Conditions: Mydriasis Dilation
Keywords: Nyxol Pharmacologically Induced Mydriasis Dilation Pediatrics Phentolamine Mydriasis Phenylephrine Tropicamide Paremyd
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 23
Healthy volunteers: yes

Brief summary:
The objectives of this study are:

* To evaluate the safety of Nyxol in pediatric subjects
* To evaluate the efficacy of Nyxol to expedite the reversal of pharmacologically induced mydriasis in pediatric subjects The Sponsor intends to use this study to evaluate Nyxol in pediatric subjects aged 3 to 11 for the indication "the treatment of pharmacologically induced mydriasis produced by adrenergic (phenylephrine) or parasympatholytic (tropicamide) agents, or a combination thereof."

Detailed description:
This is a randomized, parallel-arm, double-masked, placebo-controlled study in approximately 20 randomized pediatric subjects evaluating the safety and efficacy of Nyxol in pediatric subjects with pharmacologically induced mydriasis with three dilating agents (e.g., phenylephrine, tropicamide, and Paremyd). Pediatric subjects will be recruited for the study into 2 age groups as follows:1) 3 to 5 years of age: 10 subjects 2) 6 to 11 years of age: 10 subjects.

Arms:
arm 1: One drop of study medication in each eye. arm 2: One drop of study medication in each eye.

Interventions: intervention 1: Phentolamine Ophthalmic Solution 0.75% intervention 2: Phentolamine Ophthalmic Solution Vehicle
Intervention descriptions: intervention 1: 0.75% phentolamine ophthalmic solution (Nyxol), a non-selective alpha-1 and alpha-2 adrenergic antagonist intervention 2: Phentolamine Ophthalmic Solution Vehicle

Protocol document:
Prot_000.pdf: 
 
 
 
CONFIDENTIAL  
 
 
Clinical Study Protocol 
OPI-NYXRMP-303 
MIRA-4 
 
Randomized, Parallel-Arm, Double-Masked, Placebo-Controlled Study of the 
Safety and Efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) to 
Reverse Pharmacologically Induced Mydriasis in Healthy Pediatric Subjects 
 
 
Ocuphire Pharma, Inc. 
37000 Grand River Avenue, Suite 120 
Farmington Hills, MI 48335 
 
 
 
 
 
Original: 
26 October 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.


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SPONSOR SIGNATURE & CONTACTS 
Study Title: 
Randomized, Parallel-Arm, Double-Masked, Placebo-Controlled 
Study of the Safety and Efficacy of Nyxol (0.75% Phentolamine 
Ophthalmic Solution) to Reverse Pharmacologically Induced 
Mydriasis in Healthy Pediatric Subjects 
Study Number: 
OPI-NYXRMP-303 
Original Protocol: 
 26 October 2021 
Person authorized to sign the protocol and protocol amendment(s) for the Sponsor, Ocuphire 
Pharma, Inc. 
Mina Sooch, MBA 
President & CEO  
Ocuphire Pharma, Inc. 
Telephone: 248-681-9815 (Office) 
 
Telephone: 248-980-6538 (Mobile) 
Email: msooch@ocuphire.com 
 
____________________________________ 
Signature  
 
____________________________________ 
Date 
 
Medical Monitor 
Charles Slonim, MD 
Oculos Development Services, LLC 
Telephone: 813-690-0255 (Mobile) 
Email: chuck.slonim@oculoscr.com 
 
____________________________________ 
Signature  
 
____________________________________ 
Date 
 


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INVESTIGATOR’S AGREEMENT 
 
OPI-NYXRMP-303 
MIRA-4 
 
Randomized, Parallel-Arm, Double-Masked, Placebo-Controlled Study of the  
Safety and Efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) to  
Reverse Pharmacologically Induced Mydriasis in Healthy Pediatric Subjects 
 
Original: 
 
 
26 October 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 the protection of human subjects 
(21 CFR 50), Institutional Review Boards (21 CFR 56) and the obligations of clinical 
investigators (21 CFR 312). 
 
Signature: ____________________________ Date: _________________________ 
 
Printed Name: _________________________ 
 
 
 


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PROCEDURES IN CASE OF EMERGENCY 
 
EMERGENCY CONTACT INFORMATION 
Role in Study 
Name 
Contact Information 
Clinical Study 
Leader 
Roselyn Judd 
Director of Clinical Operations  
Oculos Development Services, LLC 
586-212-0237 (Mobile) 
Email: roselyn.judd@oculoscr.com 
Medical 
Monitor 
Charles Slonim, MD 
Chief Medical Officer 
Oculos Development Services, LLC 
813-690-0255 (Mobile) 
Email: 
chuck.slonim@oculoscr.com 
 
 
 


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ABBREVIATIONS AND TERMS 
Abbreviation 
Full term 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ARP 
All Randomized Population 
BCDVA 
best-corrected distance visual acuity 
BP 
blood pressure 
°C 
degree Centigrade 
CCLRU 
Cornea and Contact Lens Research Unit 
CI 
confidence interval 
CRA 
clinical research associate 
CRO 
clinical research organization 
°F 
degree Fahrenheit 
DCNVA 
distance-corrected near visual acuity 
CRF 
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 
IOP 
intraocular pressure 
IRB 
Institutional Review Board 
ITT 
Intent-to-Treat 
LC-MS/MS 
liquid chromatography-tandem mass spectrometry 
LSM 
least squares mean 
MedDRA 
Medical Dictionary for Regulatory Activities 


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mITT 
Modified Intent-to-Treat 
Nyxol 
0.75% Phentolamine Ophthalmic Solution or 1% Phentolamine 
Mesylate Ophthalmic Solution (Nyxol®) 
OD 
oculus dexter (right eye) 
OR 
odds ratio 
OS 
oculus sinister (left eye) 
OTC 
over-the-counter 
OU 
oculus uterque (both eyes) 
Paremyd® 
1% hydroxyamphetamine hydrobromide/0.25% tropicamide 
PD 
pupil diameter 
PK 
pharmacokinetic 
PP 
Per Protocol 
PREA 
Pediatric Research Equity Act 
RAF 
Royal Air Force 
SAE 
serious adverse event 
SAP 
Statistical Analysis Plan 
SOC 
system organ class 
SP 
Safety Population 
TEAE 
treatment-emergent adverse event 
US 
United States 
USP 
United States Pharmacopeia 
 
 


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1. STUDY SUMMARY 
 
Study Number  
OPI-NYXRMP-303  
Type of Study  
Randomized, parallel-arm, double-masked, placebo-controlled study of the 
safety and efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) to 
reverse pharmacologically induced mydriasis in healthy pediatric subjects  
Name of Investigational 
Product  
Nyxol® Eye Drops – 0.75% Phentolamine Ophthalmic Solution  
Duration of Study  
2 days, including screening/treatment and follow-up  
Rationale  
Nyxol is a once-daily preservative-free eye drop formulation of 
phentolamine, which 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. 
Pharmacologically induced mydriasis is achieved either by stimulating 
the iris dilator muscle with the use of direct or indirect alpha-1 agonists 
(eg, phenylephrine, hydroxyamphetamine) or by blocking the iris 
sphincter muscle with the use of muscarinic antagonists or cycloplegic 
drugs (eg, tropicamide) or with a combination (eg, Paremyd®, which is 
1% hydroxyamphetamine hydrobromide and 0.25% tropicamide). 
Nyxol, either by directly antagonizing the alpha-1 receptor or by 
indirectly antagonizing the pupil dilation effect of muscarinic blocking, 
can expedite the reversal of pharmacologically induced mydriasis prior 
to natural reversal.  
Study Objectives  
The objectives of this study are:  
• 
To evaluate the safety of Nyxol in pediatric subjects 
• 
To evaluate the efficacy of Nyxol to expedite the reversal 
of pharmacologically induced mydriasis in pediatric subjects  
The Sponsor intends to use this study to evaluate Nyxol in pediatric 
subjects aged 3 to 11 for the indication “the treatment of pharmacologically 
induced mydriasis produced by adrenergic (phenylephrine) or 
parasympatholytic (tropicamide) agents, or a combination thereof.”  
Study Design  
This is a randomized, parallel-arm, double-masked, placebo-controlled 
study in approximately 20 to 30 randomized pediatric subjects evaluating 
the safety and efficacy of Nyxol in pediatric subjects 
with pharmacologically induced mydriasis. Pediatric subjects will be 
recruited for the study into 2 age groups as follows:  
• 3 to 5 years of age: 10 subjects 
• 6 to 11 years of age: 10 subjects 
In this young study population, it is anticipated that some randomized 
subjects may withdraw. 
Following the successful completion of screening, each subject will be 
randomized to unmasked mydriatic agent and masked treatment (refer to 


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Appendix 2 for randomization schema). Treatment randomization will be 
1:1 (Nyxol or placebo [vehicle]) and will be stratified 1:1 by subject age 
group (3‒5 years of age:6‒11 years of age). Mydriatic agent randomization 
will be 3:1:1 (phenylephrine:tropicamide:Paremyd, respectively). 
Approximately 60% of the randomized subjects will receive 1 drop of 
2.5% phenylephrine in both eyes (OU) 1 hour before treatment (12 
subjects), approximately 20% will receive 1 drop of 1% tropicamide OU 1 
hour before treatment (4 subjects), and approximately 20% will receive 1 
drop of Paremyd OU 1 hour before treatment (4 subjects). Subjects will 
have 1 drop of masked study treatment (Nyxol or placebo) administered in 
the study eye (right eye; OD) and 1 drop administered in the fellow eye 
(left eye; OS). The study eye and the fellow eye will both be evaluated at 
all assessments unless otherwise specified.  
The study eye (OD) will always be treated and assessed first. If the subject 
is not amenable to receiving treatment in the fellow eye (OS), the subject 
can still participate in the study and only the study eye will undergo study 
assessments. 
Subject Population 
Approximately 20 to 30 randomized healthy pediatric subjects, with a 
target of 20 subjects treated with study medication 
Inclusion Criteria  
1. Males or premenstrual females 3 to 11 years of age  
2. Ability to comply with all protocol-mandated procedures 
independently and to attend all scheduled office visits  
3. Parent/Legal guardian willing to give written informed consent to 
participate in this study. Children aged 7 to 11 years to 
provide signed assent form, as well as a separate parental/Legal 
Guardian consent. 
Exclusion Criteria  
Ophthalmic (in either eye):  
1. Clinically significant ocular disease as deemed by the Investigator 
(eg, amblyopia, congenital cataract, congenital glaucoma) that 
might interfere with the study  
2. Unwilling or unable to discontinue use of contact lenses at 
screening until study completion 
3. Unwilling or unable to suspend use of topical medication at 
screening until study completion  
4. Ocular trauma or ocular surgery within the 6 months prior to 
screening  
5. Use of any topical prescription or over-the-counter (OTC) 
ophthalmic medications of any kind within 7 days of screening  
6. Recent or current evidence of ocular infection or inflammation in 
either eye (such as current evidence of clinically significant 
blepharitis, conjunctivitis, or keratitis). Subjects must be symptom 
free for at least 7 days prior to screening 


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7. Closed or very narrow angle that in the Investigator’s opinion is 
potentially occludable if the subject’s pupil is dilated  
8. History of any traumatic (surgical or nonsurgical) or nontraumatic 
condition affecting the pupil or iris  
9. Known allergy, hypersensitivity, or contraindication to any 
component of the phentolamine ophthalmic solution or to any 
component of the mydriatic agents or vehicle formulation  
Systemic:  
10. Known hypersensitivity or contraindication to α- and/or β-
adrenoceptor antagonists  
11. Clinically significant systemic disease (eg, uncontrolled diabetes, 
cancer, hepatic, renal, endocrine, or cardiovascular disorders) that 
in the opinion of the Investigator could interfere with the study  
12. Subjects with learning disabilities that in the opinion of the 
Investigator could interfere with the study  
13. 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)  
14. Participation in any investigational study within 30 days prior to 
screening  
Visit Schedule  
At Visit 1, subjects will be screened for study eligibility. After screening, 
eligible subjects will be randomized 3:1:1 to unmasked mydriatic agent 
(phenylephrine:tropicamide:Paremyd, respectively) and 1:1 to 1 of 
2 masked treatment arms (Nyxol or placebo). Treatment randomization 
will be stratified 1:1 by subject age group (3‒5 years of age:6‒11 years of 
age) (Refer to Appendix 2). Each subject will receive 1 drop of mydriatic 
agent OU 1 hour before study treatment. 
At Visit 1, measurements as defined in Table 1 will be made before 
(-1 hour/baseline) and 60 minutes after (max pupil dilation/0 minutes) the 
mydriatic agent instillation OU (ie, right before the study treatment is 
administered). Additionally, measurements will be taken at 90 minutes, and 
3 hours after study treatment dosing.  
At Visit 2 (Follow-Up Visit), which is 1 day after Visit 1, measurements as 
defined in Table 1 will be measured 24 hours after study treatment dosing.  
Number of 
Investigational Sites  
Approximately 2 sites  
Sample Size 
Justification  
This safety study is not powered for efficacy. In accordance with the 
Sponsor Pediatric Safety Plan for the indication of reversal of 
pharmacologically induced mydriasis, the sample size of 20 subjects 
divided between 2 age groups is adequate to evaluate safety in the pediatric 
population.  


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Safety Endpoints  
The safety measures are conjunctival hyperemia, impairment in visual 
acuity (BCDVA]), vital signs (HR and BP), and adverse events (AEs).  
Safety analyses will include: 
• 
Change from baseline (-1 hour) in conjunctival hyperemia 
grading (CCLRU images) at each time point (0 
minutes, 90 minutes, 3 hours, and 24 hours) for the study 
eye and fellow eye  
• 
Change from baseline (-1 hour) in BCDVA at 0 minutes, 
90 minutes, 3 hours, and 24 hours for the study eye and fellow eye 
• 
Change from screening in vital signs (HR and BP) at 3 hours and 
24 hours  
Safety measurements:  
• 
Conjunctival hyperemia will be assessed visually with a 4-point 
grading scale (0-3) using CCLRU images (Appendix 1)  
• 
BCDVA will be measured in photopic conditions by a 
standard Early Treatment Diabetic Retinopathy Study (ETDRS) 
illuminated chart (on wall or stand) at 4 m (letters recorded). For 
subjects who are unable to read the letters on a ETDRS chart, the 
Patti Pics Series ETDRS chart will be used (Appendix 3)  
Efficacy Endpoints  
Efficacy endpoints for the study eye and fellow eye will include:  
• 
Percentage of subjects returning to ≤ 0.2 mm from baseline 
(-1 hour) photopic pupil diameter at each remaining time point 
(0 minutes, 90 minutes, 3 hours, and 24 hours)  
• 
Change (in mm) in photopic pupil diameter from max pupil 
dilation (0 minutes) at each time point (90 minutes, 3 hours, and 24 
hours)  
• 
Time (hours) to return to ≤ 0.2 mm from baseline (-1 hour) 
photopic pupil diameter (time-savings analysis)  
Efficacy measurements:  
Pupil diameter will be measured with a NeurOptics VIP-300 pupillometer 
(mm) or other similar measurement.  
Study Medications, Dose 
and Mode of 
Administration  
Nyxol® Eye Drops (Phentolamine Ophthalmic Solution):  
1 drop OU administered 1 hour post mydriatic drug instillation. 
Placebo (Nyxol vehicle):  
1 drop OU administered 1 hour post mydriatic drug instillation. 
Study eye will always be treated first. If the subject is not amenable to 
receiving treatment in the fellow eye (OS) the subject can still participate 
in the study and only the study eye will undergo study assessments.  
Duration of Subject 
Participation and Study  
The total length of subject participation is 2 days, with 2 clinic visits, as 
summarized below:  
• 
Screening/Treatment Visit (Visit 1/Day 1)  


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• 
Follow-Up Visit (Visit 2/Day 2)  
The execution of the entire study (first subject screened through last 
randomized subject completed) is expected to be approximately 2 to 4 
months.  


