Conditions: Neurotrophic Keratopathy
Keywords: Neurotrophic Keratopathy NK
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 18
Healthy volunteers: no

Brief summary:
The objective of this study is to assess the safety and efficacy of RGN-259 Ophthalmic Solution compared to placebo for the treatment of NK.

Detailed description:
Neurotrophic keratopathy (NK) is a degenerative corneal disease that occurs as a result of partial or total impairment of trigeminal innervation. The resulting loss of corneal sensitivity (anesthesia) leads to a reduction in lacrimation and a decline in status, metabolism, and mitosis of corneal epithelial cells. Previous studies (physician-sponsored studies) used to treat to nine patients with NK, six of whom had discrete geographic, non-healing lesions, and three of whom had punctate lesions and the study result reported.

Arms:
arm 1: It is a preservative-free, sterile eye drop solution containing Tβ4 arm 2: It is composed of the same excipients as RGN-259 but does not contain Tβ4.

Interventions: intervention 1: RGN-259 intervention 2: Placebo
Intervention descriptions: intervention 1: A preservative-free, sterile eye drop solution containing Tβ4 for direct instillation into affected eye(s), five times a day for 4 weeks. intervention 2: It is composed of the same excipients as RGN-259 but does not contain Tβ4

Protocol document:
Prot_000.pdf: 
Clinical Trial Protocol: RGN-NK-301/Ora Protocol # 15-110-0006
Protocol Title:
A Phase 3, Multi-Center, Randomized, Double Masked, Placebo Controlled 
Clinical Study to Assess the Safety and Efficacy of 0.1% RGN-259 Ophthalmic 
Solution for the Treatment of Neurotrophic Keratopathy (SEER-1)
Protocol Number:
RGN-NK-301/15-110-0006
Study Phase:
3
Product Name:
Thymosin β4 (RGN-259)
IND Number:
73446
Indication:
Neurotrophic Keratopathy
Investigators:
Multi-center clinical investigation
Sponsor:
ReGenTree, LLC
116 Village Boulevard, Suite 200
Princeton, NJ 08540, USA
Phone number: (609)734-4328
Fax number: (609)228-5117
Contract Research 
Organization:
Ora, Inc.
300 Brickstone Square, 3rd Floor
Andover, MA 01810, USA
IRB/IEC:
Alpha IRB
1001 Avenida Pico
Suite C, #497
San Clemente, CA 92673, USA
Date
Original Protocol:
17 August 2015
Amendment 1:
25 September 2015
Amendment 2: 
26 February 2016
Amendment 3:
22 December 2016
Amendment 4:
04May 2017
Confidentiality Statement 
This protocol contains confidential, proprietary information of Ora, Inc. and/or ReGenTree, LLC.
Further 
dissemination, distribution or copying of this protocol or its contents is strictly prohibited. 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 2 of 65
SPONSOR PERSONNEL 
Sponsor:
Medical Monitor
ORA PERSONNEL 
Department Vice President
Department Senior Manager
Clinical Project Manager


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 3 of 65
SYNOPSIS 
Protocol Title:
A Phase 3, Multi-Center, Randomized, Double Masked, 
Placebo Controlled Clinical Study to Assess the Safety and 
Efficacy of 0.1% RGN-259 Ophthalmic Solution for the 
Treatment of Neurotrophic Keratopathy (SEER-1)
Protocol Number:
RGN-NK-301
Investigational Product:
1)
0.1% RGN-259 Ophthalmic Solution
2)
Placebo Ophthalmic Solution (Vehicle of RGN-259)
Study Phase:
3
Study Objective:
To compare the safety and efficacy of RGN-259 to placebo for 
the treatment of Neurotrophic Keratopathy (NK)
Overall Study Design:
Structure:
Multi-center, double-masked, randomized, placebo-controlled 
study 
Duration:
Approximately 6 weeks (43 days)
Controls:
Placebo Ophthalmic Solution (Vehicle of RGN-259)
Dosage/Dose Regimen/ 
Instillation/Application/Use:
Subjects eligible to be randomized will receive 1-2 drops of
the following treatments to be administered in the affected 
eye(s) 5 times per day for 28 days (from Visit 1 to Visit 5).
1.
0.1%  RGN-259 Ophthalmic Solution
2.
Placebo Ophthalmic Solution
Subjects will be randomized in 
ratio 
Summary of Visit Schedule:
Seven visits over the course of approximately 6 weeks
•
Visit 1 = Day 1, Screening/Enrollment/Baseline
•
Visit 2 = Day 8 ± 2 day, 1-Week Follow Up
•
Visit 3 = Day 15 ± 2 day, 2-Week Follow Up
•
Visit 4 = Day 22 ± 2 day, 3-Week Follow Up
•
Visit 5 = Day 29 ± 2 day, 4-Week Follow-Up
•
Visit 6 = Day 36 ± 3 day, 1-Week Post-Treatment Follow-
Up
•
Visit 7 = Day 43 ± 3 day, 2-Week Post-Treatment Follow-
Up
Measures Taken to Reduce Bias:
Randomization will be used to avoid bias in the assignment of 
subjects to treatment, to increase the likelihood that known and 
unknown subject attributes are evenly balanced across 
treatment groups, and to enhance the validity of statistical 
comparisons across treatment groups. Masked treatment will 
be used to reduce the potential of bias during data collection 
and evaluation of clinical endpoints.
Study Population Characteristics:
Number of Subjects:
Up to 100 subjects will be screened and approximately 46
subjects will be enrolled in this study 
at approximately 20 clinical sites in the US.


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 4 of 65
Condition/Disease:
Neurotrophic Keratopathy
Inclusion Criteria:
Subjects must:
a.
Be male or female of any race, at least 18 years of age at 
Visit 1;
b.
Have provided verbal and written informed consent.
c.
Be able and willing to follow instructions, including 
participation in all study assessments and visits;
d.
Have a persistent epithelial defect (a defect that has not 
reduced in size) for at least 
with failure of 
conventional, nonsurgical treatment using non-preserved 
ocular lubricants, non-preserved topical ophthalmic 
antibiotics, oral doxycycline, patching, serum tears and/or
therapeutic contact lenses at the time of Visit 1;
e.
Have stage 2 or 3 neurotrophic keratopathy (Mackie 
Classification) in at least one eye
and which is confirmed not to 
be simply superficial punctate keratitis, at Visit 1;
f.
Have evidence of decreased corneal sensitivity
 at Visit 1;
g.
Have at least one eye (the same eye) satisfy all criteria for 
d, e, f above
h.
If a female of childbearing potential, have a negative urine 
pregnancy test at Visit 1 and agree to use an adequate 
method of birth control throughout the study period. 
[Females are considered of childbearing potential unless 
they are surgically sterilized (bilateral tubal ligation, 
hysterectomy or bilateral oophorectomy) or post-
menopausal (at least 12 months since last menses). 
Adequate birth control is defined as use of hormonal 
contraceptives (oral, implantable, injectable or 
transdermal), spermicide in conjunction with a barrier 
such as condom or diaphragm, or an intrauterine device 
(IUD), or surgical sterilization of partner. For non-
sexually active females, abstinence may be regarded as an 
adequate method of birth control; however, if the patient 
becomes sexually active during the study, she must agree 
to use adequate birth control as defined above for the 
remainder of the study.]
Exclusion Criteria:
Subjects must not:
a.
Have any clinically significant slit lamp findings at Visit 1 
that in the opinion of the investigator may interfere with 
the study parameters;
b.
Have significant blepharitis, meibomian gland dysfunction 
(MGD), lid margin inflammation or active ocular allergy 
that requires treatment at Visit 1;
c.
Have a lid function abnormality (ex. Lagophthalmos) 
which, in the opinion of the investigator, is the primary
cause of the persistent epithelial defect;


