................................. 16 
7.1.2. 
Per Protocol Population (PP) ......................................................................................... 16 
7.1.3. 
All Randomized Population (ARP) ............................................................................... 16 
7.1.4. 
Safety Population (SP) .................................................................................................. 16 
7.2. 
Covariates and Subgroups ............................................................................................................... 16 
7.2.1. 
Planned Covariates ........................................................................................................ 16 
7.2.2. 
Planned Subgroups ........................................................................................................ 16 
7.3. 
Management of Analysis Data ........................................................................................................ 17 
7.3.1. 
Data Handling ............................................................................................................... 17 
7.3.2. 
Missing Data ................................................................................................................. 17 
7.3.2.1. 
Handling of Missing Date Values ............................................................ 17 
7.3.2.2. 
Missing Baseline Data ............................................................................. 18 
7.3.2.3. 
Imputation Methods ................................................................................. 18 
7.3.3. 
Handling of Early Termination Visit Information ......................................................... 19 
7.3.4. 
Pooling of Investigational Sites ..................................................................................... 19 
7.3.5. 
Coding Conventions for Events and Medications ......................................................... 19 
7.3.6. 
Analysis Software ......................................................................................................... 20 
7.3.7. 
Study Data ..................................................................................................................... 20 
7.4. 
Planned Study Analyses .................................................................................................................. 21 
7.4.1. 
Statistical Summaries: Descriptive and Inferential ....................................................... 21 
7.4.2. 
Interim Analyses and Data Monitoring ......................................................................... 21 
7.4.3. 
Final Analysis and Publication of Study Results .......................................................... 21 
7.5. 
Multiple Testing Procedures ........................................................................................................... 21 
7.6. 
Baseline Values ............................................................................................................................... 22 
8. 
SUMMARY OF STUDY DATA ................................................................................................................ 23 
8.1. 
Subject Disposition ......................................................................................................................... 23 
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8.2. 
Protocol Deviations ......................................................................................................................... 23 
8.3. 
Demographics and Baseline Characteristics ................................................................................... 23 
8.4. 
Medical History .............................................................................................................................. 24 
8.5. 
Prior and Concurrent Medications .................................................................................................. 24 
8.6. 
Treatment Exposure and Compliance ............................................................................................. 25 
9. 
EFFICACY ANALYSES ............................................................................................................................ 26 
9.1. 
Clinical Efficacy ............................................................................................................................. 26 
9.1.1. 
Primary Efficacy Analysis ............................................................................................ 26 
9.1.2. 
Secondary and Exploratory Efficacy Analysis .............................................................. 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. 
Vital Signs ....................................................................................................................................... 30 
10.5. 
IOP .................................................................................................................................................. 31 
10.6. 
Biomicroscopy and Ophthalmoscopy ............................................................................................. 31 
10.7. 
Other Safety Measures .................................................................................................................... 31 
11. REFERENCES ............................................................................................................................................ 31 
12. APPENDICES ............................................................................................................................................. 32 
12.1. 
List of Planned Tables ..................................................................................................................... 32 
12.2. 
List of Planned Listings .................................................................................................................. 36 
12.3. 
Hierarchy of Sequential Testing ...................................................................................................... 38 
 
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LIST OF TABLES 
 
Table 1 
Screening, Baseline, Treatment, and Follow-Up Visits and 
Procedures Schedule ..................................................................................................... 12 
LIST OF FIGURES 
Figure 1 
SDTM, ADaM, and TFL Development and Validation ................................................ 20 
 