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TABLE OF CONTENTS 
SPONSOR SIGNATURE & CONTACTS………………………………………………………………………2 
INVESTIGATOR’S AGREEMENT.................................................................................................................... 3 
ABBREVIATIONS AND TERMS ....................................................................................................................... 5 
1. 
STUDY SUMMARY .................................................................................................................................... 7 
2. 
INTRODUCTION ...................................................................................................................................... 14 
2.1. 
Investigational products ................................................................................................................... 14 
2.2. 
Findings from nonclinical and clinical studies ............................................................................... 14 
2.3. 
Design justification ........................................................................................................................... 16 
2.4. 
Route of administration, dosage regimen, and treatment period ................................................. 17 
2.5. 
Compliance ........................................................................................................................................ 17 
2.6. 
Study population ............................................................................................................................... 17 
3. 
OBJECTIVES AND PURPOSE ............................................................................................................... 19 
4. 
STUDY DESIGN ........................................................................................................................................ 20 
4.1. 
Safety and efficacy endpoints .......................................................................................................... 20 
4.2. 
Description and schedule of visits and procedures ........................................................................ 21 
4.3. 
Measures taken to minimize/avoid bias .......................................................................................... 23 
4.4. 
Study medications ............................................................................................................................. 23 
4.4.1. Packaging and labeling ...................................................................................................................... 24 
4.4.2. Storage of study medication and dispensing ...................................................................................... 24 
4.4.3. Study medication administration ........................................................................................................ 24 
4.4.4. Study medication accountability ........................................................................................................ 24 
4.4.4.1. Receipt and disposition of study medication .............................................................................. 24 
4.4.4.2. Return of study medication ........................................................................................................ 24 
4.5. 
Expected duration of subject participation .................................................................................... 24 
4.6. 
Randomization and procedure for breaking the code ................................................................... 25 
4.7. 
Collection of data .............................................................................................................................. 25 
4.7.1. Reasons for withdrawal from study .................................................................................................... 26 
4.7.2. Subject replacement ........................................................................................................................... 26 
4.7.3. Entire study terminated ...................................................................................................................... 26 
4.7.4. Actions after discontinuation ............................................................................................................. 26 
4.8. 
Completed study ............................................................................................................................... 26 
5. 
SUBJECT INCLUSION AND EXCLUSION CRITERIA ..................................................................... 27 
5.1. 
Subject inclusion criteria ................................................................................................................. 27 
5.2. 
Subject exclusion criteria ................................................................................................................. 27 
6. 
TREATMENT OF SUBJECTS ................................................................................................................ 29 
6.1. 
Treatment adherence ....................................................................................................................... 29 
6.2. 
Concomitant medications ................................................................................................................. 29 
7. 
ASSESSMENTS OF SAFETY AND EFFICACY ................................................................................... 30 
7.1. 
Specification of safety parameters .................................................................................................. 30 
7.2. 
Assessing, recording, and analyzing safety and efficacy parameters ........................................... 30 
7.2.1. Screening Visit (Day 1) ...................................................................................................................... 30 
7.2.2. Treatment Visit (Visit 1/Day 1) .......................................................................................................... 31 
7.2.3. Follow-Up Visit/Day 2 ....................................................................................................................... 31 
7.2.4. Unscheduled Visits ............................................................................................................................. 32 
7.2.5. Visit Variation .................................................................................................................................... 32 
7.3. 
Adverse events and serious adverse events ..................................................................................... 32 
7.3.1. Adverse event definitions .................................................................................................................... 32 
7.3.2. Follow-up of subjects after adverse events ........................................................................................ 36 
8. 
STATISTICS .............................................................................................................................................. 37 


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8.1. 
Sample size ........................................................................................................................................ 37 
8.2. 
Analysis populations ......................................................................................................................... 37 
8.3. 
Statistical methods ............................................................................................................................ 38 
8.3.1. General considerations ...................................................................................................................... 38 
8.3.2. Demographic and baseline characteristics ........................................................................................ 38 
8.3.3. Subject disposition ............................................................................................................................. 38 
8.3.4. Medical history and prior/concomitant medications ......................................................................... 38 
8.3.5. Analysis of safety ................................................................................................................................ 38 
8.3.6. Analysis of efficacy ............................................................................................................................. 39 
8.4. 
Procedure for accounting for missing, unused, or spurious data ................................................. 40 
8.5. 
Procedure for reporting deviations from the statistical plan ........................................................ 40 
9. 
DIRECT ACCESS TO SOURCE DATA AND DOCUMENTS ............................................................ 41 
10. 
QUALITY CONTROL AND QUALITY ASSURANCE........................................................................ 42 
11. 
ETHICAL CONSIDERATIONS AND GOOD CLINICAL PRACTICE COMPLIANCE ................ 43 
11.1. 
Good Clinical Practice compliance ................................................................................................. 43 
11.2. 
Institutional Review Board .............................................................................................................. 43 
11.3. 
Protocol deviations/violations .......................................................................................................... 43 
11.4. 
Informed consent and assent requirements .................................................................................... 43 
12. 
DATA HANDLING AND RECORD KEEPING .................................................................................... 45 
12.1. 
Data entry .......................................................................................................................................... 45 
12.2. 
Data quality control and reporting ................................................................................................. 45 
12.3. 
Archiving of data .............................................................................................................................. 45 
12.4. 
Records retention .............................................................................................................................. 45 
12.5. 
Amendments to the protocol ............................................................................................................ 45 
13. 
BIBLIOGRAPHY ...................................................................................................................................... 46 
APPENDIX 1: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING IMAGES FROM CCLRU47 
APPENDIX 2: RANDOMIZATION SCHEMA BY AGE GROUP, MYDRIATIC AGENT AND 
INVESTIGATIONAL TREATMENT .............................................................................................................. 48 
APPENDIX 3: PATTI PICS SERIES ETDRS CHART .................................................................................. 49 
APPENDIX 4: ADRENERGIC AND CHOLINERGIC DRUGS ................................................................... 50 
 
 


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2.  INTRODUCTION 
2.1. Investigational products 
The test product is Nyxol® Eye Drops – 0.75% Phentolamine Ophthalmic Solution (0.75% POS) 
(herein referred to as 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 as and used in 
place of “1% phentolamine mesylate ophthalmic solution”, which was how Nyxol had been 
described in all studies prior to the End of Phase 2 meeting in mid-2020. 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) (2021). 
Nyxol has been assessed in 9 investigator-initiated and sponsored Phase 1, 2, and 3 clinical trials. 
Across all trials, 335 of 567 subjects were exposed to at least 1 dose of Nyxol. 
In prior clinical trials in adults and pediatric (12-17 years old) subjects, Nyxol has demonstrated 
a consistent ability to decrease pupil diameter (PD) by approximately 20% (~1 to 2 mm) in both 
mesopic and photopic conditions with rapid onset and durable effects to 24 to 36 hours (OP-
NYX-01a2 CSR; OP-NYX-SNV CSR; OPI-NYXG-201 CSR; OPI-NYXRM-201 CSR; 
Karpecki 2021; Pepose MIRA-2 2021; Pepose VEGA-1 2021; Pepose ORION 2021).  
Nyxol was observed to be well tolerated in all studies at single doses and multiple daily doses for 
up to 14 days. Safety of the subjects in these trials was evaluated by adverse event (AE) 
monitoring, ophthalmic examinations, and vital sign assessments. Across all trials, there were no 
treatment-related serious adverse events (SAEs). 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). Adverse events reported were mild to moderate in 
severity, with the most common being transient conjunctival hyperemia and ocular irritation; 
however, Nyxol dosing at or near bedtime was observed to mitigate or minimize these side 
effects during the daytime. 
Phase 2 Reversal of Pharmacologically Induced Mydriasis Study (MIRA-1) 
In OPI-NYXRM-201 (MIRA-1), 32 healthy subjects were randomized at Visit 1 in a 1:1 ratio to 
receive 1 drop of 1% Nyxol (1% phentolamine mesylate ophthalmic solution) or 1 drop of 
placebo (vehicle) in both eyes (OU) at Visit 1 and 1 drop of the alternative study medication OU 
at Visit 2. One hour before administration of study medication, subjects received 1 drop OU of a 
mydriatic agent (2.5% phenylephrine or 1% tropicamide) to dilate their pupils. Each subject 
received the same mydriatic agent throughout the study. Pupil diameter was measured 
30 minutes and 1 hour, 2 hours, 4 hours, and 6 hours after dosing at Visit 1 and Visit 2 and 
compared to baseline. 
In this study, Nyxol demonstrated a statistically significant reduction in PD from max pupil 
dilation (0 minutes [60 minutes after mydriatic agent]) compared to placebo at 1 hour, 2 hours, 
4 hours, and 6 hours after treatment. These statistically significant (p<0.0001) and clinical 
meaningful (typically > 1 mm) PD reductions were observed at each time point, including when 
stratified by both parasympathetic (tropicamide) and adrenergic (phenylephrine) agents.  


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Additionally, Nyxol was effective at returning a statistically significant percent of subjects’ eyes 
to baseline PD compared with placebo treatment at every time point with estimable results, 
regardless of whether the PD threshold used was ≤ 0.5 mm from baseline (2-hour time point) or 
the more stringent, ≤ 0 mm from baseline (4-hour and 6-hour time points). The reduction in time 
needed to achieve reversal of mydriasis in either eye was statistically significantly shorter with 
Nyxol treatment compared with placebo treatment, regardless of the threshold examined or the 
mydriatic agent used. In this study, Nyxol led to an average time-savings of 2.2 hours to return 
PD to ≤ 0 mm from baseline compared to placebo. Additionally, 40% of subjects receiving 
phenylephrine and 30% of subjects receiving tropicamide achieved a time-savings of more than 
4 hours with Nyxol, based on a post-hoc analysis. 
In a post-hoc analysis, a statistically significant difference was observed in the percentage of 
subjects returning to baseline in the Nyxol group compared to the placebo group (after dilation 
with either phenylephrine or tropicamide) at 2 hours (29% vs 13%, respectively; p=0.03). This 
difference favoring Nyxol widened (68% for Nyxol and 23% for placebo) at 4 hours (p<0.0001).  
In this study, there were no severe AEs, with only mild-to-moderate conjunctival hyperemia that 
resolved in most subjects within 6 hours.  
Phase 3 Reversal of Pharmacologically Induced Mydriasis Study (MIRA-2) 
OPI-NYXRM-301 (MIRA-2) was a Phase 3, multicenter, randomized, placebo-controlled, 
double-masked clinical trial. In MIRA-2, 185 healthy subjects including 14 pediatric subjects 
aged 12 to 17 from 12 sites were randomized 1:1 to receive Nyxol or placebo (2 drops in the 
study eye and 1 drop in the fellow eye; pediatric subjects received 1 drop in each eye), 1 hour 
after pupil dilation with 1 of 3 mydriatic agents (2.5% phenylephrine, 1% tropicamide, or 
Paremyd). The primary endpoint of an increase in the percent of study eyes returning to baseline 
PD at 90 minutes after Nyxol compared to placebo was met (Figure 1A [green box]). For 
subjects in the Modified Intent-to-Treat (mITT) Population treated with Nyxol, 49% had PD 
return to ≤ 0.2 mm from baseline at 90 minutes compared to only 7% of subjects treated with 
placebo (p<0.0001). At 60 minutes, 28% of study eyes in subjects who received Nyxol had 
returned to baseline PD, compared to only 2% of participants who received placebo. The 
differences in percent of subjects returning to baseline were statistically significant across all 
time points from 60 minutes through 6 hours (p<0.0001; Figure 1A). Additionally, multiple 
secondary endpoints met statistical significance, including mean PD, which was significantly 
smaller with Nyxol compared with placebo at all time points after 30 minutes (p<0.0001; Figure 
1B). Finally, the mean time to return to baseline PD for subjects in the Per Protocol (PP) 
Population was ~2 hours in Nyxol-treated study eyes compared to ~6 hours in placebo-treated 
study eyes. Within the small pediatric subset, study eyes and non-study eyes treated with 1 drop 
of Nyxol showed greater reversal of mydriasis compared with eyes treated with 1 drop of 
placebo from 30 min through 24 hours, although the results were not powered to detect 
statistically significant differences. Efficacy was demonstrated across the mydriatic agents and 
for subjects with either light or dark irides. 
Nyxol treatment was well tolerated in this study. Adverse effects were limited to predominantly 
mild, transient hyperemia and predominantly mild discomfort on drug instillation. Reduction in 
PD with Nyxol did not result in a decrease in best-corrected distance visual acuity (BCDVA). 
There were no discontinuations due to AEs or SAEs. Vital signs were not affected by Nyxol 


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treatment. Nyxol was also well tolerated in the pediatric population, with AEs in a single subject, 
who reported transient mild pain upon Nyxol instillation OU.  
Figure 1: MIRA-2 Pupil Diameter Results. A. Percent of Subjects Returning to ≤ 0.2 mm 
From Baseline. B. Mean Pupil Diameter Across Time Points. 
A.  
 