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 5 of 65 
d. Be diagnosed with ongoing ocular infection (bacterial, 
viral or fungal) or active inflammation (e.g. follicular 
conjunctivitis) not related to NK in the affected eye at 
Visit 1; 
e. 
Have had any ocular surgical procedure that is not 
considered the primary cause of NK in the affected eye 
within 2 months prior to Visit 1, or have any scheduled 
ocular surgical procedure during the study period; 
f. 
Have had any ocular surgical procedure that is considered 
to be the cause of NK within 4 weeks prior to Visit 1; 
g. Have received Botox injection for blepharoptosis within 
the previous 90 days in the affected eye; 
h. Have started the use of topical steroids in the affected eye 
in the previous 2 weeks or systemic steroids in the 
previous 4 weeks; 
i. 
Have used contact lenses (excluding therapeutic contact 
lenses) within 14 days prior to Visit 1 or anticipates use of 
contact lenses during the study period; 
j. 
Have an uncontrolled systemic disease that in the opinion 
of the investigator may interfere with the study 
parameters; 
k. Anticipate a change in immunosuppressive therapy during 
the course of the study; 
l. 
Have a known allergy and/or sensitivity to the study drug 
or its components; 
m. Be a woman who is pregnant, nursing or planning a 
pregnancy; 
n. Be unwilling to submit a urine pregnancy test at Visit 1, 
Visit 5 and Visit 7 (or early termination visit) if of 
childbearing potential;   
o. Be currently enrolled in an investigational drug or device 
study or have used an investigational drug or device 
within 30 days of Visit 1; 
p. Have undergone surgical treatments for NK including 
tarsorrhaphy or amniotic membrane graft within the 
previous 6 weeks in the affected eye. 
q. Plan to use serum tears in the affected eye during the study 
period. 
Study Formulations and 
Formulation Numbers: 
Study Drug:  
RGN-259 Ophthalmic Solution (and Placebo) to be supplied 
by ReGenTree. 
Evaluation Criteria: 
Efficacy Measures: 
Primary Efficacy Measures: 
Percentage of subjects achieving complete healing of the 
persistent epithelial defect as determined by corneal fluorescein 
staining at 4 weeks (Visit 5). 
Secondary Efficacy Measures: 
x 
Percentage of subjects achieving complete healing of 
the persistent epithelial defect determined by corneal 
fluorescein staining at Visits 2, 3, 4, 6, and 7; 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 6 of 65
x
Epithelial Defect Measurement and Classification at 
Visits 2, 3, 4, 5, 6, and 7;    
x
Tear Film Break-up Time at Visits 5, 6, and 7;
x
Ocular Discomfort using Ora Calibra™ Discomfort 
and 4-Symptom Questionnaire at Visits 2, 3, 4, 5, 6, 
and 7;
x
Visual Acuity at Visits 2, 3, 4, 5, 6, and 7. 
Safety Measures:
x
Visual Acuity (ETDRS);
x
Slit-Lamp Biomicroscopy;
x
Cochet-Bonnet;
x
Adverse Event Query;
x
Dilated Fundoscopy;
x
Intraocular Pressure.
General Statistical Methods and Types of Analyses
Analysis Populations:
x
x
  
x
Sample Size:
  
 
  
  
Primary Efficacy Analysis:
  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 7 of 65
  
Secondary Efficacy Analyses:
  
  
 
Summary of Known and Potential Risks and Benefits to Human Subjects
There are no known risks associated with the ocular instillation of RGN-259 ophthalmic solution.
Doses up to 0.1% produced no local or systemic toxicity and did not induce any signs of ocular disease 
in a 28-day repeated-dose study in rabbits. In two Phase 2 studies evaluating 0.1% Tβ4 Ophthalmic 
solution, there were no Tβ4-related adverse events reported. 
Potential risks are minimal based on (1) the safety profile of Tβ4 in nonclinical toxicology and safety 
pharmacology studies where systemic doses 
 produced no observed adverse events. 
In a Phase 1 study in healthy volunteers, systemic doses of Tβ4 injectable solution up to 
mg/kg 
bw/day yielded no dose limiting toxicities and no serious adverse events. Three unexpected and 
possibly related adverse events; dizziness (1 subject), headache (1 subject) and pyrexia (1 subject) were 
reported as the only adverse events reported.
Potential benefits of RGN-259 include improvements in various signs and symptoms associated with 
NK.