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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 
BAT 
Brightness Acuity Tester 
BCDVA 
best-corrected distance visual acuity  
BP 
blood pressure 
CCLRU 
Cornea and Contact Lens Research Unit 
CDISC 
Clinical Data Interchange Standards Consortium 
CI 
confidence interval 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
DCNVA 
distance-corrected near visual acuity 
DD 
drug dictionary 
DLD 
dim light vision disturbances 
EDC 
electronic data capture 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
HR 
heart rate 
IOP 
Intraocular pressure 
ITT 
Intention-To-Treat 
LOCF 
last observation carried forward 
logMAR  
logarithm of the minimum angle of resolution 
LSM 
least squares mean 
MAR 
missing at random 
MCMC 
Markov Chain Monte Carlo 
MedDRA 
Medical Dictionary for Regulatory Activities 
mHCVA 
mesopic high-contrast visual acuity 
mITT 
Modified Intention-To-Treat 
Nyxol 
Phentolamine Mesylate Ophthalmic Solution 1% (Nyxol®) 
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Abbreviation/Term 
Definition 
OD 
right eye 
OPD 
optical path difference 
OR 
odds ratio 
OS 
left eye 
OU 
both eyes 
PD 
pupil diameter 
pLCVA 
photopic low-contrast visual acuity 
PP 
Per Protocol 
PT 
preferred term 
QD 
once daily 
RMS 
root-mean square 
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 
World Health Organization 
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3. 
INTRODUCTION 
3.1. 
Preface 
This document presents a statistical analysis plan (SAP) for Ocuphire Pharma, Inc. Protocol 
OPI-NYXDLD-301 (LYNX-1) (Randomized, Placebo-Controlled, Double-Masked Study of 
the Safety and Efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) in Subjects with 
Dim Light Vision Disturbances). 
Reference materials for this statistical plan include the protocol OPI-NYXDLD-301 
amendment 3 (29JUN2021) and Case Report Forms (CRFs) Version 3.0 (02AUG2021).  
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 LYNX-1 study is a randomized, parallel arm, double-masked, placebo-controlled study 
of the safety and efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) to evaluate 
the efficacy of Nyxol to improve mesopic low-contrast visual acuity (mLCVA) in subjects 
with dim light vision disturbances (DLD). 
The Sponsor intends to use this first Phase 3 registration study to evaluate Nyxol for the 
 
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 
A hierarchical analysis was added to formally test a family of endpoints beyond the primary 
efficacy endpoint. Otherwise, the study protocol includes the subject questionnaire as both an 
efficacy and safety endpoint; in this analysis plan, all subject questionnaire endpoints are 
treated as efficacy variables. Also, two secondary efficacy endpoints were added:
 
 
 Otherwise, the analyses described in this analysis plan are 
consistent with the analyses described in the 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: 
• To evaluate the efficacy of Nyxol to improve mLCVA in subjects with DLD 
• To evaluate the efficacy of Nyxol to improve visual performance  
• To evaluate the safety of Nyxol 
• To evaluate any additional benefits of Nyxol in treating DLD subjects 
4.2. 
Study Endpoints 
4.2.1. 
Primary Endpoints 
The primary efficacy endpoint is the percent of subjects with ≥ 15 Early Treatment Diabetic 
Retinopathy Study (ETDRS) letters (≥ 3 lines) of improvement in the study eye compared to 
baseline in monocular mLCVA at Day 8. 
4.2.2. 
Secondary and Exploratory Endpoints 
Secondary endpoints for efficacy and safety assessments include the following: 
Efficacy: 
Secondary efficacy endpoints (for the study eye, the non-study eye, the best of either eye, and 
binocular)  include: 
• 
 
 
 
 
 
 
 
 
 
 
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• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Exploratory efficacy endpoints (for the study eye, the non-study eye, and the best of either 
eye) are collected and analyzed for the sites that have an optical path difference (OPD) 
Scanner and include but are not limited to: 
• 
 
 
In addition, 
 
 
 
 
Efficacy Measurements: 
• PD is measured with a 
 
• mLCVA is measured under 
(letters recorded, converted to 
logMAR).  
• pLCVA is measured under
(letters recorded, converted to 
logMAR).  
• DCNVA is measured under mesopic conditions by a high-contrast Near Visual 
 
 
• Best-corrected distance visual acuity (BCDVA) is measured under mesopic 
conditions by a 
 at 
 (letters recorded, converted to logMAR). 
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• 
 
   
• Subject questionnaire is a brief symptom survey. 
Safety and Tolerability: 
• Conjunctival hyperemia 
• Adverse events (AEs) 
• Vital signs (heart rate [HR] and blood pressure [BP]) 
• Intraocular pressure (IOP) 
• Biomicroscopy 
• Ophthalmoscopy 
• Dry eye measurements 
• Urine pregnancy tests for females of childbearing potential 
Safety Measurements: 
• Conjunctival hyperemia is assessed visually with a grading scale (0-3) using images 
from the Cornea and Contact Lens Research Unit (CCLRU).  
• IOP is measured with the Tono-Pen. 
• BP and HR is measured manually or with available digital devices. 
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5. 
STUDY METHODS 
5.1. 
General Study Design and Plan 
A sample size of approximately 160 DLD subjects ≥ 18 years of age were randomized in a 
1:1 ratio to Nyxol or placebo, with the expectation that approximately 136 subjects were 
evaluable for efficacy, where evaluable means receiving at least 1 dose of study medication 
and having a Baseline and a Day 8 mLCVA measurement. 
 