 
 
 
 
        
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.  
2.3. Design justification 
Pupil size is under the control of 2 opposing sets of muscles – the circular constrictor muscles 
controlled by the cholinergic nervous system and the radial dilator muscles, controlled by the 
adrenergic nervous system (Steinhauer 2004; Yoshitomi 1985). The radial dilator muscles 
contain predominantly α-1 adrenergic receptors that can be inhibited by α-1 antagonists (Yu 
2002); therefore, it is possible to inhibit dilation of the pupil through blockade of the radial 
dilator muscles.  
Pharmacologically induced mydriasis is achieved either by stimulating the iris dilator muscle 
with the use of direct or indirect alpha-1 agonists (eg, phenylephrine, hydroxyamphetamine) or 
by blocking the iris sphincter muscle with the use of muscarinic antagonists or cycloplegic drugs 
(eg, tropicamide) or with a combination (eg, Paremyd, which is 1% hydroxyamphetamine 
hydrobromide and 0.25% tropicamide). Pharmacologically induced mydriasis is commonly used 
to facilitate clinical examination of the retina and other intraocular structures. Typically, 
pharmacologically induced mydriasis dilates the pupil to 6 to 8 mm, with effects lasting 6 to 
24 hours. However, factors such as an individual’s dark irides and older age cause variations in 
effect duration, which ranges from hours to days. During this time, individuals may experience 
sensitivity to light, blurred vision, or cycloplegia (loss of accommodation via the temporary 
paralysis of the muscle that allows the eye to focus on near objects). Accelerating mydriatic 
reversal after an eye exam may reduce the duration of these side effects and be beneficial for 
many patients, including pediatric patients. 
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. To counteract the dilatory effects of mydriatic agent, Nyxol is proposed to be instilled 
in the eyes post eye exam, allowing a rapid reversal of mydriasis, thereby minimizing the 
duration of side effects and discomfort post exam.  
In previous reversal of pharmacologically induced mydriasis clinical studies (MIRA-1 and 
MIRA-2), Nyxol was effective at inducing reversal of mydriasis with both adrenergic 
(phenylephrine) and parasympathetic (tropicamide) mydriatic agents, as described previously. 
The placebo outcomes in these studies demonstrated that the natural reversal of mydriasis takes 
longer with tropicamide than with phenylephrine. Despite this difference, Nyxol was able to 


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reverse mydriasis faster in the vast majority of eyes regardless of the mydriatic agent used, but it 
worked faster for phenylephrine (an alpha-1 agonist), as expected given the pharmacology of 
Nyxol (an alpha-1 and alpha-2 antagonist). A similar pattern of results was seen in the pediatric 
subjects studied in MIRA-2. 
Alpha-1 adrenergic antagonists have been shown to be safe and effective for the pharmacological 
reversal of mydriasis in adults and pediatric patients. In 1990, the United States (US) Food and 
Drug Administration (FDA) approved Dapiprazole Hydrochloride Ophthalmic Solution 0.5% 
(Rev-Eyes) for this indication; however, the product was withdrawn and discontinued by the 
manufacturer for reasons not related to safety or efficacy. Many people who undergo pupil 
dilation for an annual ophthalmic examination or other ophthalmic procedure continue to request 
an option for rapid reversal of the mydriasis. If Nyxol is a safe and effective treatment for the 
reversal of mydriasis, this may provide a new option for adult and pediatric patients who 
experience side effects of pupil dilation. 
The aforementioned completed MIRA-2 study was the first of 2 registration Phase 3 studies 
evaluating Nyxol for the reversal of pharmacologically induced mydriasis. MIRA-3 the second 
Phase 3 study, which is similar in design to MIRA-2, is ongoing. This current study, MIRA-4, 
conducted in pediatric subjects aged 3 to 11 years, supports the Phase 3 Nyxol registration 
program for the treatment of pharmacologically induced mydriasis using 3 common mydriatic 
agents (phenylephrine, tropicamide, and Paremyd). This study is primarily for safety and not 
powered to evaluate efficacy. In accordance with the Sponsor Pediatric Safety Plan for the 
indication of reversal of pharmacologically induced mydriasis, the sample size of 20 subjects is 
adequate to evaluate safety in the pediatric population. 
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 dosing of Nyxol selected for this study, 0.75%, was based upon 1) preclinical safety studies, 
2) previous ophthalmic clinical studies of Nyxol described above and in the IB, and 3) clinical 
studies conducted with varying doses of drugs in the same class.  
Note that 0.75% POS, 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 MIRA-1. 
2.5. Compliance 
This study will be conducted in compliance with the protocol and in accordance with 
International Council for Harmonisation (ICH) Guidelines for Good Clinical Practice (GCP), the 
ethical principles set forth in the Declaration of Helsinki, and with the US Title 21 Code of 
Federal Regulations. 
2.6. Study population 
Approximately 20 to 30 healthy pediatric subjects 3 to 11 years of age, inclusive, will be 
randomized in a 1:1 ratio to 1 of 2 masked treatment arms (Nyxol or placebo), to achieve a target 
of 20 randomized subjects (ages 3‒5, n=10; ages 6‒11, n=10) who receive treatment with study 
medication. In this young study population, it is anticipated that some randomized subjects may 


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withdraw (Section 4.7.1). Treatment randomization will be stratified 1:1 by subject age group 
(3‒5 years of age:6‒11 years of age). Mydriatic agent randomization will be 3:1:1 
(phenylephrine:tropicamide:Paremyd, respectively). Refer to Appendix 2 for randomization 
schema. Subjects will be recruited from 2 investigational sites. 
 
 


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3. OBJECTIVES AND PURPOSE 
The MIRA-4 study is a randomized, parallel-arm, double-masked, placebo-controlled study of 
the safety and efficacy of Nyxol (0.75% POS) to reverse pharmacologically induced mydriasis in 
healthy pediatric subjects. 
The objectives of this study are: 
• To evaluate the safety of Nyxol in pediatric subjects 
• To evaluate the efficacy of Nyxol to expedite the reversal of pharmacologically induced 
mydriasis in pediatric subjects 
The Sponsor intends to use this study to evaluate Nyxol for the indication “the treatment of 
pharmacologically induced mydriasis produced by adrenergic (phenylephrine) or 
parasympatholytic (tropicamide) agents, or a combination thereof.” 
 
 


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4. STUDY DESIGN 
This is a randomized, parallel-arm, double-masked, placebo-controlled study in 
approximately 20 to 30 randomized pediatric subjects evaluating the safety and efficacy 
of Nyxol in pediatric subjects with pharmacologically induced mydriasis. Pediatric subjects will 
be recruited for the study into 2 age groups as follows:  
• 3 to 5 years of age: 10 subjects 
• 6 to 11 years of age: 10 subjects 
In this young study population, it is anticipated that some randomized subjects may withdraw. 
Following the successful completion of screening, each subject will be randomized to unmasked 
mydriatic agent and masked treatment (refer to Appendix 2 for randomization schema). 
Treatment randomization will be 1:1 (Nyxol or placebo [vehicle]) and will be stratified 1:1 by 
subject age group (3‒5 years of age:6‒11 years of age). Mydriatic agent randomization will be 
3:1:1 (phenylephrine:tropicamide:Paremyd, respectively). Approximately 60% of the 
randomized subjects will receive 1 drop of 2.5% phenylephrine OU 1 hour before treatment (12 
subjects), approximately 20% will receive 1 drop of 1% tropicamide OU 1 hour before treatment 
(4 subjects), and approximately 20% will receive 1 drop of Paremyd OU 1 hour before treatment 
(4 subjects). Subjects will have 1 drop of masked study treatment (Nyxol or placebo) 
administered in the study eye (right eye; OD) and 1 drop administered in the fellow eye (left eye; 
OS). The study eye and the fellow eye will both be evaluated at all assessments unless otherwise 
specified. The study eye (OD) will always be treated and assessed first. If the subject is not 
amenable to receiving treatment in the fellow eye (OS), the subject can still participate in the 
study and only the study eye will undergo study assessments.  
4.1. Safety and efficacy endpoints 
The study eye is defined as the right eye (OD). The fellow eye is defined as the left eye (OS). 
The study eye and fellow eye will only be evaluated if mydriatic and study drug were 
successfully administered. The study eye will always be treated and assessed first.  
Safety: 
The safety measures are conjunctival hyperemia, impairment in visual acuity (BCDVA), 
vital signs (HR and BP), and AEs.  
Safety analyses will include: 
• Change from baseline (-1 hour) in conjunctival hyperemia grading (Cornea and Contact 
Lens Research Unit [CCLRU] images) at each time point (0 minutes, 90 minutes, 
3 hours, and 24 hours) for the study eye and fellow eye  
• 
Change from baseline (-1 hour) in BCDVA at 0 minutes, 90 minutes, 3 hours, and 
24 hours for the study eye and fellow eye  
• 
Change from baseline (-1 hour) in vital signs (HR and BP) at 3 hours and 24 hours  
Safety measurements: 
• Conjunctival hyperemia will be assessed visually with a 4-point grading scale (0-3) using 
CCLRU images (Appendix 1)  


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• Best-corrected distance visual acuity will be measured in photopic conditions by a 
standard Early Treatment Diabetic Retinopathy Study (ETDRS) illuminated chart (on 
wall or stand) at 4 m (letters recorded). For subjects who are unable to read the letters on 
a standard ETDRS chart, the Patti Pics Series ETDRS chart will be used (Appendix 3)  
Efficacy: 
Efficacy endpoints for the study eye and fellow eye will include: 
• Percentage of subjects returning to ≤ 0.2 mm from baseline (-1 hour) photopic PD at each 
remaining time point (0 minutes, 90 minutes, 3 hours, and 24 hours) 
• Change (in mm) in photopic PD from max pupil dilation (0 minutes) at each time point 
(90 minutes, 3 hours, and 24 hours) 
• Time (hours) to return to ≤ 0.2 mm from baseline (-1 hour) photopic PD (time-savings 
analysis)  
Efficacy measurements: 
• Pupil diameter and pupillary light reflex will be measured with a NeurOptics VIP-300 
pupillometer (mm) or other similar measurement 
Efficacy endpoints will be summarized by age group and by mydriatic agent but will not be 
analyzed due to the small sample size for each subgroup. Each mydriatic agent will be 
summarized individually, and an additional summary combining 1% tropicamide and Paremyd 
subjects into a “tropicamide or Paremyd” group will be presented. Other subgroups such as sex 
and race may be summarized as well.  
4.2. Description and schedule of visits and procedures 
Study procedures are shown in detail in Table 1. For this study, the following time points apply: 
• Baseline (-1 hour): just prior to administration of mydriatic agent at -1 hour and time 
point at which PD measurement is considered normal 
• Max pupil dilation (0 minutes): just prior to administration of study medication and time 
point at which maximum PD is expected  
 
 


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Table 1: Screening and Mydriatic/Treatment Schedule 
 
Screening [a] 
Mydriasis/Treatment 
Follow-Up 
Day 
1 
1 
2 
Visit 
1 
1 
2 
Start time [b] 
 
-1 hour 
(Baseline) 
0 ±5 min 
(Max) 
90 ±10 
min 
3 hours 
±10 min 24 +6 hours 
Informed consent/assent 
X 
 
 
 
 
 
Subject identification number assigned 
X 
 
 
 
 
 
Medical/Ophthalmic history 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Prior/Concomitant medications [c] 
X 
X 
X 
X 
X 
X 
HR/BP 
X 
 
 
 
X 
X 
PD 
 
X 
X 
X 
X 
X 
BCDVA [d] 
 
X 
X 
X 
X 
X 
Conjunctival hyperemia  
X 
X 
X 
X 
X 
X 
Biomicroscopy [e] 
X 
 
 
 
 
 
Study medication kit number assigned 
(randomization) 
X 
  
 
 
 
 
Mydriatic agent administration 
 
X 
 
 
 
 
Treatment: Nyxol/Placebo administration 
 
 
X 
 
 
 
AEs [c] 
X 
X 
X 
X 
X 
X 
AE, adverse event; BCDVA, best-corrected distance visual acuity; BP, blood pressure; ETDRS, Early Treatment Diabetic 
Retinopathy Study; HR, heart rate; PD, pupil diameter. 
[a] Screening Visit: if subject qualifies, this becomes Treatment Visit 1. The mydriatic agent will be given at -1 hour. 
[b] Start time refers to the time that first assessment is performed. Assessments at each time point are shown in the order performed.  
[c] Investigators to note changes to concomitant medications and AEs at any time throughout the visit. 
[d] BCDVA will be measured in photopic conditions by a standard ETDRS illuminated chart (on wall or stand) at 4 m (letters 
recorded). For subjects who are unable to read the letters on a ETDRS chart, the Patti Pics Series ETDRS chart will be used. The 
same method will be used consistently for each subject (when possible). 
[e] It is recommended that a table-top slit lamp be used if at all possible. The use of a portable slit lamp, or a hand-held light source 
should be reserved for subjects who cannot comply with the table-top slit lamp exam. The same method will be used consistently for 
each subject (when possible). 
 
 
 


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4.3. Measures taken to minimize/avoid bias 
This is a placebo-controlled, double-masked, 1:1 randomized (Nyxol or placebo) study. Nyxol 
vehicle is used as placebo. 
4.4. Study medications 
Study Medication Identification 
Established name 
Phentolamine mesylate – parent phentolamine 
CAS registry number 
65-28-1 – parent 50-60-2 
Chemical class 
An α-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, sterile, non-preserved, 
isotonic, ophthalmic solution  
Concentration active 
1% – parent 0.75% (free acid) 
Manufacturer drug substance 
Industriale Chimica s.r.l. (Italy) 
Manufacturer drug product, placebo Woodstock Sterile Solutions, Inc. 
Storage requirements 
Shipped and stored refrigerated (2°C to 8°C; 
36°F to 46°F). Stored at the site at room 
temperature (15ºC to 25ºC; 59ºF to 77ºF) in a 
secured location (locked) with no access for 
unauthorized personnel per ICH GCP 
Guidelines.  
Formulation 
Nyxol (0.75% POS) is a clear, colorless to slightly brown, sterile, non-preserved, isotonic, 
buffered aqueous solution containing 1% phentolamine mesylate (equivalent to 0.75% 
phentolamine free base), mannitol, and sodium acetate. Placebo for Nyxol is a clear, colorless, 
sterile, non-preserved, isotonic, buffered aqueous solution containing mannitol and sodium 
acetate. The pH of the study medications may be adjusted with NaOH (United States 
Pharmacopeia [USP]) and/or HCl (USP) to pH 4.8.  