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 8 of 65 
TABLE OF CONTENTS 
SYNOPSIS ...........................................................................................................................3 
List of Abbreviations .........................................................................................................11 
1 
INTRODUCTION ....................................................................................................12 
1.1 
The Pharmacology of Thymosin β4 (Tβ4) .....................................................12 
1.2 
Toxicology Overview .....................................................................................12 
1.3 
Pharmacokinetics and Dose-Range Selection ................................................13 
1.4 
Study Rationale ..............................................................................................14 
2 
STUDY OBJECTIVES .............................................................................................15 
3 
CLINICAL HYPOTHESIS ......................................................................................15 
4 
OVERALL STUDY DESIGN ..................................................................................15 
5 
STUDY POPULATION ...........................................................................................16 
5.1 
Number of Subjects (approximate) ................................................................16 
5.2 
Study Population Characteristics ....................................................................17 
5.3 
Inclusion Criteria ............................................................................................17 
5.4 
Exclusion Criteria ...........................................................................................17 
6 
STUDY PARAMETERS ..........................................................................................19 
6.1 
Efficacy Measures ..........................................................................................19 
6.1.1 
Primary Efficacy Measure(s) ............................................................19 
6.1.2 
Secondary Efficacy Measure(s) ........................................................19 
6.2 
Safety Measures .............................................................................................19 
7 
STUDY MATERIALS .............................................................................................19 
7.1 
Study Treatment(s) .........................................................................................19 
7.1.1 
Study Treatments/Formulations/Composition ..................................19 
7.1.2 
Instructions for Use and Administration ...........................................20 
7.2 
Other Study Supplies ......................................................................................20 
8 
STUDY METHODS AND PROCEDURES ............................................................20 
8.1 
Subject Entry Procedures ...............................................................................20 
8.1.1 
Overview ...........................................................................................20 
8.1.2 
Informed Consent..............................................................................20 
8.1.3 
Washout Intervals .............................................................................21 
8.1.4 
Procedures for Final Study Entry ......................................................21 
8.1.5 
Methods for Assignment to Treatment Groups: ...............................21 
8.2 
Concurrent Therapies .....................................................................................21 
8.2.1 
Prohibited Medications/Treatments ..................................................21 
8.2.2 
Allowed Concomitant Medications/ Treatments/ Procedures ..........21 
8.2.3 
Escape Medications ..........................................................................22 
8.2.4 
Escape Surgical Intervention ............................................................22 
8.2.5 
Special Diet or Activities ..................................................................22 
8.3 
Examination Procedures .................................................................................22 
8.3.1 
Procedures to be Performed at Each Study Visit with Regard to 
Study Objective(s) ..........................................................................................22 
8.4 
Schedule of Visits, Measurements and Dosing ..............................................27 
8.4.1 
Scheduled Visits................................................................................27 
8.4.2 
Unscheduled Visits ...........................................................................27 
8.4.3 
Early Termination Visits ...................................................................28 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 9 of 65 
8.5 
Compliance with Protocol ..............................................................................28 
8.6 
Subject Disposition .........................................................................................29 
8.6.1 
Completed Subjects ..........................................................................29 
8.6.2 
Discontinued Subjects .......................................................................29 
8.7 
Study Termination ..........................................................................................29 
8.8 
Study Duration ...............................................................................................29 
8.9 
Monitoring and Quality Assurance ................................................................30 
9 
ADVERSE EVENTS ................................................................................................30 
9.1 
Adverse Event ................................................................................................30 
9.1.1 
Severity .............................................................................................31 
9.1.2 
Relationship to Investigational Product ............................................31 
9.1.3 
Expectedness .....................................................................................31 
9.2 
Serious Adverse Events ..................................................................................32 
9.3 
Procedures for Reporting Adverse Events .....................................................33 
9.3.1 
Reporting a Suspected Unexpected Adverse Reaction .....................33 
9.3.2 
Reporting a Serious Adverse Event ..................................................33 
9.4 
Procedures for Unmasking (if applicable) ......................................................34 
9.5 
Type and Duration of the Follow-up of Subjects after Adverse Events ........34 
9.6 
Study Populations ...........................................................................................35 
9.7 
Efficacy measures: .........................................................................................35 
9.8 
Safety measures: .............................................................................................35 
9.9 
Statistical Hypothesis and Sample Size ..........................................................36 
9.10 Statistical Analysis .........................................................................................36 
9.10.1 General Considerations .....................................................................36 
9.10.2 Unit of Analysis ................................................................................37 
9.10.3 Missing Data .....................................................................................37 
9.10.4 Primary Efficacy Analyses ...............................................................37 
9.10.5 Secondary Efficacy Analyses ...........................................................38 
9.10.6 Adverse Events .................................................................................38 
9.10.7 Laboratory Data ................................................................................39 
9.10.8 Interim Analyses ...............................................................................39 
9.10.9 Quality Assurance of Data Processing ..............................................39 
10 
COMPLIANCE WITH GOOD CLINICAL PRACTICES, ETHICAL 
CONSIDERATIONS, AND ADMINISTRATIVE ISSUES ...................................40 
10.1 Protection of Human Subjects ........................................................................40 
10.1.1 Subject Informed Consent.................................................................40 
10.1.2 Institutional Review Board (IRB) Approval .....................................40 
10.2 Ethical Conduct of the Study ..........................................................................40 
10.3 Subject Confidentiality ...................................................................................41 
10.4 Documentation ...............................................................................................41 
10.4.1 Retention of Documentation .............................................................41 
10.5 Labeling, Packaging, Storage, Accountability, and Return or Disposal of 
Investigational Product ...................................................................................42 
10.5.1 Labeling/Packaging ...........................................................................42 
10.5.2 Storage of Investigational Product ....................................................42 
10.5.3 Accountability of Investigational Product ........................................42 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 10 of 65 
10.5.4 Return or Disposal of Investigational Product ..................................42 
10.6 Recording of Data on Source Documents and Electronic Case Reports Forms 
(eCRFs) ..........................................................................................................43 
10.7 Handling of Biological Specimens .................................................................43 
10.8 Publications ....................................................................................................43 
11 
REFERENCES .........................................................................................................44 
Appendix 1:  Schedule of Visits and Measurements .......................................................45 
Appendix 2:  Examination Procedures, Tests, Equipment and Techniques ....................46 
Appendix 3:  World Medical Association Declaration of Helsinki (2000) .....................56 
Appendix 4:  Amendment Summary of Changes ............................................................61 
Appendix 5:  Sponsor and Ora Approvals .......................................................................63 
Appendix 6:  Investigator’s Signature .............................................................................65 
 
 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 11 of 65 
List of Abbreviations 
AE 
Adverse Event 
ANCOVA 
Analysis of covariance 
CI 
Confidence interval 
CL 
Clearance 
CRF 
Case Report Form 
CRO 
Clinical (or Contract) Research Organization 
ETDRS 
Early Treatment of Diabetic Retinopathy Study 
GCPs 
Good Clinical Practices 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IMP 
Investigational Medicinal Product 
IRB 
Institutional/Independent Review Board 
ITT 
Intent-to-Treat 
i.v. 
Intravenously; Intravenous 
LOCF 
Last Observation Carried Forward 
MedDRA 
Medical Dictionary for Regulatory Activities 
NEI 
National Eye Institute 
NK 
Neurotrophic Keratopathy 
NGF 
Nerve Growth Factor 
PK 
Pharmacokinetics 
PP 
Per Protocol 
ROPI 
Report of Prior Investigations 
SAE 
Serious Adverse Event 
SD 
Standard deviation 
RGN-259 
Thymosin Beta-4 
TEAE 
Treatment-Emergent Adverse Events 
TFBUT 
Tear-Film Break-Up Time 
Vd 
Volume of distribution 
WHO Drug 
World Health Organization Drug Dictionary 
 
 
 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 12 of 65 
1 INTRODUCTION 
Neurotrophic keratopathy (NK) is a degenerative corneal disease that occurs as a 
result of partial or total impairment of trigeminal innervation. The resulting loss 
of corneal sensitivity (anesthesia) leads to a reduction in lacrimation and a decline 
in status, metabolism, and mitosis of corneal epithelial cells. Ultimately NK 
results in a deficiency in epithelial repair, stromal edema, loss of microvilli, and 
abnormal basal lamina homeostasis.  Patients with NK develop poorly-healing, 
recurrent corneal abrasions or defects that are slow to respond to current 
therapies.  NK is most commonly associated with diabetes or herpes zoster-
infected patients, but can also be the result of trauma to the eye or the trigeminal 
nerve.  While the condition is rare, there is a significant need for improvement in 
therapeutic options, as the wounds associated with NK can be threatening to the 
eyesight.   
1.1 The Pharmacology of Thymosin β4 (Tβ4) 
Tβ4 is a synthetically produced copy of a naturally occurring 43-amino acid 
peptide. The natural peptide is found in high concentrations in the majority of 
tissue types, with the highest concentrations in blood platelets and white blood 
cells.  It is also found extracellularly in blood plasma or in wound fluid (Huff, 
2001).  It is one of a family of at least 16 highly conserved peptides, collectively 
called the β-thymosins, which are present in high concentrations in almost every 
cell type.  
 