. The subjects were recruited from approximately 15 
investigational sites. 
Following the successful completion of screening,
 
 The Screening Visit 
should occur the same day as the Baseline Visit. 
 
 
 
 
 
 The study eye and non-study eye, as well as binocular, was evaluated 
at all appropriate assessments. 
The subject instilled one drop once daily (QD) at or near bedtime (8PM to 10PM) in both 
eyes (OU) for 14 days (+ 1 day). Each subject received sufficient study medication for the 
duration of the trial. A study medication box was dispensed at Visit 1 (Screening/ Baseline; 
Day 1). The first dose of study medication was taken on the evening of the Baseline Visit 
(Day 1).  
All opened bottles as well as any unopened study medication were returned at Visit 2 
(Treatment; Day 8) and Visit 3 (Treatment; Day 15). At Visit 2, the box was re-dispensed to 
the subject following study drug accountability and removal of opened study medication 
materials. Study medication accountability was conducted at Visit 3, but the box was not re-
dispensed. 
Up to 3 days after Visit 3, a follow-up phone call (Visit 4) was completed. Subjects were 
asked if they had any problems with their eyes from the last visit, including whether their 
eyes are red, and if there have been any changes in their medical condition or concomitant 
medications since their last visit. There was no treatment administered at Visit 4. 
The schedule for assessments and timing of events is presented in Table 1. 
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Table 1 
Screening, Baseline, Treatment, and Follow-Up Visits and 
Procedures Schedule 
  
Day 
Visit 
Informed consent 
Subject ID number assigned 
Medical/ophthalmic history [b] 
Demographics 
Drug accountability and review of subject diary 
Concomitant medications [c] 
Urine pregnancy test [d] 
Heart rate/Blood pressure [e] 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse events 
Randomization number assigned 
Medications dispensed/Re-dispensed 
Diary dispensed/Re-dispensed 
Abbreviations: BAT, Brightness Acuity Tester; BCDVA, best-corrected distance visual acuity; CCLRU, Cornea and Contact Lens Research 
Unit; DCNVA, distance-corrected near visual acuity; mLCVA, mesopic low-contrast visual acuity 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
 
 
 
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5.2. 
Inclusion – Exclusion Criteria and General Study Population 
The study population will be approximately 160 DLD subjects ≥18 years of age, with 
approximately 136 evaluable subjects. Written informed consent will be obtained from each 
subject.  
The inclusion and exclusion criteria defined in the protocol apply to all subjects and are not 
repeated herein the SAP. Reference is made to the final protocol for the specific inclusion 
and exclusion criteria for study subjects. 
5.3. 
Randomization and Blinding 
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 identification number in numerical order 
within site. Once a subject is qualified for the study, the subject is assigned a randomization 
number in the order provided by the biostatistician, and randomized into the study. 
Treatment randomization will be 1:1, Nyxol or Placebo (vehicle), 
 
 It is assumed that there will be approximately 15% drop-out between Day 1 
and Day 15. 
The study medication will be masked to investigators, study staff, study subjects, the clinical 
research organization, and Ocuphire. Only in case of medical emergency or occurrence of 
serious adverse events (SAEs) will the randomization code be unmasked by the study 
pharmacist and made available to the Investigator, Ocuphire, and/or other personnel involved 
in the monitoring or conduct of this study. Rules for unmasking a subject for safety reasons 
are fully described in the protocol and not repeated herein this SAP. 
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: 
• Mesopic low-contrast BCDVA (mLCVA) 
• Photopic low-contrast BCDVA (pLCVA) 
• Mesopic high-contrast BCDVA (mHCVA) 
• Mesopic high-contrast DCNVA 
• Photopic pupil diameter 
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• Mesopic pupil diameter 
• Total RMS error (score) and higher-order RMS (spherical, coma, trefoil) error 
• Subject questionnaire 
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.   
• 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 136 subjects that are evaluable for efficacy (68 per treatment 
group) is needed for the study.  
 