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4.4.1. Packaging and labeling 
The investigational products, active and placebo, are packaged in a 0.5-mL low-density 
polyethylene Blow-Fill-Seal vial containing 0.3 mL solution for single-dose use. Five individual 
vials are included in a molded strip. Each strip containing 5 vials is wrapped with a multicolor 
“rainbow” aluminum foil overwrap that has been purged with nitrogen. The foil is 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 locked location with 
limited access documented by signature of authorized persons who may dispense investigational 
materials. The products are shipped refrigerated (2°C to 8°C; 36°F to 46°F) and stored at the site 
at room temperature (15ºC to 25ºC; 59ºF to 77ºF). Study medication must not be frozen and must 
be protected from light.  
4.4.3. Study medication administration 
Study medication will be administered by the Investigator or designee at the site on Treatment 
Visit 1. To administer the study medication, the foil pouch is opened and the strip of 5 vials 
removed from the pouch. A single vial is removed from the strip by twisting the vial, which 
exposes the dropper tip and contents of the vial. The vial should be removed immediately prior 
to dosing to protect the contents of the vial. After administration, all used (1 vial) and unused 
vials (4 vials [5 vials total]) should be returned to the labeled foil pouch and the original kit box 
and retained at the site, for proper accountability and reconciliation.  
4.4.4. Study medication accountability 
4.4.4.1. Receipt and disposition of study medication 
The Investigator or designee (eg, study coordinator or pharmacist) will 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 medication using the inventories supplied for 
the study. The Investigator or designee will account for all study medication. The monitor will 
review dispensing and study medication accountability records during study visits and at the 
completion of the study and note any discrepancies.  
4.4.4.2. Return of study medication 
When the study is completed or is terminated by Ocuphire, all study material, including used and 
unused study medication vials and strips in the original kits, will be returned to Ocuphire (or its 
designee) or destroyed under the specific direction of same. All study medication accounting 
procedures must be completed before the study is considered completed. A final study 
medication disposition will be completed by the study coordinator. 
4.5. Expected duration of subject participation 
The total length of subject participation is 2 days, with 2 clinic visits, as summarized below: 
• Screening/Treatment Visit (Visit 1/Day 1)  
• Follow-Up Visit (Visit 2/Day 2) 


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The execution of the entire study (first subject screened through last randomized subject 
completed) is expected to be approximately 2 to 4 months. 
4.6. Randomization and procedure for breaking the code 
A randomization code for allocating subjects to treatment will be prepared by an unmasked 
biostatistician not directly connected with the study. Subjects will be randomized in a 1:1 ratio to 
1 of 2 masked treatment arms (Nyxol or placebo). Treatment randomization will be stratified 1:1 
by subject age group (3‒5 years of age:6‒11 years of age). Mydriatic agent randomization will be 
3:1:1 (phenylephrine:tropicamide:Paremyd, respectively). Refer to Appendix 2. Note that the 
mydriatic agent administered is unmasked. 
At the initiation of study-related procedures, every potential subject will be assigned a subject 
identification number.  
The study medication will be masked to both Investigator and study subjects, as well as 
Ocuphire. Only in case of medical emergency or occurrence of SAEs will the 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. 
Subjects may be replaced.  
4.7. Collection of data 
The source documentation for all data collected in the study will maintained by the Investigator 
in the subject files at the study site. All original data collected during this trial is to be recorded 
on paper case report forms (CRFs) and then electronically entered into the database following 
study completion. The paper CRF is considered to be the source documentation for this study. 
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 data collected in the CRF against any 
applicable source documentation during remote review or scheduled monitoring visits. The study 
monitor will work closely with the site staff to address any discrepancies 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.. 
Study medication discontinuation 
The study medication may be discontinued for the following reasons: 
• Adverse events: Adverse events include clinically significant laboratory abnormalities 
and intercurrent diseases reported by the subject or observed by the Investigator with 
documentation on the CRF 
• Death: If a subject dies, the AE that caused the death should be documented on the CRF 
and be noted as serious and fatal 
• Disallowed concurrent medication: Any medication not allowed by the protocol will be 
a protocol violation 
• Lack of efficacy: A subject may elect to discontinue participation in the study for a 
perceived lack of efficacy 


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• 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: Any other reason for subject discontinuation should be noted on the CRF 
The reason for premature study medication discontinuation should be entered onto the 
appropriate CRF.  
4.7.1. Reasons for withdrawal from study 
• Subject withdraws consent 
• Subject is lost to follow-up 
• Subject withdraws for other reason 
• After being randomized, subject refuses mydriatic dosing 
• Subject receives mydriatic agent but refuses to continue with study drug dosing 
4.7.2. Subject replacement 
Subjects who withdraw from the study may be replaced in an attempt to have the planned 
number of subjects per age group. 
4.7.3.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. 
4.7.4. 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 noncompliant, every possible effort 
should be made by the Investigator to assure the 3-hour measurements are assessed prior to 
discontinuation, in addition to a follow-up telephone call that includes assessments for AEs, 
concomitant medications, and subject-evaluated conjunctival hyperemia.  
4.8. Completed study 
The study is completed when all randomized subjects have completed the study, all CRFs have 
been completed, and all CRF data have been 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.  
 
 


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5. SUBJECT INCLUSION AND EXCLUSION CRITERIA 
5.1. Subject inclusion criteria 
Included in the study will be individuals with the following characteristics: 
1. Males or premenstrual females 3 to 11 years of age  
2. Ability to comply with all protocol-mandated procedures independently and to attend all 
scheduled office visits  
3. Parent/Legal guardian willing to give written informed consent to participate in this 
study. Children aged 7 to 11 years to provide signed assent form, as well as a separate 
parental/Legal Guardian consent 
5.2. Subject exclusion criteria 
Excluded from the study will be individuals with the following characteristics: 
Ophthalmic (in either eye):  
1. Clinically significant ocular disease as deemed by the Investigator (eg, amblyopia, 
congenital cataract, congenital glaucoma) that might interfere with the study  
2. Unwilling or unable to discontinue use of contact lenses at screening until study 
completion 
3. Unwilling or unable to suspend use of topical medication at screening until study 
completion  
4. Ocular trauma or ocular surgery within the 6 months prior to screening  
5. Use of any topical prescription or over-the-counter (OTC) ophthalmic medications of any 
kind within 7 days of screening  
6. Recent or current evidence of ocular infection or inflammation in either eye (such as 
current evidence of clinically significant blepharitis, conjunctivitis, or keratitis). Subjects 
must be symptom free for at least 7 days prior to screening 
7. Closed or very narrow angle that in the Investigator’s opinion is potentially occludable if 
the subject’s pupil is dilated  
8. History of any traumatic (surgical or nonsurgical) or nontraumatic condition affecting the 
pupil or iris  
9. Known allergy, hypersensitivity, or contraindication to any component of the 
phentolamine ophthalmic solution or to any component of the mydriatic agents or vehicle 
formulation  
Systemic:  
10. Known hypersensitivity or contraindication to α- and/or β-adrenoceptor antagonists  
11. Clinically significant systemic disease (eg, uncontrolled diabetes, cancer, hepatic, renal, 
endocrine, or cardiovascular disorders) that might interfere with the study  
12.  Subjects with learning disabilities that in the opinion of the Investigator could interfere 
with the study 


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13. 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)  
14. Participation in any investigational study within 30 days prior to screening  
 
 


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6. TREATMENT OF SUBJECTS 
Approximately 20 to 30 healthy pediatric subjects 3 to 11 years of age, inclusive, will be 
randomized in a 1:1 ratio to 1 of 2 masked treatment arms (Nyxol or placebo), to achieve a target 
of 20 randomized subjects (ages 3‒5, n=10; ages 6‒11, n=10) who receive treatment with study 
medication. In this young study population, it is anticipated that some randomized subjects may 
withdraw (Section 4.7.1). Treatment randomization will be stratified 1:1 by subject age group 
(3‒5 years of age:6‒11 years of age). Mydriatic agent randomization will be 3:1:1 
(phenylephrine:tropicamide:Paremyd, respectively). Refer to Appendix 2 for stratification 
schema. Subjects will receive their mydriatic agent OU 1 hour before treatment with study 
medication on the Treatment Visit (Visit 1/Day 1). There will be no treatment administered on 
the Follow-Up Visit (Visit 2/Day 2). 
The study eye (OD) will always be treated and assessed first. If the subject is not amenable to 
receiving treatment in the fellow eye (OS), the subject can still participate in the study and only 
the study eye will undergo study assessments.  
6.1. Treatment adherence 
All subjects will be treated by the Investigator or designee at the study clinic on Visit 1.  
6.2. Concomitant medications 
As noted in the exclusion criteria (Section 5.2), use of any topical prescription or OTC 
ophthalmic medications of any kind within 7 days of screening is prohibited.  
Additionally, initiation of treatment with or any changes to the current dosage, drug, or regimen 
of any systemic adrenergic or cholinergic drugs (Appendix 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 should be documented on the appropriate CRF. 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 CRF. The name of the drug, dose, route of administration, duration 
of treatment, and indication will be recorded for each medication. For combination products (eg, 
Contac®, Cosopt®), the brand name is required. For non-combination products, the generic name 
is desired. The use of routine ophthalmic diagnostic pharmaceutical agents (eg, fluorescein and 
local anesthetic) will be allowed and should be documented. Any change in dosing parameters 
should also be recorded in the CRF. 
 


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7. ASSESSMENTS OF SAFETY AND EFFICACY  
7.1. Specification of safety parameters 
Safety and efficacy endpoints are detailed in Section 4.1.  
The assessment of safety and tolerability will be performed by: 
• Conjunctival hyperemia measured with CCLRU images using a 4-point scale (0-3) 
(Appendix 1). 
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  
• Best-corrected distance visual acuity will be measured in photopic conditions by a 
standard ETDRS illuminated chart (on wall or stand) at 4 m (letters recorded)  
• Heart rate and BP (as per the site’s normal equipment and procedures) 
• Adverse events (refer to Section 7.3 for AE and SAE assessment) 
The efficacy assessment, PD, will be measured as follows: 
• Pupil diameter and pupil reactivity will be measured with the NeurOptics VIP-300 
pupillometer (mm) or other similar measurement  
7.2. Assessing, recording, and analyzing safety and efficacy parameters 
The photopic lighting conditions in the room will be defined as lights off with ambient light 
coming from the distance ETDRS illuminated chart (on wall or stand) 4 m away at a luminance 
level of 85 cd/m2 (or 85 lux). Subjects will be allowed to acclimate to these lighting conditions 
(with the eyes open normally for a minimum of 2 minutes) prior to the PD and BCDVA 
measurements at all scheduled time points. Subjects will sit in the exam chair facing the 
illuminated chart during the acclimation period and for all assessments. Room lights will be off 
with the distance ETDRS chart illuminated. Room lights should be turned on for the remaining 
safety assessments (eg, conjunctival hyperemia, AEs, and BP/HR). 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 time points. 
7.2.1.Screening Visit (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 Treatment Visit, when the 
dose of study treatment is given.  
Once subjects arrive 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. 


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Children aged 7 to 11 years must provide assent. Screening includes an explanation of the study, 
a medical and ophthalmic history, demographics, a review of prior/concomitant medications, and 
HR/BP assessment. The second step in screening includes an ophthalmic examination consisting 
of biomicroscopy and external examinations.  
Investigators are cautioned to appropriately note all observations of conjunctival hyperemia (also 
called conjunctival erythema) on the biomicroscopy CRF at screening. 
7.2.2. Treatment Visit (Visit 1/Day 1) 
Treatment Visit should be the same day as the Screening Visit. Once the subject has completed 
the screening assessments part of the visit and it is confirmed that he/she meets all of the 
inclusion criteria but none of the exclusion criteria, the visit will then transition to the Treatment 
Visit assessments. The study eye is OD and the fellow eye is OS. The study eye (OD) will 
always be treated before the fellow eye (OS). If the subject is not amenable to receiving 
treatment in the fellow eye (OS), the subject can still participate in the study and only the study 
eye will undergo study assessments.  
As part of the Treatment Visit, the subject will:  
• Be randomized to 1 of 2 treatment arms, with further randomization by mydriatic agent and 
stratification by age group 
• Receive 1 of 3 approved mydriatic agents (2.5% phenylephrine, 1% tropicamide, or Paremyd) 
at -1 hour (baseline). The mydriatic agent will be administered as a single drop in each eye. If a 
drop of mydriatic agent is missed, the Investigator should give the drop again. Note: Mydriatic 
agent is administered after assessments conducted at -1 hour (baseline)  
• Receive the masked study medication (Nyxol or placebo) based on his/her randomized 
treatment arm (kit assignment) at 0 minutes (max pupil dilation). Note: Study medication is 
administered after assessments conducted at 0 minutes (max pupil dilation). Study medication 
will be administered OU 1 hour after mydriatic drug instillation. Subjects will receive 1 drop in 
the study eye (OD) and 1 drop in the fellow eye (OS). If a drop of study medication is missed, 
the Investigator should give the drop again. The study eye (OD) will always be treated and 
assessed first. If the subject is not amenable to receiving treatment in the fellow eye (OS), the 
subject can still participate in the study and only the study eye (OD) will undergo study 
assessments  
• Be assessed at -1 hour (baseline), 0 minutes (max pupil dilation), 90 minutes, and 3 hours 
relative to study treatment for the following: PD (OD, OS), BCDVA (OD, OS), and 
conjunctival hyperemia (OD, OS), as well as concomitant medications and any AEs 
• Be assessed at -3 hours, relative to study treatment for HR/BP 
7.2.3.Follow-Up Visit/Day 2  
On Day 2, the subject will return to the clinic for the Follow-Up Visit.  
As part of the Follow-Up Visit, the subject will be assessed at 24 hours + 6 hours relative to 
study treatment administration during the Treatment Visit for the following: concomitant 
medications, HR/BP, PD, BCDVA, conjunctival hyperemia, and AEs. 