Tβ4 has wound healing and anti-inflammatory properties. It is thought to exert its 
therapeutic effect through promotion of keratinocyte and endothelial cell 
migration, increased collagen deposition, and stimulation of angiogenesis (Huff, 
2002).  It is released from platelets and is selectively cross-linked by factor 
XIIIa, a tissue transglutaminase, to molecules including actin, collagen, fibrin, 
and fibrinogen (Malinda, 1999).  Tβ4 also up-regulates expression of laminin-5, 
a protein involved in adhesion and migration of cells that function in the wound-
healing process (Sosne 2004).  It also inhibits the activation of the transcription 
factor (nuclear factor κB, NFκB), a key regulator of inflammatory responses 
(Sosne 2007). 
1.2 Toxicology Overview 
Tβ4 plays a role in vascular biology by acting as a chemo-attractant for 
endothelial cells, and stimulating angiogenesis, promoting cell migration, and 
attenuating apoptotic activities in some cells.  These properties suggest a 
potential for Tβ4 to play a role in tumor metastasis, but the data supporting such 
potential is equivocal.  While various reports have shown that Tβ4 is upregulated 
in a number of metastatic cells, a correlation between Tβ4 expression and 
malignancy has not been established (Yamamoto, 1993).  Forced over-
expression of Tβ4, either in vitro or in vivo, does not induce tumorogenesis (Cha, 
2003; 2010). 
 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 13 of 65
There is no structure-activity relationship suggesting carcinogenic risk, and no 
evidence of pre-neoplastic lesions in any repeat-dose toxicity study of Tβ4.  In 
twenty-four nonclinical toxicology and safety pharmacology studies, ReGenTree 
has established the safety of Tβ4 for its current and planned uses in man.  
Because of its high concentration and ubiquitous distribution, it has been 
suggested that Tβ4 functions as both an intracellular structural element and as a 
regulatory molecule when released from cells either by secretion, cell death, or 
lysis (Goldstein et al., 2005).  It is likely that its high endogenous concentration 
is, at least in part, the driving force behind the high safety factor and negligible 
toxicologic effects observed with pharmacological doses of Tβ4.  
 
Developmental toxicology was examined in a series of studies using both rats and 
rabbits as test species (study numbers 20024503, 20024504, 20024505, and 
20024506).  For each species, a dose-finding screen was followed by a study 
with daily dosing of animals throughout pregnancy.  There were no adverse 
effects observed to either maternal or embryonic/fetal animals in any of these 
studies.  The no observable adverse effect level (NOAEL) for rats was 
 
.  These were the highest doses used in 
each species.  
 
An ocular toxicity study (BGY00015) was also performed to assess the potential 
ocular and systemic toxicity of Tβ4 ophthalmic drops.  Rabbits received 4 drops, 
4 times daily for 28 consecutive days.  None of the 3 doses used 
 
caused any local or systemic toxicity, and none elicited any other 
adverse ocular effects. 
  
A total of 108 patients have been enrolled in 4 clinical studies employing the 
0.1% concentration of Tβ4 ophthalmic drops, and the drug has been shown to be 
safe and well-tolerated.  The largest of these, a 72-patient trial of 0.1% Tβ4 
ophthalmic drops for treatment of dry eye, reported no significant adverse effects 
and no clinically significant changes in any safety parameters.   
1.3
Pharmacokinetics and Dose-Range Selection 
The plasma pharmacokinetics (PK) of daily doses of 
mg/kg/day Tβ4 
for 28 days were characterized following single and repeat bolus intravenous (iv) 
injections to male and female Sprague-Dawley rats (Study 20018593). The T1/2 
estimate ranged from 
 hours for males and from 
 hours 
for females. The volume of distribution (Vd) estimates for both males and females 
indicated distribution beyond the vasculature. The clearance (CL) ranged from 
 mL/h/kg and 
mL/h/kg and 
 mL/h/kg for males and 
females, respectively. The increases in exposure between 
 mg/kg/day 
were approximately dose proportional on Days 1 and 28. Exposure to Tβ4 tended 
to be higher for males than for females. No notable accumulation of Tβ4 was 
observed following repeated administration for 28 days. In conclusion, 
administration of Tβ4 Injectable Solution by once daily iv injection was well 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 14 of 65
tolerated in rats at levels up to 
mg/kg/day.  Based on these results, the no-
observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day. 
 
To further assess PK activity of Tβ4, radiolabled 14C-Tβ4 was administered iv to 
male and female Sprague-Dawley rats (Study 2116-001). The peak of 
radioactivity (Tmax) in plasma was observed at 5 minutes post-dose (first time 
point assessed) for both males and females. For both sexes, the Cmax at 5 minutes 
post-dose was followed by a steep decline to 4 hours (distribution phase), with a 
much more gradual decline through 48 hours (elimination phase). There were no 
differences between the plasma PK curves for males and females. Whole blood 
was also measured for both sexes, and as with plasma, there were essentially no 
differences between the blood PK curves for males and females. No significant 
distribution to the erythrocytes was noted through 24 hours for either sex.  
 
While there were no Phase 1 studies of ophthalmic formulations of Tβ4, a double-
blind, randomized, placebo-controlled, dose response study was conducted using 
four cohorts with 10 healthy subjects each given a single intravenous dose of Tβ4 
or placebo. Cohorts received ascending doses of either 
 mg 
of Tβ4 (Phase 1A). Following safety review of the Phase 1A data, subjects in 
Phase 1B received the same doses administered to those in Phase 1A but daily for 
14 consecutive days. Safety evaluations, incidence of unexpected adverse events 
(AEs) and PK parameters were evaluated. AEs were infrequent and mild and 
moderate in intensity. There were no deaths, no dose limiting toxicities or SAEs. 
The PK profile showed a dose proportional response, increasing half-life with 
increasing dose and minimal drug accumulation.  
 
For the previous dry eye clinical trial (RGN-DE-202), the 0.1% concentration of 
Tβ4 was chosen and was shown to be safe, well tolerated, and produced clinically 
significant effects in ocular discomfort and corneal staining.  Following this, 
physician-sponsored studies used 0.1% Tβ4 under a compassionate IND to treat 
nine patients with NK, six of whom had discrete geographic, non-healing lesions, 
and three of whom had punctate lesions. The physicians reported that the six with 
discrete geographic lesions completely healed or almost completely healed during 
the treatment course or shortly thereafter using the 0.1% concentration four times 
daily (Dunn, 2010a).  For these reasons, we propose to use this same 
concentration of 0.1% Tβ4 for the studies of NK.  
 
1.4
Study Rationale 
ReGenTree’s plan is to develop 0.1% Tβ4, formulated as RGN-259 sterile, 
preservative-free eye drops, as an agent to treat ocular surface defects associated 
with dry eye, neurotrophic keratopathy, chemical injury, or other underlying 
pathologies or injuries to the eye. This course of development is suggested by the 
limited pharmacological interventions for enhancing corneal wound healing, 
repair and regeneration and by the known activities of RGN-259. The selection of 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 15 of 65
NK as a condition to assess treatment efficacy is based on the known spectrum of 
RGN-259 activity and the lack of available treatments for NK. 
2
STUDY OBJECTIVES 
The objective of this study is to assess the safety and efficacy of RGN-259 Ophthalmic 
Solution compared to placebo for the treatment of NK after a 4 week, 5 times per day, 
treatment period and a 2-week post-treatment follow up.
3
CLINICAL HYPOTHESIS 
The clinical hypothesis for this study is the following: 
x
Significantly more subjects receiving RGN-259 ophthalmic solution will 
have a clearing of a persistent epithelial defect at Visit 5 (Day 29) when 
compared to subjects receiving placebo.
4
OVERALL STUDY DESIGN 
This is a Phase 3, multicenter, randomized, double-masked study designed to evaluate the 
efficacy and safety of RGN-259 ophthalmic solution compared to placebo in patients 
with neurotrophic keratopathy. Approximately 46 male and female patients at least 18 
years of age with stage 2 or stage 3 neurotrophic keratopathy (Mackie Classification) and 
meeting all other study eligibility criteria will be randomized to receive treatment with 
. This study will consist of two periods: a 28-day 
treatment period and a 14-day post-treatment period. A study flow chart appears below: 
Visit 1
(Day 1):
Screening/Enrollment/Baseline
Visit 2
(Day 8 ± 2):
One-Week Follow-Up
Visit 3