 
 
 
  
 
 
It is assumed that there will be approximately 15% drop-out between Day 1 and Day 15. To 
account for this drop-out, a total of approximately 160 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 will include all randomized subjects who received at least 1 dose of study 
medication and have a Baseline and any Day 8 efficacy measurement. The mITT will be used 
for the primary endpoint analysis and to analyze 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 includes all subjects in the mITT who received at least 5 doses of study 
medication during the first 7 days of dosing including the day prior to Day 8, have a Baseline 
and a Day 8 mLCVA measurement, and have no major protocol deviations considered to 
have significant impact on treatment outcome. The PP population will be used to analyze 
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 dose of study 
medication. 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 
 
 
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select efficacy endpoints. Other possible subgroups include sex and race. 
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 percent of subjects with ≥ 15 ETDRS letters (≥ 3 lines) 
of improvement in the study eye compared to baseline in monocular mLCVA at Day 8. 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. Confirmatory 
analyses will 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.’  
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3) 
 If the day is unknown, then: 
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 
 
 
 
 
    
 
 
 
 
 
 
   
The imputations will be done separately for each treatment group and will include the 
following variables in the imputation model: mLCVA at baseline and Day 8.  
The number of imputations 
 The change from baseline will be calculated 
from these imputed datasets. The observed outcome (
) for each imputed dataset will be 
 See Section 9.1 for details on these models. The estimates 
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and standard errors (SEs) of the odds ratios based on the
 
  
Example SAS code is provided below: 
 
 
 
 
 
      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
         
 
 
          
 
          
 
          
 
          
  
         
 
 
 
 
 
 
 
    
 
 
 
      
 
      
 
 
 
   
   
7.3.3. 
Handling of Early Termination Visit Information 
In the event that a subject is terminated early from this study, the early termination data for 
safety and efficacy variables will be assigned to the closest visit (Day 8 or Day 15). If the 
closest visit has valid data, the early termination data will be assigned to the next available 
visit. 
7.3.4. 
Pooling of Investigational Sites 
The data from all study centers will be pooled together for all planned analyses. 
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). 
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Prior and concomitant medications will be coded using WHO-DD (World Health 
Organization Drug Dictionary) (Global Version 2020-09-01). 
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) MedNet version 1.212.2.  
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 1. 
Figure 1 
SDTM, ADaM, and TFL Development and Validation 
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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 or 
Placebo).  
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. 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. 
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7.6. 
Baseline Values 
Baseline values are the values obtained prior to any drug administration on the evening of the 
Baseline Visit (Day 1). If the Day 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, , 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 considered to have significant impact on treatment outcome, 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.  
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), irides type 
(light or dark), and eyeglasses-wearing status (yes or no; distance vision or near vision). 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, irides type, and distance vision/near vision 
correction status, 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 and non-study eye, 
and for binocular for visual acuity assessments, using descriptive statistics: 
• mLCVA 
• pLCVA  
• mHCVA 
• DCNVA 
• Pupil diameter (photopic and mesopic) 
• IOP 
• Total RMS error (score)  
• Total wavefront higher-order error (score) 
• Higher-order RMS (spherical, coma, trefoil) error 
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. 
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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 Exposure and Compliance 
 
 
 
 
 
 
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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. If the analysis using the mITT Population shows 
a positive effect for Nyxol at the 0.05 level of significance, the primary endpoint will be 
considered met.  
Confirmatory analysis of the primary efficacy endpoint will be performed using the ARP, 
also using imputation for missing data. For the analysis of the secondary efficacy endpoints, 
only observed case data will be used.  
To formally test the significance of endpoints of interest beyond primary efficacy, endpoints 
will be tested in a predefined sequence using observed data only, each at the significance 
level 0.05, until the first nonsignificant test. The primary efficacy endpoint will be first in this 
sequence. The endpoints in the sequence are for the mITT population and study eye, and 
either Day 8 or Day 15. The sequence is specified in Section 12.3. 
All efficacy assessment data, regardless of whether they are included in the analysis, will be 
presented in by-subject listings. 
9.1. 
Clinical Efficacy   
All efficacy data will be summarized by treatment group (Nyxol or placebo) and visit (Day 1, 
Day 8, and Day 15). 
9.1.1. 
Primary Efficacy Analysis 
 