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7.2.4. Unscheduled Visits 
An Unscheduled Visit may be any visit to the Investigator other than the specific visits requested 
in the protocol as possibly required for the subject’s ophthalmic condition. The Investigator will 
perform all procedures necessary to evaluate the subject at these visits and record any AEs in the 
CRF. 
As noted in Section 4.7.4, every possible effort should be made by Investigators to assure that 
subjects who discontinue early from the study have a telephone follow-up that includes 
assessments of AEs, concomitant medications, and subject-evaluated conjunctival hyperemia. 
7.2.5. Visit Variation 
Visits on Day 2 may occur between 24 to 30 hours after the baseline (-1 hour time point on 
Treatment Day 1). 
7.3. 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 CRF page. Only treatment-emergent adverse events 
(TEAEs)/adverse reactions will be summarized (Section 8.3.5). 
All AEs/adverse reactions occurring during the study (ie, once the subject has signed the 
informed consent/assent) must be documented, regardless of the assumption of causal 
relationship, on the respective CRF. 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 TEAEs/adverse reactions at the time of study 
completion or discontinuation from the study, the ongoing TEAEs/adverse reactions are to be 
followed and provided appropriate medical care until the signs and symptoms have remitted or 
stabilized or until medical assessments have returned to acceptable or pre-study limits.  
Documentation of AEs/adverse reactions includes start date and end date, severity, relationship 
to study medications, action(s) taken, seriousness, and outcome.  
7.3.1. 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 
temporally 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 


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question: “Have you had any problems since your last assessment/visit?”. Adverse events also 
may be detected when they are volunteered by the subject during or between visits or through 
study assessments. 
Life-threatening adverse event or life-threatening suspected adverse reaction. An AE or 
suspected adverse reaction is considered “life-threatening” if, in the view of either the 
Investigator or Ocuphire, its occurrence places the subject at immediate risk of death. It does not 
include an AE or suspected adverse reaction that, had it occurred in a more severe form, might 
have caused death.  
Serious adverse event or serious suspected adverse reaction. An AE or suspected adverse 
reaction is considered ‘‘serious’’ if, in the view of either the Investigator or Ocuphire, it results 
in any of the following outcomes:  
• Death 
• Life-threatening AE 
• Inpatient hospitalization or prolongation of existing hospitalization 
• Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions  
• Congenital anomaly/birth defect  
• Other medically important serious event 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the patient or 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 Application 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. 


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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”.  
If the relationship between the AE/SAE and the study medication is determined by Ocuphire to 
be anything other than “Not related” or “Unlikely related” the event will be considered to be 
related to the study medication 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 


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• Dose interrupted: An indication that a medication schedule was modified by temporarily 
terminating a prescribed regimen of medication 
• Drug withdrawn: An indication that a medication schedule was modified through 
termination of a prescribed regimen of medication 
• Not applicable: Determination of a value is not relevant in the current context 
• Unknown: Not known, not observed, not recorded, or refused 
Additional other action taken: 
• Concomitant medication 
• Hospitalization 
Outcome of an AE is coded as: 
• Fatal: The termination of life as a result of an AE 
• Not recovered/not resolved: One of the possible results of an AE outcome that indicates 
that the event has not improved or recuperated 
• Recovered/resolved: One of the possible results of an AE outcome that indicates that the 
event has improved or 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. 
Investigators are cautioned to use the appropriate verbatim term on the AE form to 
describe this observation:  
• Redness related to instillation that is transient (ie, is no longer present 2 hours after 
instillation) = “conjunctival erythema upon instillation” 
• Redness that is NOT transient (ie, 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 (Visit 2/Day 2). 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 clinical research organization (CRO) within 
24 hours of its occurrence or within 24 hours of learning of its occurrence. Recurrent episodes, 
complications, or progression of the initial SAE must be reported to the CRO as follow-up to the 
original episode within 24 hours of the Investigator receiving the information. Information about 


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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 MIRA4_Safety@Oculoscr.com. The Investigator must assess the 
SAE relationship and complete the SAE form. The CRO may request additional information. 
Follow-up information (eg, discharge summary) will be retained in the subject’s chart and a copy 
will be emailed to MIRA4_Safety@Oculoscr.com. In addition, all SAEs should be recorded on 
the AE CRF 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, not drug related, 
expected, or unexpected (based on the available information in the IB). 
Any death occurring during the study or 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 Visit, that is not reasonably associated 
with study medication administration, does not require completion of the SAE form. 
7.3.2. 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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8. STATISTICS 
A detailed presentation of the statistical approach is outlined in the Statistical Analysis Plan 
(SAP). 
8.1. Sample size 
A sample size of approximately 20 subjects (approximately 10 treated with Nyxol) in this study 
will result in a total of > 300 subjects treated with Nyxol in the reversal of mydriasis program 
(including prior studies); this number of subjects is needed to meet the minimum number of 
subjects exposed to Nyxol to assess safety in this population as specified in the Pediatric 
Research Equity Act (PREA) plan. This safety study is not powered for efficacy. In accordance 
with the Sponsor Pediatric Safety Plan for the indication of reversal of pharmacologically 
induced mydriasis, the sample size of 20 subjects divided between 2 age groups is adequate to 
evaluate safety in the 3- to 11-year-old pediatric population. 
All subjects will be randomized into the study in a 1:1 ratio to 1 of the 2 treatment arms (Nyxol 
or placebo), with a 1:1 stratification by subject age group (3‒5 years of age:6‒11 years of age). 
Furthermore, subjects will be randomized into the study at a ratio of 3:1:1 to mydriatic agent 
(phenylephrine:tropicamide:Paremyd, respectively). Therefore, if 12 subjects are randomized to 
2.5% phenylephrine, then 4 subjects will be assigned to 1% tropicamide, and 4 subjects will be 
assigned to Paremyd, resulting in 20 total subjects (Appendix 2).  
8.2. Analysis populations 
Modified Intent-to-Treat Population: The mITT Population will include all randomized 
subjects who received 1 drop of study treatment in the study eye and completed at least 
1 scheduled post-treatment PD measurement during Visit 1 in the study eye. The mITT 
Population will be used to analyze selected efficacy endpoints, with subjects included in their 
randomized treatment regardless of the treatment they actually received. 
Per Protocol Population: The PP Population includes all subjects in the mITT Population who 
had 1 drop of study treatment in the study eye, had all scheduled PD measurements during the 
Treatment Visit in the study eye, had an increase of > 0.2 mm in PD in the study eye at 0 minutes 
compared to baseline (-1 hour), and had no major protocol deviations considered to have 
significant impact on treatment outcome. The PP Population will be used to analyze selected 
efficacy endpoints, with subjects included in their randomized treatment regardless of the 
treatment they actually received.  
All Randomized Population (ARP): The ARP will include all randomized subjects. This 
population is also known as the Intent-to-Treat Population. The ARP will be used in 
confirmatory efficacy analyses, with subjects included in their randomized treatment regardless 
of the treatment they actually received. 
Safety Population (SP): The SP will include all randomized subjects who received at least 
1 drop of study treatment. The SP will be used to summarize safety variables, using the treatment 
they actually received. 


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8.3. Statistical methods  
8.3.1. General considerations 
All continuous variables will be summarized by treatment and time point (as applicable) using 
descriptive statistics (n, mean, median, standard deviation, minimum, and maximum). All 
categorical variables will be summarized by treatment and time point (as applicable) using 
frequency counts and percentages.  
All study data will be listed by treatment, subject, and time point (as applicable). 
All statistical tests will be performed using a significance level of 5% (two-tailed). The p-values 
for the analysis of efficacy endpoints will be considered descriptive. 
8.3.2. Demographic and baseline characteristics 
Demographic and baseline characteristics such as age group, race, and sex will be summarized 
by treatment group using the mITT Population, PP Population, SP, and the ARP. These data will 
also be provided in by-subject listings. 
8.3.3. Subject disposition 
Subject disposition, including randomization, completion, and withdrawal from the study, will be 
summarized using the ARP. These data will also be provided in by-subject listings. 
8.3.4. 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 WHODrug and will be summarized by treatment group using the SP. 
Medical history and prior and concomitant medications will also be provided in by-subject 
listings.  
8.3.5. 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. 
Safety endpoints are indicated in Section 4.1.  
For HR and BP, baseline is defined as the screening value. Heart rate and BP values and change 
from baseline in the values will be summarized by treatment group and time point (screening, 
3 hours, and 24 hours).  
Observed values and change from baseline (-1 hour) in conjunctival hyperemia at each time 
point (0 minutes, 90 minutes, 3 hours, and 24 hours) will be summarized separately for the study 
eye and the fellow eye. Treatments will be compared using the same analysis of covariance 
(ANCOVA) model proposed for the continuous efficacy endpoints. Conjunctival hyperemia will 
also be summarized categorically. 


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The visual acuity assessments (BCDVA) will be summarized for the study eye and fellow eye at 
time points 0 minutes, 90 minutes, 3 hours, and 24 hours using the number of letters correctly 
identified. Letters will be recorded and may later be converted to logMAR by programming for 
such analyses. One letter is equivalent to 0.02 logMAR. As a reference, 5 letters are represented 
by 1 line. Treatments will be compared using the same ANCOVA model proposed for the 
continuous efficacy endpoints. 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. Treatment-emergent AEs 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 be 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. Safety tables and listings for pediatric 
subjects may be provided. 
8.3.6.Analysis of efficacy 
Efficacy will be assessed using the mITT and PP Populations, with subjects included in the 
treatment arm into which they were randomized. Confirmatory analyses may be performed using 
the ARP. Generally, for the analysis of the efficacy endpoints, only observed case data will be 
used.  
For all efficacy endpoints, baseline is defined as -1 hour prior to treatment on the Treatment Visit 
(Visit 1/Day 1). Max pupil dilation is defined as 0 minutes, during which maximum PD is 
expected.  
All efficacy data will be summarized by treatment group, study day, and time point (-1 hour 
[baseline], 0 minutes, 90 minutes, 3 hours, and 24 hours), as appropriate. 
The returning to ≤ 0.2 mm from baseline (-1 hour) photopic PD endpoint will be analyzed 
separately for the study eye and fellow eye at each time point using a logistic regression model 
with treatment, mydriatic agent, and age group (3‒5 years or 6‒11 years) as factors and the 
baseline PD as a covariate. The percentage of subjects in each treatment group meeting the 
criteria and the odds ratio (OR) with 95% confidence interval (CI) and p-value will be provided. 
The analysis will be performed using the mITT and PP Populations, with subjects included in 
their randomized treatment regardless of the treatment they actually received. Subjects who did 
not have an increase of > 0.2 mm in PD in the study eye or fellow eye at the max pupil dilation 
(0 minutes) will not be counted as returning to ≤ 0.2 mm from baseline PD for that eye. 
A sensitivity analysis of the returning to ≤ 0.2 mm from baseline (-1 hour) photopic PD endpoint 
will be completed using a logistic regression model with treatment as a factor and the baseline 
PD as a covariate; the mydriatic agent and age group are excluded from this model. 
The continuous change (in mm) in photopic PD from max pupil dilation (0 minutes) endpoint 
will be analyzed separately for the study eye and fellow eye at each time point using ANCOVA, 
with change from baseline as the dependent variable, treatment, mydriatic agent, and age group 
as factors, and the baseline PD as the covariate. The ANCOVA will be performed using the 
mITT or PP Populations (as specified in the SAP), with subjects included in their randomized 


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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.  
The analysis of the time (hours) to return to ≤ 0.2 mm from baseline PD (time-savings analysis) 
endpoint will be performed separately for the study eye and fellow eye using a Cox proportional 
hazards regression model, with treatment, mydriatic agent, and age group as factors, and the 
baseline PD as a covariate. 
In addition, efficacy endpoints will be summarized by age group and by mydriatic agent but will 
not be analyzed due to the small sample size for each subgroup. Each mydriatic agent will be 
summarized individually, and an additional summary combining 1% tropicamide and Paremyd 
subjects into a “tropicamide or Paremyd” group will be presented. Other subgroups, such as 
light/dark irides, sex, and race, may be summarized as well.  
8.4.  Procedure for accounting for missing, unused, or spurious data 
Imputation will be performed for missing data in limited circumstances as described in the SAP. 
For the summarization and analysis of efficacy endpoints and safety data, observed case data 
generally will be used. 
8.5. Procedure for reporting deviations from the statistical plan 
Any deviations from the SAP will be described and a justification given in the final Clinical 
Study Report.  
 
 


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9. 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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10. QUALITY CONTROL AND QUALITY ASSURANCE 
The progress of the study will be monitored by on-site, 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 CRFs, 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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11. ETHICAL CONSIDERATIONS AND GOOD CLINICAL PRACTICE 
COMPLIANCE  
11.1. Good Clinical Practice 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, ICH GCP Guidelines, obtaining 
prospective informed consent, monitoring of the conduct of the study and the completeness of 
the CRFs by Ocuphire or its designee(s), and appropriate record retention by the Investigator.  
11.2. 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. 
11.3. 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 (eg, pupillometer), and/or use 
of a prohibited medication during the study. However, treatment in study eye only and missed 
assessments as outlined in this protocol will not be considered protocol deviations. 
All protocol deviations will be reported by entering the event in the appropriate CRF 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 its 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. 
11.4.  Informed consent and assent requirements 
Written informed consent will be obtained from the legal guardian of each subject. A signed 
assent form will be obtained for all minors ages 7 to 11, as well as a separate parental/Legal 
Guardian consent. A copy of the signed and dated consent/assent document will be given to each 
subject or parent guardian. The 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 


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give written consent/assent after the receipt of detailed information. The verbal explanation will 
cover all the elements specified in the written information provided to the subject. 
It should be emphasized that the subject and legal guardian are 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 on their subsequent care.  
The Investigator will inform the subject of the aims, methods, anticipated benefits, and potential 
hazards of the study, including any discomfort it may entail. The subject must be given every 
opportunity to clarify any points he/she does not understand and, if necessary, ask for more 
information. At the end of the interview, the subject may be given time to reflect if this is 
required, or if the subject requests more time. Subjects 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/legal 
guardian. 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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12. 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. 
12.1.  Data entry 
Study-specific data that have been outlined in the protocol will be entered into the clinical 
database by the Data Manager.  
12.2. Data quality control and reporting 
After data have been entered into the study database, a system of computerized data validation 
checks will be implemented and applied to the database. Query reports pertaining to data 
omissions and discrepancies will be forwarded to the Clinical Investigator and Ocuphire, or its 
designee, for resolution. The study database will be updated in accordance with the resolved 
query reports. All changes to the study database will be documented.  
12.3. 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. 
12.4.  Records retention 
The Investigator’s site and clinical laboratory will retain all records related to the study in 
compliance with ICH GCP Guidelines. 
12.5.  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 (eg, change in monitor, change of 
telephone number). 
Protocol amendments will be submitted to the appropriate authority(ies) as required by the 
applicable regulatory requirement(s). 
 