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 16 of 65
(Day 15 ± 2):
Two-Week Follow-Up
Visit 4
(Day 22 ± 2):
Three-Week Follow-Up
Visit 5
(Day 29 ± 2):
Four-Week Follow-Up
Visit 6
(Day 36 ± 3):
One-Week Post-
Treatment Follow-Up
Visit 7
(Day 43 ± 3):
Two-Week Post-
Treatment Follow-Up
Patients who terminate early during the treatment period or post-treatment period will be 
asked to complete safety assessments. Patients who are terminated early from the study 
will not be replaced. 
In this study, the vehicle of RGN-259 ophthalmic solution has been selected as the 
placebo control to objectively evaluate the efficacy and safety of RGN-259 ophthalmic 
solution. In addition, randomized assignment and double-masked design features have 
been implemented to minimize any bias for patient selection and data evaluation. 
5
STUDY POPULATION 
5.1
Number of Subjects (approximate) 
It is estimated that approximately 100 subjects will be screened to enroll 
approximately 46 subjects at approximately 20 clinical sites in the US.  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 17 of 65
5.2
Study Population Characteristics 
Subjects diagnosed with neurotrophic keratopathy in at least one eye and who 
meet all inclusion and exclusion criteria. 
5.3
Inclusion Criteria
Subjects must: 
a.
Be male or female of any race, at least 18 years of age at Visit 1. 
b. Have provided verbal and written informed consent; 
c.
Be able and willing to follow instructions, including participation in all 
study assessments and visits; 
d. Have a persistent epithelial defect (a defect that has not reduced in 
size) for at least 
 with failure of conventional, nonsurgical 
treatment using non-preserved ocular lubricants, non-preserved topical 
ophthalmic antibiotics, oral doxycycline, patching, serum tears and/or 
therapeutic contact lenses at the time of Visit 1; 
e.
Have stage 2 or 3 neurotrophic keratopathy (Mackie Classification) in 
at least one eye of which the longest dimension (length or width) of 
the defect 
and which is 
confirmed not to be simply superficial punctate keratitis, at Visit 1; 
f.
Have evidence of decreased corneal sensitivity
 at Visit 1; 
g. Have at least one eye (the same eye) satisfy all criteria for d, e, f above 
h. If a female of childbearing potential, have a negative urine pregnancy 
test at Visit 1 and agree to use an adequate method of birth control 
throughout the study period. [Females are considered of childbearing 
potential unless they are surgically sterilized (bilateral tubal ligation, 
hysterectomy or bilateral oophorectomy) or post-menopausal (at least 
12 months since last menses). Adequate birth control is defined as use 
of hormonal contraceptives (oral, implantable, injectable or 
transdermal), spermicide in conjunction with a barrier such as condom 
or diaphragm, or an intrauterine device (IUD), or surgical sterilization 
of partner. For non-sexually active females, abstinence may be 
regarded as an adequate method of birth control; however, if the 
patient becomes sexually active during the study, she must agree to use 
adequate birth control as defined above for the remainder of the study.] 
5.4
Exclusion Criteria
Subjects must not: 
a.
Have any clinically significant slit lamp findings at Visit 1 that in the 
opinion of the investigator may interfere with the study parameters; 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 18 of 65 
b. Have significant blepharitis, meibomian gland dysfunction (MGD), lid 
margin inflammation or active ocular allergy that requires treatment at 
Visit1; 
c. Have a lid function abnormality (ex. Lagophthalmos) which, in the 
opinion of the investigator, is the primary cause of the persistent 
epithelial defect;  
d. Be diagnosed with ongoing ocular infection (bacterial, viral or fungal) 
or active inflammation (e.g. follicular conjunctivitis) not related to NK 
in the affected eye at Visit 1; 
e. Have had any ocular surgical procedure that is not considered the 
primary cause of NK in the affected eye within 2 months prior to Visit 
1, or have any scheduled ocular surgical procedure during the study 
period; 
f. Have had any ocular surgical procedure that is considered to be the 
cause of NK within 4 weeks prior to Visit 1; 
g. Have received Botox injection for blepharoptosis within the previous 
90 days in the affected eye; 
h. Have started the use of topical steroids in the affected eye in the 
previous 2 weeks or systemic steroids in the previous 4 weeks; 
i. Have used contact lenses (excluding therapeutic contact lenses) within 
14 days prior to Visit 1 or anticipates use of contact lenses during the 
study period; 
j. Have an uncontrolled systemic disease that in the opinion of the 
investigator may interfere with the study parameters; 
k. Anticipate a change in immunosuppressive therapy during the course 
of the study; 
l. Have a known allergy and/or sensitivity to the study drug or its 
components; 
m. Be a woman who is pregnant, nursing or planning a pregnancy; 
n. Be unwilling to submit a urine pregnancy test at Visit 1, Visit 5 and 
Visit 7 (or early termination visit) if of childbearing potential;  
o. Be currently enrolled in an investigational drug or device study or 
have used an investigational drug or device within 30 days of Visit 1; 
p. Have undergone surgical treatments for NK including tarsorrhaphy or 
amniotic membrane graft within the previous 6 weeks in the affected 
eye; 
q. Plan to use serum tears in the affected eye during the study period. 
 
If at any time during the study the Investigator determines that a subject’s 
safety has been compromised, the subject may be withdrawn from the study. 
Subjects may withdraw consent from the study at any time. ReGenTree and/or 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 19 of 65
the Investigator may discontinue any subject for non-compliance or any valid 
medical reason (see Section 8.6.2). 
6
STUDY PARAMETERS 
6.1
Efficacy Measures  
6.1.1
Primary Efficacy Measure(s) 
x
   
6.1.2
Secondary Efficacy Measure(s) 
x
x
   
x
x
x
6.2
Safety Measures 
x
Visual Acuity (Early Treatment of Diabetic Retinopathy Study, 
ETDRS); 
x
Slit-Lamp Biomicroscopy; 
x
Cochet-Bonnet; 
x
Adverse Event Query; 
x
Dilated Fundoscopy; 
x
Intraocular Pressure. 
7
STUDY MATERIALS 
7.1
Study Treatment(s)
7.1.1  Study Treatments/Formulations/Composition  
1)
0.1% RGN-259 Ophthalmic Solution 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 20 of 65 
2) 
Placebo Ophthalmic Solution (Vehicle for RGN-259 Ophthalmic 
Solution) 
7.1.2  Instructions for Use and Administration 
x At the end of Visit 1, qualified subjects will be randomized and the 
first dose of Study Drug will be administered in the Clinic. A 1-
week supply of RGN-259 Ophthalmic Solution or Placebo 
Ophthalmic Solution will be dispensed to use 5 times per day;   
x At Visits 2, 3 and 4, remaining/used study drug will be collected 
from subjects for drug accountability.  Subjects will receive an 
additional 1 week supply of the same study drug they were 
previously randomized to (at Visit 1), to use 5 times per day for 1 
week; 
x At Visit 5, remaining/used study drug will be collected from 
subjects for drug accountability.  
x Subjects will be instructed to use study drug on the day of 
applicable visits (Visits 2, 3, 4, and 5) prior to the visit.  
7.2 Other Study Supplies 
HCG urine pregnancy tests, Wratten #2 yellow filter, sodium fluorescein or 
BioGloTM Fuorescein Strips, 1% Tropicamide, and Fluoress. 
 