 
 
 
 
  
  
 
 
By including
 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]. Additionally, a sensitivity 
analysis will be applied to the primary efficacy endpoint for the mITT and PP populations, 
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which uses a logistic regression model with only treatment as a 
 
).  
For the analysis of the PP Population, subjects who did not dose the night before, or the 
morning of, their Day 15 visit will be excluded from the Day 15 visit summary. 
In addition, the primary efficacy endpoint 
 using the 
same model indicated above 
. For these subgroup analyses, 
observed case data only will be used; that is, missing values will not be imputed.  
9.1.2. 
Secondary and Exploratory Efficacy Analysis 
Secondary and exploratory efficacy endpoints are indicated in Section 4.2.2. Secondary and 
exploratory efficacy endpoints will be analyzed by study eye, non-study eye, the best of 
either eye, and binocular, unless otherwise indicated.  
Visual acuity assessments (mLCVA, pLCVA, mHCVA, and DCNVA) will be summarized 
using letters. For mLCVA, pLCVA, and mHCVA, the assessments will also be summarized 
using logMAR units. Only letters will be recorded in the CRF. Letters will be converted to 
logMAR using the following formula: logMAR = 0.02*(S - # Letters), where S = 55. The 
values of S are the number of letters read equivalent to a Snellen Acuity of 20/20. For 
example, for mLCVA, if the # letters = 55, then logMAR = 0.02*(55 - 55) = 0.00; if # letters 
> 55, then logMAR is negative.  
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 
 and the respective baseline value included as the covariate.  
Each ANCOVA will be performed using the mITT and PP populations. The output from 
each ANCOVA will include the LSM and SE for both treatment groups, along with the 
placebo-corrected LSM, its 95% CI and associated p-value.  
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 
 
s, and the respective baseline as a covariate. For each analysis, the percentage 
of subjects in each treatment group meeting the criteria, the OR with 95% CI and p-value 
will be provided.  
In addition, each secondary efficacy endpoint will be analyzed by 
 the 
same model indicated above but without irides as a factor. 
For Day 8 and Day 15, the time since last dose of study treatment will be calculated as 
follows: date/time of visit minus date/time of the most recent dose of study medication in the 
subject diary.
 
 For each subject, the category could be different for Day 8 
and Day 15. Summaries of for improvement in mLCVA, and change and percent change in 
pupil diameter, will be presented based on these categorizations.  
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Questions from the subject questionnaire, including those questions for which it is not 
appropriate to be analyzed using the methods above, will be summarized categorically for 
each treatment group and visit, as well as by
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10. 
SAFETY ANALYSES 
All safety analyses will be conducted using the SP. 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/follow-up phone call, 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  
• 
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 
• 
Summary table of TEAEs leading to study medication discontinuation by SOC and 
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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 visit (Day 1, Day 8, and Day 15) for the observed value and change from 
baseline (Day 1). Separate summaries will be created for the study eye, the non-study eye, 
and the best of either 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. 
Vital Signs 
Descriptive statistics of observed values will be presented for vital sign data at each visit 
(Day 1, Day 8, and Day 15), including systolic BP (mmHg), diastolic BP (mmHg), and HR 
(bpm) by treatment group and overall. Changes from baseline (Day 1) to each scheduled 
post-baseline time point will be presented.  
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10.5. 
IOP 
Observed values and change from baseline in IOP will be summarized descriptively using 
counts and percentages for each treatment group at each visit (Day 1, Day 8, and Day 15) for 
the observed value and change from baseline (Day 1). Separate summaries will be created for 
the study eye, the non-study eye, and the best of either eye. Treatments will be compared 
using the same ANCOVA model proposed for the continuous secondary efficacy endpoints. 
10.6. 
Biomicroscopy and Ophthalmoscopy 
Results from biomicroscopic and ophthalmoscopic examinations at Day 1 and Day 15 will be 
summarized by treatment group. 
10.7. 
Other Safety Measures 
Urine pregnancy tests for females of childbearing potential will be presented in by-subject 
listings. Dry eye tests (TearLabs osmolarity test, tear break-up time and cornea fluorescein 
staining), performed only at Day 1, will also be listed. 
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. 
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12.2. 
List of Planned Listings 
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12.3. 
Hierarchy of Sequential Testing 
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