 


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13. BIBLIOGRAPHY 
1. 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. 
2. Holladay J, Meyer A, Pitlick B. Phentolamine mesylate ophthalmic solution 1% once daily 
reduces pupil diameter and improves night vision disturbances. American Academy of 
Ophthalmology 2018 annual meeting. Presented at Chicago, IL; October 27-30, 2018. 
3. Karpecki PM, Foster SA, Montaquila SM, et al. Phentolamine eye drops reverse 
pharmacologically induced mydriasis in a randomized Phase 2b trial. Optom Vis Sci. 
2021;98(3):234‒242.  
4. McDonald M. Phentolamine mesylate treatment of severe night vision complaints. American 
Society of Cataract and Refractive Surgery 2011 annual meeting. Presented at San Diego, 
CA; March 25-29, 2011. 
5. OP-NYX-01a2 CSR. Ocuphire Pharma, Inc. 24 May 2013. 
6. OP-NYX-SNV CSR. Ocuphire Pharma, Inc. 07 February 2008. 
7. OPI-NYXG-201 CSR. Ocuphire Pharma, Inc. 23 April 2020. 
8. OPI-NYXRM-201 CSR. Ocuphire Pharma, Inc. 25 April 2020. 
9. Pepose JS, Hartman PJ, DuBiner HB, et al. Phentolamine mesylate ophthalmic solution 
provides lasting pupil modulation and improves near visual acuity in presbyopic glaucoma 
patients in a randomized Phase 2b clinical trial. Clin Ophthalmol. 2021;15:79–91. 
10. Pepose JS, Sooch MP, Charizanis K, et al. Phase 3 clinical trial to evaluate the efficacy of 
phentolamine ophthalmic solution on the reversal of pharmacologically induced mydriasis. 
ASCRS ASOA 2021 annual meeting. Presented at Las Vegas, NV; July 23-27, 2021.  
11. Pepose JS, Sooch MP, Charizanis K, et al. Phase 2 clinical trial to evaluate the efficacy of 
phentolamine ophthalmic solution and low-dose pilocarpine for the treatment of presbyopia. 
ASCRS ASOA 2021 annual meeting. Presented at Las Vegas, NV; July 23-27, 2021.  
12. 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. 
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: CONJUNCTIVAL HYPEREMIA GRADING SCALE USING IMAGES 
FROM CCLRU 
 
None (0) 
Mild (+1) 
Moderate (+2) 
Severe (+3) 
Normal; appears 
white with a small 
number of 
conjunctival blood 
vessel easily 
observed 
Prominent, pinkish-
red color of both the 
bulbar and palpebral 
conjunctiva 
Bright, scarlet red 
color of the bulbar 
and palpebral 
conjunctiva 
Beefy red with 
petechiae, dark red 
bulbar and palpebral 
conjunctiva with 
evidence of 
subconjunctival 
hemorrhage  
 
 
 


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APPENDIX 2: RANDOMIZATION SCHEMA BY AGE GROUP, MYDRIATIC AGENT 
AND INVESTIGATIONAL TREATMENT 
All subjects 
1:1  
age group 
3:1:1  
mydriatic agent 
1:1 
treatment 
20 
10  
3-5 years of 
age 
6 Phenylephrine 
3 Nyxol 
3 Placebo 
2 Tropicamide 
1 Nyxol 
1 Placebo 
2 Paremyd 
1 Nyxol 
1 Placebo 
10  
6-11 years of 
age 
6 Phenylephrine 
3 Nyxol 
3 Placebo 
2 Tropicamide 
1 Nyxol 
1 Placebo 
2 Paremyd 
1 Nyxol 
1 Placebo 
 
 
 


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APPENDIX 3: PATTI PICS SERIES ETDRS CHART 
The Patti Pic Series ETDRS Chart is to be used by subjects who are unable to read the letters on 
a standard ETDRS 
charxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxt 
. 
 
 
 


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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 
 
 
 
 
 
 
 

SAP_001.pdf: 
  
 
CONFIDENTIAL 
 
 
 
 
Ocuphire Pharma, Inc. 
 
STATISTICAL ANALYSIS PLAN 
 
 
 
Protocol Title: 
Randomized, Parallel-Arm, Double-Masked, Placebo-Controlled 
Study of the Safety and Efficacy of Nyxol (0.75% Phentolamine 
Ophthalmic Solution) to Reverse Pharmacologically Induced 
Mydriasis in Healthy Pediatric Subjects 
Study Number: 
OPI-NYXRMP-303 (MIRA-4) 
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: 
2022-04-13 
Status: 
FINAL V1.0 
 
 
 


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1. 
TABLE OF CONTENTS 
1. 
TABLE OF CONTENTS ............................................................................................................................... 2 
2. 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS................................................................... 5 
3. 
INTRODUCTION ......................................................................................................................................... 7 
3.1. 
Preface .............................................................................................................................................. 7 
3.2. 
Purpose of Analyses .......................................................................................................................... 7 
3.3. 
Summary of Statistical Analysis Changes to the Protocol ................................................................ 7 
4. 
STUDY OBJECTIVES AND ENDPOINTS ................................................................................................. 8 
4.1. 
Study Objectives ............................................................................................................................... 8 
4.2. 
Study Endpoints ................................................................................................................................ 8 
5. 
STUDY METHODS .................................................................................................................................... 10 
5.1. 
General Study Design and Plan....................................................................................................... 10 
5.2. 
Inclusion – Exclusion Criteria and General Study Population ........................................................ 12 
5.3. 
Randomization and Blinding........................................................................................................... 12 
5.4. 
Analysis Variables .......................................................................................................................... 12 
6. 
SAMPLE SIZE ............................................................................................................................................ 14 
7. 
GENERAL CONSIDERATIONS ............................................................................................................... 15 
7.1. 
Analysis Populations ....................................................................................................................... 15 
7.1.1. 
Modified Intention-to-Treat (mITT) ............................................................................. 15 
7.1.2. 
Per Protocol Population (PP) ......................................................................................... 15 
7.1.3. 
All Randomized Population (ARP) ............................................................................... 15 
7.1.4. 
Safety Population (SP) .................................................................................................. 15 
7.2. 
Covariates and Subgroups ............................................................................................................... 15 
7.2.1. 
Planned Covariates ........................................................................................................ 15 
7.2.2. 
Planned Subgroups ........................................................................................................ 16 
7.3. 
Management of Analysis Data ........................................................................................................ 16 
7.3.1. 
Data Handling ............................................................................................................... 16 
7.3.2. 
Missing Data ................................................................................................................. 16 
7.3.2.1. 
Handling of Missing Date Values ............................................................ 16 
7.3.2.2. 
Missing Baseline Data ............................................................................. 17 
7.3.3. 
Handling of Early Termination Visit Information ......................................................... 17 
7.3.4. 
Pooling of Investigational Sites ..................................................................................... 17 
7.3.5. 
Coding Conventions for Events and Medications ......................................................... 17 
7.3.6. 
Analysis Software ......................................................................................................... 17 
7.3.7. 
Study Data ..................................................................................................................... 18 
7.4. 
Planned Study Analyses .................................................................................................................. 19 
7.4.1. 
Statistical Summaries: Descriptive and Inferential ....................................................... 19 
7.4.2. 
Interim Analyses and Data Monitoring ......................................................................... 19 
7.4.3. 
Final Analysis and Publication of Study Results .......................................................... 19 
7.5. 
Multiple Testing Procedures ........................................................................................................... 19 
7.6. 
Baseline Values ............................................................................................................................... 19 
8. 
SUMMARY OF STUDY DATA ................................................................................................................ 20 
8.1. 
Subject Disposition ......................................................................................................................... 20 
8.2. 
Protocol Deviations ......................................................................................................................... 20 
8.3. 
Demographics and Baseline Characteristics ................................................................................... 20 
8.4. 
Medical History .............................................................................................................................. 21 


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8.5. 
Prior and Concurrent Medications .................................................................................................. 21 
8.6. 
Treatment Administration ............................................................................................................... 22 
9. 
EFFICACY ANALYSES ............................................................................................................................ 23 
9.1. 
Clinical Efficacy ............................................................................................................................. 23 
10. SAFETY ANALYSES................................................................................................................................. 25 
10.1. 
Adverse Events ............................................................................................................................... 25 
10.2. 
Deaths, Serious Adverse Events and Other Significant Adverse Events ........................................ 26 
10.2.1. 
Deaths............................................................................................................................ 26 
10.2.2. 
Serious Adverse Events ................................................................................................. 26 
10.2.3. 
Adverse Events Leading to Withdrawal from the Study ............................................... 26 
10.2.4. 
Adverse Events Leading to Discontinuation of Study Medication ............................... 26 
10.3. 
Conjunctival Hyperemia ................................................................................................................. 26 
10.4. 
Visual Acuity .................................................................................................................................. 26 
10.5. 
Vital Signs ....................................................................................................................................... 27 
10.6. 
Other Safety Measures .................................................................................................................... 27 
11. REFERENCES ............................................................................................................................................ 28 
12. APPENDICES ............................................................................................................................................. 29 
12.1. 
List of Planned Tables ..................................................................................................................... 29 
12.2. 
List of Planned Listings .................................................................................................................. 31 
12.3. 
Hierarchy of Sequential Testing ...................................................................................................... 33 
 
 


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LIST OF TABLES 
 
Table 1 
Screening and Mydriatic/Treatment Schedule .............................................................. 11 
 
 
LIST OF FIGURES 
 
Figure 1 
SDTM, ADaM, and TFL Development and Validation ................................................ 18 
 
 
 


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2. 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Only abbreviations and terms relevant to the SAP are repeated herein. The reader is referred 
to the protocol for the complete and comprehensive list of abbreviations and definitions of 
terms for the study. 
Abbreviation/Term 
Definition 
ADaM 
Analysis Data Model 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ARP 
All Randomized Population 
ATC 
Anatomical Therapeutic Chemical 
BCDVA 
best-corrected distance visual acuity  
BP 
blood pressure 
CCLRU 
Cornea and Contact Lens Research Unit 
CDISC 
Clinical Data Interchange Standards Consortium 
CI 
confidence interval 
CRF 
case report form 
CSR 
clinical study report 
DD 
Drug Dictionary 
HR 
heart rate 
ITT 
Intention-to-treat 
logMAR  
logarithm of the minimum angle of resolution 
LSM 
least squares mean 
max 
maximum 
MedDRA 
Medical Dictionary for Regulatory Activities 
mITT 
Modified intention-to-treat 
Nyxol 
Phentolamine Mesylate Ophthalmic Solution 1% (Nyxol®) 
OD 
right eye 
OR 
odds ratio 
OS 
left eye 
PD 
pupil diameter 
PP 
Per Protocol 
PREA 
Pediatric Research Equity Act 
PT 
preferred term 
SAE 
serious averse event 
SAP 
Statistical Analysis Plan 


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Abbreviation/Term 
Definition 
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-NYXRMP-303 (MIRA-4) (Randomized, Parallel-Arm, Double-Masked, Placebo-
Controlled Study of the Safety and Efficacy of Nyxol [0.75% Phentolamine Ophthalmic 
Solution] to Reverse Pharmacologically Induced Mydriasis in Healthy Pediatric Subjects). 
Reference materials for this statistical plan include the protocol OPI-NYXRMP-303 
(26OCT2021) and Case Report Forms (CRFs; Version 03DEC2021).  
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 MIRA-4 study is a randomized, parallel arm, double-masked, placebo-controlled study 
of the safety and efficacy of Nyxol (0.75% Phentolamine Ophthalmic Solution) to reverse 
pharmacologically-induced mydriasis in healthy pediatric subjects. 
The Sponsor intends to use this study to evaluate Nyxol in pediatric subjects aged 3 to 11 for 
the indication “the treatment of pharmacologically induced mydriasis produced by adrenergic 
(phenylephrine) or parasympatholytic (tropicamide) agents, or a combination thereof.” 
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 CSR. Additional analyses not prospectively identified in this SAP may also be 
completed for publications, or regulatory or funding inquiries. These analyses, if performed, 
may not be reported in the CSR but will be fully detailed in the document containing the 
additional analyses. 
3.3. 
Summary of Statistical Analysis Changes to the Protocol 
The efficacy analyses described in the protocol included several factors in the modeling, 
including mydriatic agent and age group. These factors have been removed from the efficacy 
analysis due to small sample sizes. As a result, the sensitivity analysis, which is an analysis 
of the primary endpoint without mydriatic agent and age group in the model, is no longer 
required as a separate analysis. Also, a hierarchical analysis was added to formally test a 
family of efficacy endpoints. Otherwise, the analyses described in this analysis plan are 
consistent with the analyses described in the study protocol. 


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4. 
STUDY OBJECTIVES AND ENDPOINTS 
Study objectives and endpoints defined in the protocol include safety and efficacy endpoints. 
Objectives and pre-specified endpoints are as follows: 
4.1. 
Study Objectives 
The objectives of this study are as follows: 
• To evaluate the safety of Nyxol in pediatric subjects 
• To evaluate the efficacy of Nyxol to expedite the reversal of pharmacologically 
induced mydriasis in pediatric subjects  
4.2. 
Study Endpoints 
Efficacy endpoints will be analyzed by study eye and fellow eye and will include: 
• Percentage of subjects returning to ≤ 0.2 mm from baseline (-1 hour) photopic pupil 
diameter (PD) at each remaining time point (0 minutes, 90 minutes, 3 hours, and 24 
hours) 
• Change (in mm) in photopic PD from max pupil dilation (0 minutes) at each time 
point (90 minutes, 3 hours, and 24 hours) 
• Time (hours) to return to ≤ 0.2 mm from baseline (-1 hour) photopic PD (time-
savings analysis)  
Efficacy measurements: 
• Pupil diameter will be measured with a NeurOptics VIP-300 pupillometer (mm) or 
other similar measurement 
Safety endpoints will include: 
• Change from baseline (-1 hour) in conjunctival hyperemia grading (Cornea and 
Contact Lens Research Unit [CCLRU] images) at each time point (0 minutes, 
90 minutes, 3 hours, and 24 hours) for the study eye and fellow eye  
• 
Change from baseline (-1 hour) in BCDVA at 0 minutes, 90 minutes, 3 hours, and 
24 hours for the study eye and fellow eye  
• 
Change from baseline (-1 hour) in vital signs (HR and BP) at 3 hours and 24 hours  
Safety measurements: 
• Conjunctival hyperemia will be assessed visually with a 4-point grading scale (0-3) 
using CCLRU images (Appendix 1 of the protocol)  


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• Best-corrected distance visual acuity will be measured in photopic conditions by a 
standard Early Treatment Diabetic Retinopathy Study (ETDRS) illuminated chart (on 
wall or stand) at 4 m (letters recorded). For subjects who are unable to read the letters 
on a standard ETDRS chart, the Patti Pics Series ETDRS chart will be used 
(Appendix 3 of the protocol)  
 


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5. 
STUDY METHODS 
5.1. 
General Study Design and Plan 
This is a randomized, parallel-arm, double-masked, placebo-controlled study in 
approximately 20 to 30 randomized pediatric subjects evaluating the safety and efficacy of 
Nyxol in pediatric subjects with pharmacologically induced mydriasis. Pediatric subjects will 
be recruited for the study into 2 age groups as follows:  
• 3 to 5 years of age: 10 subjects 
• 6 to 11 years of age: 10 subjects 
In this young study population, it is anticipated that some randomized subjects may 
withdraw. 
Following the successful completion of screening, each subject will be randomized to 
unmasked mydriatic agent and masked treatment. Treatment randomization will be 1:1 
(Nyxol or placebo [vehicle]) and will be stratified 1:1 by subject age group (3‒5 years of age: 
6‒11 years of age). Mydriatic agent randomization will be 3:1:1 (phenylephrine: tropicamide: 
Paremyd, respectively). Approximately 60% of the randomized subjects will receive 1 drop 
of 2.5% phenylephrine in both eyes (OU) 1 hour before treatment (12 subjects), 
approximately 20% will receive 1 drop of 1% tropicamide OU 1 hour before treatment (4 
subjects), and approximately 20% will receive 1 drop of Paremyd OU 1 hour before 
treatment (4 subjects).  
Subjects will have 1 drop of masked study treatment (Nyxol or placebo) administered in the 
study eye (right eye [OD]) and 1 drop administered in the fellow eye (left eye [OS]). The 
study eye and the fellow eye will both be evaluated at all assessments unless otherwise 
specified.  
The study eye (OD) will always be treated and assessed first. If the subject is not amenable to 
receiving treatment in the fellow eye (OS), the subject can still participate in the study and 
only the study eye will undergo study assessments. The study eye and fellow eye will both be 
evaluated at all assessments unless otherwise specified.   
At Visit 1, measurements will be made before (-1 hour/baseline) and 60 minutes after (max 
pupil dilation/0 minutes) the mydriatic agent instillation OU (i.e., right before the study 
treatment is administered). Additionally, measurements will be taken at 90 minutes, and 3 
hours after study treatment dosing.  
At Visit 2 (Follow-Up Visit), which is 1 day after Visit 1, measurements will be taken 24 
hours after study treatment dosing..  
The schedule for assessments and timing of events is presented in  
Table 1. 