8 STUDY METHODS AND PROCEDURES 
8.1 Subject Entry Procedures 
8.1.1 
Overview 
Subjects as defined by the criteria in Sections 5.2, 5.3, and 5.4 will be 
considered for entry into this study. 
8.1.2 
Informed Consent 
Prior to a subject’s participation in the trial (i.e., prior to changes in a 
subject’s medical treatment and/or study related procedures), the study 
will be discussed with each subject, and subjects wishing to participate 
must give written informed consent  using an informed consent form.  
The informed consent form must be the most recent version that has 
received approval/favorable review by a properly constituted 
Institutional Review Board. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 21 of 65 
8.1.3 
Washout Intervals 
Prohibited medications, treatments, and activities are outlined in the 
Exclusion Criteria (Section 5.4). 
8.1.4 
Procedures for Final Study Entry 
Meet all inclusion and exclusion criteria. 
8.1.5 
Methods for Assignment to Treatment Groups: 
At Visit 1, subjects who provide written informed consent will be 
assigned a unique 5-digit screening number, which includes the 2-digit 
site number plus a unique 3-digit screening number beginning with 
001 (e.g., 11001, 11002, 11003, etc.). Screening numbers must be 
assigned in ascending consecutive order. 
If all inclusion and exclusion criteria are met at Visits 1, each 
qualifying subject will then be assigned a 4-digit randomization 
number at the end of Visit 1. All randomization numbers will be 
assigned in strict numerical sequence and no numbers will be skipped 
or omitted.  Assignment of qualified subjects to a treatment group 
will be accomplished by using a randomization code generated by an 
independent biostatistician.  Investigators and study staff who will 
conduct the assignment procedure will be masked to the randomization 
code.  
8.2 Concurrent Therapies 
The use of any concurrent medication, prescription or over-the-counter, is to 
be recorded on the subject’s source document and corresponding electronic 
case report form (eCRF) along with the reason the medication was taken. 
Concurrent enrollment in another investigational drug or medical device study 
is not permitted. 
8.2.1 Prohibited Medications/Treatments 
Disallowed medications/ treatments during the study are outlined in the 
Exclusion Criteria (Section 5.4). 
8.2.2 Allowed Concomitant Medications/ Treatments/ Procedures 
Subjects who are taking antibiotic eye drops or ointments or non-preserved 
artificial tears for at least two weeks at the time of screening may continue 
to use them throughout the study at the discretion of the Investigator. 
However, these medications should not be started after the subject begins the 
study. 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 22 of 65
If a subject is taking one of the above listed allowed concomitant eye drops 
or ointments during the study the subject should be instructed to dose with 
the study eye drops first and wait at least 5 minutes before dosing with the 
other medication. 
The use of these medications will be collected and recorded in the 
concomitant medications page of the eCRF. 
8.2.3
Escape Medications 
At the discretion of the investigator, subjects may use topical steroid as an 
escape medication. Subjects receiving the escape medications will be 
exited, and will be counted as subjects who did not have complete healing 
of the epithelial defect in the primary efficacy analysis.  
8.2.4
Escape Surgical Intervention
At the discretion of the investigator, subjects may undergo surgical 
intervention (ex. tarsorraphy, amniotic membrane graft) to address their 
persistent epithelial defect. Subjects receiving surgical intervention will be 
exited, and will be counted as subjects who did not have complete healing 
of the epithelial defect in the primary efficacy analysis. 
8.2.5
Special Diet or Activities 
No special diets or activities are required for this study. 
8.3
Examination Procedures 
8.3.1
Procedures to be Performed at Each Study Visit with Regard to Study 
Objective(s) 
Visit 1 (Day 1):  Screening/Enrollment/Baseline
The following procedures will be performed: 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 23 of 65
Visit 2 (Day 8 ± 2):  One-Week Follow-Up
The following procedures will be performed: 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 24 of 65
 
Visit 3 (Day 15 ± 2):  Two-Week Follow-Up
The following procedures will be performed: 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 25 of 65
Visit 4 (Day 22 ± 2):  Three-Week Follow-Up
The following procedures will be performed: 
Visit 5 (Day 29 ± 2):  Four-Week Follow-Up
The following procedures will be performed: 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 26 of 65
  
 
Visit 6 (Day 36 ± 3):  One-Week Post-Treatment Follow-Up
The following procedures will be performed: 
Visit 7 (Day 43 ± 3):  Two-Week Post-Treatment Follow-Up
The following procedures will be performed: 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 27 of 65
  
Adverse Events (both elicited and observed) will be monitored throughout the 
study.  All adverse events (both elicited and observed) will be promptly 
reviewed by the Investigator for accuracy and completeness.  All adverse events 
will be documented on the appropriate Source Document and electronic case
report form (eCRF).
If a female has a positive pregnancy test during the study, then the Investigator 
will notify Ora immediately.  The Investigator shall request from the subject 
and/or the subject’s physician copies of all related medical reports during the 
pregnancy and shall document the outcome of the pregnancy.  The Investigator 
will retain these reports together with the subject’s source documents and will 
provide a copy of all documentation to Ora.
8.4
Schedule of Visits, Measurements and Dosing 
8.4.1
Scheduled Visits 
8.4.2
Unscheduled Visits


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 28 of 65
8.4.3
Early Termination Visits 
These visits will be performed for any subject that is discontinued from 
the study prior to completion (for reasons listed in Section 8.6.2). All 
procedures performed at an early termination visit will be recorded in the 
source documents and on the appropriate eCRF pages. Any procedure 
indicated in the eCRF that is not performed should be indicated as “Not 
done.”
Evaluations that may be conducted at an Early Termination Visit include: 
8.5
Compliance with Protocol 
Subjects will be instructed on proper instillation and storage of study drug 
at the end of Visits 1, 2, 3, and 4 and given written instructions. Study 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 29 of 65
drug will be collected at each visit up to and including Visit 5 to assess 
dosing compliance. Dosing compliance will be based on the used vial 
count. If the subject uses 
 study drug 
during a dosing period, a subject will be deemed non-compliant and a 
dosing deviation will be recorded. These guidelines will be used by the 
Investigator for determining the subject’s necessary compliance for the 
study and for recording deviations from this compliance.  
8.6
Subject Disposition 
8.6.1
Completed Subjects 
A completed subject is one who has not been discontinued from the 
study.
8.6.2
Discontinued Subjects 
Subjects may be discontinued prior to their completion of the study due to: 
x
subject no longer willing to comply with study protocol; 
x
adverse events; 
x
protocol violations;
x
lack of efficacy;
x
administrative reasons (e.g. inability to continue, lost to follow-up); 
x
Sponsor termination of study; 
x
any sound medical reason in the Investigator’s opinion.
Notification of a subject discontinuation and the reason for discontinuation 
will be made to Ora, Inc. and/or ReGenTree and will be clearly 
documented on the eCRF. 
8.7
Study Termination 
The study may be stopped at any time by the Investigator, ReGenTree, and/or 
Ora with appropriate notification.
8.8
Study Duration 
An individual subject’s participation will involve 7 visits over approximately 
a 6-week period. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 30 of 65 
8.9 Monitoring and Quality Assurance 
During the course of the study an Ora, Inc. will make routine site visits to 
review protocol compliance, ensure subject safety, assess study drug 
accountability, and ensure the study is being conducted according to the 
pertinent regulatory requirements.  The review of the subjects’ medical 
records will be performed in a manner that adequately maintains subject 
confidentiality.  Further details of the study monitoring will be outlined in a 
monitoring plan. 
Regulatory authorities of domestic and foreign agencies, Ora, Inc. Quality 
Assurance, ReGenTree, and/or its designees may carry out on-site inspections 
and/or audits which may include source data checks.  Therefore direct access 
to the original source data will be required for inspections and/or audits.  All 
inspections and audits will be carried out giving consideration to data 
protection as well as subject confidentiality to the extent that local, state, and 
federal laws apply. 
9 ADVERSE EVENTS 
9.1 Adverse Event 
An adverse event (AE) is defined as any untoward medical occurrence 
associated with the use of an investigational medicinal product (IMP) in 
humans, whether or not considered IMP-related. An AE can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an investigational 
product, without any judgment about causality. An AE can arise from any use 
of the investigational product (e.g. off-label use, use in combination with 
another drug or medical device) and from any route of administration, 
formulation, or dose, including an overdose.  
All AEs spontaneously reported by the subject and/or in response to an open 
question from study personnel or revealed by observation, physical 
examination or other diagnostic procedures will be recorded in the source 
document and on the appropriate sections of the eCRF. Any clinically relevant 
deterioration in clinical finding is considered an AE and must be recorded. 
When possible, signs and symptoms indicating a common underlying 
pathology should be noted as one comprehensive event. 
Documentation regarding the AE should be made as to the nature, date of 
onset, end date, severity, and relationship to investigational product, action(s) 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 31 of 65 
taken, seriousness, and outcome of any sign or symptom observed by the 
physician or reported by the patient upon indirect questioning.  
9.1.1 
Severity 
Severity of an AE is defined as a qualitative assessment of the degree 
of intensity of an AE as determined by the Investigator or reported to 
him/her by the patient/subject.  The assessment of severity is made 
irrespective of relationship to investigational product or seriousness of 
the event and should be evaluated according to the following scale: 
x Mild: Event is noticeable to the subject, but is easily tolerated and 
does not interfere with the subject’s daily activities. 
x Moderate:  Event is bothersome, possibly requiring additional 
therapy, and may interfere with the subject’s daily activities. 
x Severe:  Event is intolerable, necessitates additional therapy or 
alteration of therapy, and interferes with the subject’s daily 
activities. 
9.1.2 
Relationship to Investigational Product 
The relationship of each AE to the investigational product should be 
determined by the investigator using these explanations: 
x Definite: When there are good reason and sufficient 
documentation to demonstrate a direct causal relationship 
between investigational product and AE; 
x Probable: When there are good reasons and sufficient 
documentation to assume a causal relationship in the sense of 
plausible, conceivable, likely but not necessarily highly probable. 
x Possible: When there is sufficient information to accept the 
possibility of a causal relationship in the sense of not impossible 
and not unlikely, although the connection is uncertain or doubtful, 
for example; due to missing data or insufficient evidence. 
x None: When there is sufficient information to accept a lack of a 
causal relationship, in the sense of impossible and improbable. 
x Unclassified: When the causal relationship is not assessable for 
whatever reason due to insufficient evidence, conflicting data or 
poor documentation. 
9.1.3 
Expectedness 
The expectedness of an AE should be determined based upon existing 
safety information about the investigational product using these 
explanations: 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 32 of 65 
x Unexpected:  an AE that is not listed in the Investigator’s 
Brochure (IB) or Report of Prior Investigations (ROPI) or is not 
listed at the specificity or severity that has been observed. 
x Expected:  an AE that is listed in the IB or ROPI at the specificity 
and severity that has been observed. 
x Not applicable: an AE unrelated to the investigational product. 
Adverse events that are mentioned in the IB as occurring with a class 
of products or as anticipated from the pharmacological/ mechanical (or 
other) properties of the product, but are not specifically mentioned as 
occurring with the particular product under investigation are to be 
considered unexpected. 
The Investigator should initially classify the expectedness of an AE, 
but the final classification is subject to the Medical Monitor’s, 
ReGenTree’s, and Ora’s determination. 
9.2 Serious Adverse Events 
An AE is considered serious if, in the view of either the Investigator or 
Sponsor, it results in any of the following outcomes: 
x Death; 
x A life-threatening AE; 
o Note:  An AE is considered “life-threatening” if, in the view of either 
the Investigator or Sponsor, its occurrence places the patient or subject 
at immediate risk of death.  It does not include an AE that, had it 
occurred in a more severe form, might have caused death. 
x Inpatient hospitalization or prolongation of existing hospitalization; 
o Note:  The term “inpatient hospitalization” refers to any inpatient 
admission (even if less than 24 hours).  For chronic or long-term 
inpatients, inpatient admission includes transfer within the hospital to 
an acute/intensive care inpatient unit.  Inpatient hospitalization does 
not include:  emergency room visits; outpatient/same-day/ambulatory 
procedures; observation/short stay units; rehabilitation facilities; 
hospice facilities; nursing homes; or clinical research/Phase 1 units. 
o Note:  The term “prolongation of existing hospitalization” refers to 
any extension of an inpatient hospitalization beyond the stay 
anticipated or required for the reason for the initial admission as 
determined by the Investigator or treating physician. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 33 of 65 
x A persistent or significant incapacity or substantial disruption of the ability 
to conduct normal life functions; 
o Note: A serious AE (SAE) specifically related to visual threat would 
be interpreted as any potential impairment or damage to the subject’s 
eyes (eg, hemorrhage, retinal detachment, central corneal ulcer or 
damage to the optic nerve). 
x A congenital anomaly/birth defect. 
Important medical events that may not result in death, are 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. 
9.3 Procedures for Reporting Adverse Events 
All AEs and their outcomes must be reported to Ora, ReGenTree, and the 
IRB/IEC as required by the IRB/IEC, federal, state, or local regulations and 
governing health authorities and recorded on the appropriate eCRF. 
9.3.1 
Reporting a Suspected Unexpected Adverse Reaction 
All AEs that are ‘suspected’ and ‘unexpected’ are to be reported to 
Ora, ReGenTree, and the IRB/IEC as required by the IRB/IEC, 
federal, state, or local regulations and governing health authorities. 
9.3.2 
Reporting a Serious Adverse Event 
To ensure subject safety, all SAEs, regardless of relationship to the 
investigational product, must be immediately reported.  All 
information relevant to the SAE must be recorded on the appropriate 
case report forms.  The Investigator is obligated to pursue and obtain 
information requested by Ora and/or ReGenTree in addition to that 
information reported on the case report form.  All subjects 
experiencing an SAE must be followed up and the outcome reported. 
In the event of an SAE, the Investigator must notify Ora and 
ReGenTree immediately; obtain and maintain in his/her files all 
pertinent medical records, information, and medical judgments from 
colleagues who assisted in the treatment and follow-up of the subject; 
provide Ora and ReGenTree with a complete case history, which 
includes a statement as to whether the event was or was not suspected 
to be related to the use of the investigational product; and inform the 
IRB of the AE within their guidelines for reporting SAEs. 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 34 of 65
Contact information for reporting SAEs: 
9.4
Procedures for Unmasking (if applicable) 
All subjects, investigators, and study personnel involved with the conduct of 
the study will be masked with regard to treatment assignments. When 
medically necessary, the investigator may need to determine what treatment 
has been assigned to a patient. When possible (i.e., in non-emergent 
situations), Ora and/or ReGenTree should be notified before unmasking study 
drug.  
Serious unexpected suspected adverse reactions, which are subject to 
expedited reporting, will be unmasked before submission to the Regulatory 
Authorities. The procedure for unmasking will be described in a separate 
Safety Plan for this study. 
9.5
Type and Duration of the Follow-up of Subjects after Adverse 
Events 
The Investigator will follow unresolved AEs to resolution until the patient is 
lost to follow-up or until the AE is otherwise re-classified.  Resolution means 
the patient has returned to baseline state of health or the Investigator does not 
expect any further improvement or worsening of the AE. If the patient is lost 
to follow-up, the Investigator should make 3 reasonable attempts to contact 
the patient via telephone, post, or certified mail. All follow-ups will be 
documented in the patient’s source document. Non-serious AEs identified on 
the last scheduled contact must be recorded on the AE eCRF with the status 
noted.  
If the Investigator becomes aware of any new information regarding an SAE 
(i.e., resolution, change in condition, or new treatment), a new 
SAE/Unanticipated Report Form must be completed and faxed to Ora and 
ReGenTree within 24 hours of the site’s awareness of the event. The original 
SAE form is not to be altered. The report should describe whether the event 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 35 of 65
has resolved or continues and how the event was treated. STATISTICAL 
HYPOTHESES AND METHODS OF ANALYSES 
9.6
Study Populations 
The following analysis populations are considered:
x
   