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Table 1 
Screening and Mydriatic/Treatment Schedule 
 
 
Screening [a] 
Mydriasis/Treatment 
Follow-Up 
Day 
1 
1 
2 
Visit 
1 
1 
2 
Start time [b] 
 
-1 hour 
(Baseline) 
0 ±5 min 
(Max) 
90 ±10 
min 
3 hours 
±10 min 24 +6 hours 
Informed consent/assent 
X 
 
 
 
 
 
Subject identification number assigned 
X 
 
 
 
 
 
Medical/Ophthalmic history 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
Prior/Concomitant medications [c] 
X 
X 
X 
X 
X 
X 
HR/BP 
X 
 
 
 
X 
X 
PD 
 
X 
X 
X 
X 
X 
BCDVA [d] 
 
X 
X 
X 
X 
X 
Conjunctival hyperemia  
X 
X 
X 
X 
X 
X 
Biomicroscopy [e] 
X 
 
 
 
 
 
Study medication kit number assigned 
(randomization) 
X 
  
 
 
 
 
Mydriatic agent administration 
 
X 
 
 
 
 
Treatment: Nyxol/Placebo administration 
 
 
X 
 
 
 
AEs [c] 
X 
X 
X 
X 
X 
X 
AE, adverse event; BCDVA, best-corrected distance visual acuity; BP, blood pressure; ETDRS, Early Treatment Diabetic 
Retinopathy Study; HR, heart rate; PD, pupil diameter. 
[a] Screening Visit: if subject qualifies, this becomes Treatment Visit 1. The mydriatic agent will be given at -1 hour. 
[b] Start time refers to the time that first assessment is performed. Assessments at each time point are shown in the order performed.  
[c] Investigators to note changes to concomitant medications and AEs at any time throughout the visit. 
[d] BCDVA will be measured in photopic conditions by a standard ETDRS illuminated chart (on wall or stand) at 4 m (letters 
recorded). For subjects who are unable to read the letters on a ETDRS chart, the Patti Pics Series ETDRS chart will be used. The 
same method will be used consistently for each subject (when possible). 
[e] It is recommended that a table-top slit lamp be used if at all possible. The use of a portable slit lamp, or a hand-held light source 
should be reserved for subjects who cannot comply with the table-top slit lamp exam. The same method will be used consistently 
for each subject (when possible).


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5.2. 
Inclusion – Exclusion Criteria and General Study Population 
The study population will be approximately 20 to 30 healthy pediatric subjects 3 to 11 years 
of age, inclusive. Written informed consent will be obtained from the legal guardian of each 
subject. A signed assent form will be obtained for all minors ages 7 to 11, as well as a 
separate parental/Legal Guardian consent.  
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 potential subject is assigned a Screening number in numerical order per strata. Once a 
subject is qualified for the study, the subject is assigned a randomization number in the order 
provided by the biostatistician. 
Treatment randomization will be 1:1 (Nyxol or placebo [vehicle]) and will be stratified 1:1 
by subject age group (3‒5 years of age: 6‒11 years of age). Mydriatic agent randomization 
will be 3:1:1 (phenylephrine: tropicamide: Paremyd, respectively). Approximately 60% of 
the randomized subjects will receive 1 drop of 2.5% phenylephrine in both eyes (OU) 1 hour 
before treatment (12 subjects), approximately 20% will receive 1 drop of 1% tropicamide OU 
1 hour before treatment (4 subjects), and approximately 20% will receive 1 drop of Paremyd 
OU 1 hour before treatment (4 subjects). 
The study medications will be masked to both Investigator and study subjects, as well as 
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.  
The only efficacy variable is Pupil diameter. 
Safety variables include: 
• Conjunctival hyperemia (eye redness) measured with a Cornea and Contact Lens 
Research Unit (CCLRU) card 4-point scale: 


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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.   
• BCDVA (i.e., Distance VA) 
• AEs 
• Vital signs (HR and BP) 


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6. 
SAMPLE SIZE 
A sample size of approximately 20 subjects (approximately 10 treated with Nyxol) in this 
study will result in a total of > 300 subjects treated with Nyxol in the reversal of mydriasis 
program (including prior studies); this number of subjects is needed to meet the minimum 
number of subjects exposed to Nyxol to assess safety in this population as specified in the 
Pediatric Research Equity Act (PREA) plan. This safety study is not powered for efficacy. In 
accordance with the Sponsor Pediatric Safety Plan for the indication of reversal of 
pharmacologically induced mydriasis, the sample size of 20 subjects divided between 2 age 
groups is adequate to evaluate safety in the 3- to 11-year-old pediatric population.  
All subjects will be randomized into the study in a 1:1 ratio to 1 of the 2 treatment arms 
(Nyxol or placebo), with a 1:1 stratification by subject age group (3‒5 years of age: 6‒11 
years of age). Furthermore, subjects will be randomized into the study at a ratio of 3:1:1 to 
mydriatic agent (phenylephrine: tropicamide: Paremyd, respectively). Therefore, if 12 
subjects are randomized to 2.5% phenylephrine, then 4 subjects will be assigned to 1% 
tropicamide, and 4 subjects will be assigned to Paremyd, resulting in 20 total subjects. 


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7. 
GENERAL CONSIDERATIONS 
7.1. 
Analysis Populations 
The following analysis populations will be defined for this study.  
7.1.1. 
Modified Intention-to-Treat (mITT) 
The mITT Population will include all randomized subjects who received 1 drop of study 
treatment in the study eye and completed at least 1 scheduled post-treatment PD 
measurement during Visit 1 in the study eye. The mITT Population will be used to analyze 
selected efficacy endpoints, with subjects included in their randomized treatment regardless 
of the treatment they actually received. 
7.1.2. 
Per Protocol Population (PP) 
The PP Population includes all subjects in the mITT Population who had 1 drop of study 
treatment in the study eye, had all scheduled PD measurements during the Treatment Visit in 
the study eye, had an increase of > 0.2 mm in PD in the study eye at 0 minutes compared to 
baseline (-1 hour), and had no major protocol deviations considered to have significant 
impact on treatment outcome. The PP Population will be used to analyze selected efficacy 
endpoints, with subjects included in their randomized treatment regardless of the treatment 
they actually received. 
7.1.3. 
All Randomized Population (ARP) 
The ARP will include all randomized subjects. This population is also known as the Intent-
to-Treat (ITT) Population. The ARP may 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 received at least 1 drop of study treatment. 
The SP will be used to summarize safety variables, using the treatment they actually 
received. 
7.2. 
Covariates and Subgroups 
7.2.1. 
Planned Covariates 
Planned covariates include baseline values for the given assessment.  


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7.2.2. 
Planned Subgroups 
Subgroup analyses by age group (3-5 years and 6-11 years), and by mydriatic agent 
(phenylephrine, tropicamide, Paremyd) will be completed for efficacy endpoints. An 
additional mydriatic agent subgroup, combining 1% tropicamide and the Paremyd subjects 
into a “tropicamide” group, will be used for efficacy endpoints.  
Other subgroups, such as light/dark irides, sex, and race, may be summarized as well. 
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 
There will be no substitutions made to accommodate missing data points for efficacy data. 
All data recorded on the CRF will be included in data listings that will accompany the CSR. 
Safety data will be imputed in limited situations. If the severity of an AE is missing, then the 
severity will remain missing. If relationship of the AE to study drug is missing, the 
relationship will remain missing. Missing or partial dates for AEs or concomitant 
medications will be imputed as described in Section 7.3.2.1. Otherwise, all summaries of 
safety endpoints will be completed using observed cases in the SP; no imputation will be 
completed. 
7.3.2.1. 
Handling of Missing Date Values 
Partial or Missing Dates 
The following conventions will be used to impute missing portions of dates for AEs and 
concomitant medications, if warranted. Note that the imputed values outlined here may not 
always provide the most conservative date. In those circumstances, the imputed value may be 
replaced by a date that will lead to a more conservative analysis. 
A. 
Start Dates 
1) 
If the year is unknown, then the date will not be imputed and will 
be assigned a missing value.  
2) 
If the month is unknown, then: 
i) If the year matches the first dose date year, then impute 
the month and day of the first dose date. 
ii) Otherwise, assign ‘January.’  
3) 
 If the day is unknown, then: 


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i) If the month and year match the first dose date month 
and year, then impute the day of the first dose date. 
ii) Otherwise, assign the first day of the month. 
B. 
Stop Dates 
1) 
If the year is unknown, then the date will not be imputed and will 
be assigned a missing value. 
2) 
If the month is unknown, then assign ‘December.’ 
3) 
If the day is unknown, then assign the last day of the month. 
7.3.2.2. 
Missing Baseline Data 
Every effort will be made to ensure that accurate baseline information on the subjects is 
collected. In the event that a subject is missing relevant baseline information, the subject will 
be included in the SP for assessment of safety but excluded from the efficacy analyses. Each 
case of missing baseline data will be evaluated for potential inclusion in the analysis. All 
baseline data will be observed cases, without imputation. 
7.3.3. 
Handling of Early Termination Visit Information 
In the event that a subject is terminated early from this study on Day 1, the early termination 
data for safety variables will be assigned to the closest scheduled time point on Day 1. If the 
closest time point has valid data, the early termination data will be assigned to the next 
available time point. 
7.3.4. 
Pooling of Investigational Sites 
The data from all study centers will be pooled together for all planned analyses. 
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 25.0) system for reporting (preferred term and body system). 
Prior and concomitant medications will be coded using WHO-DD (World Health 
Organization Drug Dictionary) (Global Version 2022-01). 
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. 


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7.3.7. 
Study Data 
Study data identified in the schedule for time and events ( 
Table 1) are collected, and source verified, on paper CRFs. 
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 
 
Where: 
1. Development, validation, and maintenance of SDTM domains 
2. Development and validation of ADaM data sets, with input source the appropriate 
SDTM domains. 
3. Development and validation of tables, figures, and listings (TFL), with input data 
source the SDTM domains and analysis specific ADaM data sets. 


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7.4. 
Planned Study Analyses 
7.4.1. 
Statistical Summaries: Descriptive and Inferential 
Categories for data presentation and analysis will consist of each treatment group (Nyxol 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. 
7.6. 
Baseline Values 
Baseline values are the values obtained prior to any drug administration on Day 1 (study drug 
or mydriatic agent), usually the -1 hour time point. If the Day 1 value is missing or the 
assessment is not completed at the -1 hour time point, the value at Screening will be treated 
as the baseline. 
 


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8. 
SUMMARY OF STUDY DATA 
8.1. 
Subject Disposition 
A summary of the analysis sets includes the number and percentage of subjects by treatment 
group and overall for the following categories: subjects in the ARP, subjects in the SP, 
subjects in the mITT Population, and subjects in the PP Population. All percentages will be 
based on the number of subjects in the ARP.  
End of trial information will also be summarized in this table, including the number of 
subjects completing the study, the number of subjects who prematurely discontinued the 
study with reasons for withdrawal, the number of subjects completing the study medication 
dosing, and the number of subjects who prematurely discontinued the study medication with 
reasons for study medication discontinuation.   
A by-subject data listing of study completion information including the reason for premature 
study withdrawal, if applicable, will be presented. 
8.2. 
Protocol Deviations 
Major protocol deviations, as determined by a Sponsor blinded review of the data prior to 
database lock and unblinding of the study, may result in the removal of a subject’s data from 
the PP Population. The Sponsor or designee will be responsible for producing the final 
deviation file; this file will include a description of the protocol deviation and clearly identify 
whether this violation warrants exclusion from the PP Population. This file will be finalized 
prior to database lock. 
All protocol deviations will be presented in a by-subject data listing, with a flag to indicate if 
a deviation was considered major.  
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 (OD), 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 mydriatic agent (phenylephrine, tropicamide, or Paremyd). 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 mydriatic agent will also be 


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reported. Percentages will be based on the total number of subjects in the study population 
presentation. 
The following baseline characteristics will be summarized for study eye and fellow eye, 
using descriptive statistics: 
• Pupil diameter (-1 Hour) 
• Max pupil diameter (0 Minutes)  
• BCDVA (-1 Hour) 
• BCDVA (0 Minutes) 
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 25.0 The number and percentage 
of subjects with any medical history will be summarized overall and for each system organ 
class (SOC) and preferred term (PT). Percentages will be calculated based on number of 
subjects in the SP.   
Subject medical history data including specific details will be presented in by-subject 
listings. 
8.5. 
Prior and Concurrent Medications 
The number and percentages of all concomitant medications will be summarized by 
treatment group, Anatomical Therapeutic Chemical (ATC) level 4, and PT. The total number 
of concomitant medications and the number and percentages of subjects with at least 1 
concomitant medication will be summarized by treatment group. All summaries will be 
performed using the SP.   
A concomitant medication is defined as any medication taken on or after the day of first 
exposure to study drug. 
Prior medications are defined as any medication that has a start and stop date prior to the day 
of first exposure to any study drug, collected from up to 30 days prior to Screening. The total 
number of prior medications and the number and percentages of subjects with at least 1 prior 
medication will be summarized by treatment group. 


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8.6. 
Treatment Administration 
Treatment administration data for both the study drug and the mydriatic agent on Day 1 will 
be presented in by-subject listings.  


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9. 
EFFICACY ANALYSES 
Unless otherwise noted, efficacy will be assessed using the mITT and PP populations, with 
subjects included in their randomized treatment regardless of the treatment they actually 
received. Observed data will be used for efficacy summaries; imputation will not be 
performed.  
To formally test the significance of efficacy endpoints of interest, endpoints will be tested in 
a predefined sequence, each at the significance level 0.05, until the first nonsignificant test. 
The endpoints in the sequence are for the study eye only, but the analysis populations and 
time points will vary. 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   
For all efficacy endpoints, Baseline is defined as -1 hour prior to treatment on Day 1. This is 
the time when the mydriatic agent is administered, and the pupil diameter measurement is 
considered normal. Max timepoint is defined as time 0 minutes, during which maximum 
pupil diameter is expected; this is also the timepoint at which the treatment is administered 
(Nyxol or Placebo).  
All efficacy data will be summarized by treatment group, study day, and time point (-1 hour 
[baseline], 0 minutes, 90 minutes, 3 hours, and 24 hours), as appropriate. 
The returning to ≤ 0.2 mm from baseline (-1 hour) photopic pupil diameter endpoint will be 
analyzed separately for the study eye and fellow eye at each time point using a logistic 
regression model with treatment as a factor and the baseline pupil diameter as a covariate. 
The percentage of subjects in each treatment group meeting the criteria and the odds ratio 
(OR) with 95% confidence interval (CI) and p-value will be provided. The analysis will be 
performed using the mITT and PP Populations, with subjects included in their randomized 
treatment regardless of the treatment they actually received.  
Example SAS code is as follows: 
proc logistic data=efficacydata;  
class trtname;  
model outcome = trtname base/cl rl firth; 
run; 
 
Subjects who did not have an increase of > 0.2 mm in pupil diameter in the study eye or 
fellow eye at the max pupil dilation (0 minutes) will not be counted as returning to ≤ 0.2 mm 
from baseline pupil diameter for that eye. 
The continuous change (in mm) in photopic pupil diameter from max pupil dilation (0 
minutes) endpoint will be analyzed separately for the study eye and fellow eye at each time 


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point using ANCOVA, with change from baseline as the dependent variable, treatment as a 
factor, and the baseline pupil diameter as the covariate.  
The ANCOVA will be performed using the mITT and PP populations. The output from the 
ANCOVA will include the LSM and SE for both treatment groups, along with the placebo-
corrected LSM, its 95% CI and associated p-value.  
The analysis of the time (hours) to return to ≤ 0.2 mm from baseline pupil diameter (time 
savings analysis) endpoint will be performed using a Cox proportional hazards regression 
model with treatment as a factor and the baseline pupil diameter as a covariate. Subjects who 
do not return to ≤ 0.2 mm from baseline pupil diameter by the 6 hour time point will have 
their time to return censored at 8 hours. Example SAS code is as follows: 
proc phreg data=efficacydata plots(overlay)=survival; 
 
class trtname; 
 
model timetoreturn*cnsr(1) = trtname base; 
 
hazardratio trtname; 
 
baseline covariates=efficacydata out=Pred1 survival=_all_/diradj group=trtname; 
 
ods output  hazardratios=hr; 
run; 
The output from the model will include the hazard ratio comparing treatment groups, its 95% 
CI and associated p-value. Time to return to baseline (-1 hour) pupil diameter will be 
measured beginning at the Max (0 hour) time point. 
In addition, efficacy endpoints will be summarized by age group, by mydriatic agent, and by 
irides type, but will not be analyzed due to the small sample size for each subgroup. The time 
savings analysis will not be summarized by age group. Each mydriatic agent will be 
summarized individually, and an additional summary combining 1% tropicamide and 
Paremyd subjects into a “tropicamide or Paremyd” group will be presented. 


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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 25.0.   
Treatment-emergent adverse events (TEAEs) are defined as any AE that begins or worsens 
after initiation of the investigational product and through the subject’s last study visit (study 
completion or early termination). 
If the onset of an AE is on or after the date of first dose of study medication or is increasing 
in severity after first dose of study medication, then the AE will be considered treatment 
emergent. 
Only TEAEs will be summarized; all AEs (TEAE, non-TEAE) will be included in a by-
subject listing.  
The number and percent of subjects with any TEAEs will be summarized by SOC and PT by 
treatment group and overall. At each level of tabulation (e.g., at the PT level), subjects will 
be counted only once if they had more than one such event reported during the AE collection 
period. A separate summary by SOC, PT, and treatment group will be completed by 
mydriatic agent. 
 
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 


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• 
Summary table of TEAEs leading to study medication discontinuation by SOC and 
PT 
If no TEAEs are observed during the study, only the overall summary of TEAEs will be 
generated. 
 
10.2. 
Deaths, Serious Adverse Events and Other Significant Adverse Events 
10.2.1. 
Deaths 
The AE listing will include all AEs, including deaths, regardless of causality; one of the 
columns in the listing will specify whether the AE was fatal.     
10.2.2. 
Serious Adverse Events 
The AE listing will include all AEs, including SAEs; one of the columns in the listing will 
specify whether the AE was an SAE.     
10.2.3. 
Adverse Events Leading to Withdrawal from the Study 
The AE listing will include all AEs, including AEs leading to withdrawal from the study; one 
of the columns in the listing will specify whether the AE led to withdrawal from the study.   
10.2.4. 
Adverse Events Leading to Discontinuation of Study Medication 
The AE listing will include all AEs, including AEs leading to discontinuation of study 
medication; one of the columns in the listing will specify whether the AE led to 
discontinuation of study medication.   
10.3. 
Conjunctival Hyperemia 
Results from the conjunctival hyperemia assessment, measured with a CCLRU card 4-point 
scale, will be summarized descriptively using counts and percentages for each treatment 
group at each time point (-1 hour [baseline], 0 minutes, 90 minutes, 3 hours, and 24 hours) 
for the observed value and change from baseline (-1 hour). Separate summaries will be 
created for the study eye and the fellow eye. 
Additionally, conjunctival hyperemia will be summarized as a continuous variable. 
Treatments will be compared using the same ANCOVA model proposed for the change in 
photopic pupil diameter from max pupil dilation efficacy endpoint. A descriptive summary of 
conjunctival hyperemia will be presented by treatment and age group. 
10.4. 
Visual Acuity 
Assessments will be summarized at the timepoints -1 hour, 0 minutes, 90 minutes, 3 hours, 
and 24 hours for BCDVA using letters. As a reference 5 letters is equivalent to 1 line. 
Separate summaries will be created for the study eye, the fellow eye, and both eyes. 


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Treatments will be compared using the same ANCOVA model proposed for the change in 
photopic pupil diameter from max pupil dilation efficacy endpoint. A descriptive summary of 
BCDVA will be presented by treatment and age group. 
Additionally, a categorical summary of BCDVA will be presented, with counts and 
percentages of subjects with Improvement or loss from baseline by treatment and time point. 
10.5. 
Vital Signs 
Descriptive statistics of observed values will be presented for vital sign data at each time 
point (Screening, 3 hours, and 24 hours), including systolic BP (mmHg), diastolic BP 
(mmHg), and HR (bpm) by treatment group and overall. Changes from baseline to each 
scheduled post-baseline time point will be presented.  
10.6. 
Other Safety Measures 
Results from the biomicroscopic examination, which is completed only at Screening, will 
also be presented in by-subject listings. 
 
 


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


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12. 
APPENDICES 
12.1. 
List of Planned Tables 
The list of planned tables includes all of the main tables to be presented for the study. 
Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.1.1 
Subject Disposition  
X 
 
 
 
14.1.2.1/2/3/4 
Demographics and Baseline 
Characteristics 
X 
X 
X 
X 
14.1.3.1 
Non-ocular Medical History  
 
X 
 
 
14.1.3.2 
Ocular Medical History  
 
X 
 
 
14.1.4.1 
Prior Medications  
 
X 
 
 
14.1.4.2 
Concomitant Medications  
 
X 
 
 
14.2.1.1/2 
Percent of Subjects Returning to ≤ 0.2 
mm of Baseline (-1 Hour) Pupil 
Diameter by Time Point 
 
 
X 
X 
14.2.1.3/4 
Percent of Subjects Returning to ≤ 0.2 
mm of Baseline (-1 Hour) Pupil 
Diameter by Time Point and Mydriatic 
Agent 
 
 
X 
X 
14.2.1.5/6 
Percent of Subjects Returning to ≤ 0.2 
mm of Baseline (-1 Hour) Pupil 
Diameter by Time Point and Age 
Group 
 
 
X 
X 
14.2.1.7/8 
Percent of Subjects Returning to ≤ 0.2 
mm of Baseline (-1 Hour) Pupil 
Diameter by Time Point, Mydriatic 
Agent, and Age Group 
 
 
X 
X 
14.2.1.9/10 
Percent of Subjects Returning to ≤ 0.2 
mm of Baseline (-1 Hour) Pupil 
Diameter by Time Point and Irides 
Type 
 
 
X 
X 
14.2.2.1/2 
Change From Max (0 Minute) Pupil 
Diameter by Time Point 
 
 
X 
X 
14.2.2.3/4 
Change From Max (0 Minute) Pupil 
Diameter by Time Point and Mydriatic 
Agent 
 
 
X 
X 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.2.2.5/6 
Change From Max (0 Minute) Pupil 
Diameter by Time Point and Age 
Group 
 
 
X 
X 
14.2.2.7/8 
Change From Max (0 Minute) Pupil 
Diameter by Time Point, Mydriatic 
Agent, and Age Group 
 
 
X 
X 
14.2.2.9/10 
Change From Max (0 Minute) Pupil 
Diameter by Time Point and Irides 
Type 
 
 
X 
X 
14.2.3.1/2 
Time (Hours) to Return to ≤ 0.2 mm 
of Baseline (-1 Hour) Pupil Diameter 
 
 
X 
X 
14.2.3.3 
Time (Hours) to Return to ≤ 0.2 mm 
of Baseline (-1 Hour) Pupil Diameter 
by Mydriatic Agent 
 
 
 
X 
14.2.3.4 
Time (Hours) to Return to ≤ 0.2 mm 
of Baseline (-1 Hour) Pupil Diameter 
by Irides Type 
 
 
 
X 
14.3.1.1 
Overall Summary of Treatment 
Emergent Adverse Events (TEAE) 
 
X 
 
 
14.3.1.2.1 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class and 
Preferred Term 
 
X 
 
 
14.3.1.2.2 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class, 
Preferred Term, and Mydriatic Agent 
 
X 
 
 
14.3.1.3 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class, 
Preferred Term, and Severity 
 
X 
 
 
14.3.1.4 
Treatment-Emergent Adverse Events 
(TEAE) by System Organ Class, 
Preferred Term, and Relationship to 
Study Treatment 
 
X 
 
 
14.3.1.5 
Serious Treatment-Emergent Adverse 
Events (TEAE) by System Organ 
Class and Preferred Term 
 
X 
 
 
14.3.1.6 
Treatment-Emergent Adverse Events 
(TEAE) Leading to Withdrawal From 
the Study by System Organ Class and 
Preferred Term 
 
X 
 
 


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Table 
Description of Table 
ARP Safety 
mITT 
PP 
14.3.1.7 
Treatment-Emergent Adverse Events 
(TEAE) Leading to Study Medication 
Discontinuation by System Organ 
Class and Preferred Term 
 
X 
 
 
14.3.3.1 
Categorical Summary of Conjunctival 
Hyperemia  
 
X 
 
 
14.3.3.2 
Summary of Mean Conjunctival 
Hyperemia Score by Time Point 
 
X 
 
 
14.3.3.3 
Summary of Mean Conjunctival 
Hyperemia Score by Time Point and 
Age Group 
 
X 
 
 
14.3.4.1 
Best Corrected Distance Visual Acuity 
(BCDVA) by Time Point (Letters 
Read) 
 
X 
 
 
14.3.4.2  
Best Corrected Distance Visual Acuity 
(BCDVA) by Time Point and Age 
Group (Letters Read) 
 
X 
 
 
14.3.4.3 
Percent of Subjects With 
Improvement or Loss From Baseline 
in BCDVA by Time Point 
 
X 
 
 
14.3.5 
Vital Signs by Time Point 
 
X 
 
 
 
12.2. 
List of Planned Listings 
Listing 
Description of Listing 
16.2.1.1 
Subject Disposition 
16.2.1.2 
Eligibility and Randomization 
16.2.2 
Protocol Deviations 
16.2.3 
Treatment Assignment and Analysis Populations 
16.2.4.1 
Demographics 
16.2.4.2 
General Medical History (Non-ocular) 
16.2.4.3 
Ocular Medical History 
16.2.4.4 
Biomicroscopy Examination 
16.2.4.5 
Prior and Concomitant Medications 
16.2.5 
Mydriatic Agent and Study Drug Administration 
16.2.6 
Photopic Pupil Diameter 
16.2.7 
Adverse Events 


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Listing 
Description of Listing 
16.2.8 
Conjunctival Hyperemia 
16.2.9 
Best-Corrected Distance Visual Acuity (BCDVA) 
16.2.10 
Vital Signs 
 
 
 


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12.3. 
Hierarchy of Sequential Testing 
Table Number 
Table Title 
Time Point 
Population 
Subgroup 
14.2.2.1 
Change from Max (0 Minute) Pupil Diameter by 
Time Point 
90 min 
mITT 
Overall 
14.2.2.1 
Change from Max (0 Minute) Pupil Diameter by 
Time Point 
3 hours 
mITT 
Overall 
14.2.3.2 
Time to Return to <= 0.2 mm of Baseline (-1 
Hour) Pupil Diameter 
N/A 
PP 
Overall 
14.2.1.1 
Percent of Subjects returning to <= 0.2 mm of 
Baseline (-1 Hour) Pupil Diameter by Time Point 
90 min 
miTT 
Overall 
14.2.1.1 
Percent of Subjects returning to <= 0.2 mm of 
Baseline (-1 Hour) Pupil Diameter by Time Point 
3 hours 
mITT 
Overall 
14.3.4.1 
Best Corrected Distance Visual Acuity 
(BCDVA) by Time Point (Letters Read) 
90 min 
Safety 
Overall 
14.3.4.1 
Best Corrected Distance Visual Acuity 
(BCDVA) by Time Point (Letters Read) 
3 hours 
Safety 
Overall