x
  
   
x
9.7
Efficacy measures: 
Primary Efficacy Measures: 
x
   
Secondary Efficacy Measure(s) 
x
x
   
x
x
x
9.8
Safety measures: 
x
Visual Acuity (ETDRS); 
x
Slit-Lamp Biomicroscopy; 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 36 of 65
x
Cochet-Bonnet;
x
Adverse Event Query; 
x
Dilated Fundoscopy; 
x
Intraocular Pressure. 
9.9
Statistical Hypothesis and Sample Size 
H0: 
There is no difference between study eyes treated with RGN-259 and study 
eyes treated with placebo, RGN-259 – placebo, in the percentage of study eyes 
achieving complete healing of epithelial defect at Day 29 (Visit 5). 
HA: 
There is a difference between study eyes treated with RGN-259 and study eyes 
treated with placebo, RGN-259 – placebo, in the percentage of study eyes 
achieving complete healing of epithelial defect at Day 29 (Visit 5). 
The study will be considered a success if H0 is rejected in favor of HA 
 
  
  
 
  
9.10 Statistical Analysis 
9.10.1 General Considerations 
  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 37 of 65
9.10.2 Unit of Analysis 
  
  
  
  
9.10.3 Missing Data 
9.10.4  Primary Efficacy Analyses 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 38 of 65
9.10.5 Secondary Efficacy Analyses 
  
 
   
 
 
 
  
 
  
9.10.6 Adverse Events 
Adverse events will be coded using the MedDRA dictionary. Frequencies 
and percentages of subjects with treatment-emergent adverse events 
(TEAEs), serious TEAEs, and TEAEs causing premature discontinuation 
will be provided by treatment group. An AE is treatment emergent if it 
occurs or worsens after the first dose of study treatment. Furthermore, 
frequencies will be given of subjects with TEAEs by  
x
System organ class and preferred term,  
x
System organ class, preferred term and maximal severity,  
x
System organ class and preferred term for related TEAEs (at least 
possibly related or unknown relationship), and  
x
System organ class, preferred term and maximal severity for 
related TEAEs.  
Separate summaries will be provided for ocular (study eye and any treated 
eye) and non-ocular TEAEs.


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 39 of 65 
9.10.7 Laboratory Data 
N/A 
9.10.8 Interim Analyses 
No interim analyses are planned for this study. 
9.10.9 Quality Assurance of Data Processing 
To ensure accurate, complete, and reliable data, ReGenTree or its 
representatives will provide instructional material to the study sites, as 
appropriate; instruct the investigators and study personnel on the protocol, 
the completion of the eCRFs, and study procedures; communicate 
regularly with site personnel via mail, email, telephone, and/or fax; and 
make periodic visits to the study site. During those visits, ReGenTree or 
its representatives will monitor the subject data recorded in the eCRF 
against source documents at the study site. ReGenTree or its 
representatives will review and evaluate eCRF data and use standard 
computer edits to detect errors in data collection.  When the database has 
been declared to be complete and accurate, the database will be locked and 
the treatment codes will be unmasked. Any changes to the database after 
that time can only be made by joint written agreement between 
ReGenTree, Ora, Inc. and the Study Biostatistician. 
AEs and medical history will be coded using MedDRA version 17.0 or 
higher, and concomitant medications will be coded using WHO Drug, 
Enhanced B2, September 2015 version or higher.   
 
 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 40 of 65 
10 COMPLIANCE WITH GOOD CLINICAL PRACTICES, 
ETHICAL CONSIDERATIONS, AND ADMINISTRATIVE 
ISSUES 
This study will be conducted in compliance with the protocol, current Good Clinical 
Practices (GCPs), including the International Conference on Harmonization (ICH) 
Guidelines, and in general, consistent with the Declaration of Helsinki.  In addition, 
all applicable local, state, and federal requirements relevant to the use of 
investigational products in the countries involved will be adhered to. 
10.1 Protection of Human Subjects 
10.1.1 
Subject Informed Consent 
Informed consent/assent must take place before any study specific 
procedures are initiated.  Signed and dated written informed consent 
must be obtained from each subject.  
All informed consent/assent forms must be approved for use by 
ReGenTree and receive approval/favorable opinion from an IRB/IEC 
prior to their use.  If the consent form requires revision (eg, due to a 
protocol amendment or significant new safety information), it is the 
Investigator’s responsibility to ensure that the amended informed 
consent is reviewed and approved by Ora prior to submission to the 
governing IRB/IEC and that it is read, signed and dated by all subjects 
subsequently enrolled in the study as well as those currently enrolled 
in the study. 
10.1.2 
Institutional Review Board (IRB) Approval 
This study is to be conducted in accordance with Institutional Review 
Board regulations (U.S. 21 CFR Part 56.103).  The Investigator must 
obtain appropriate IRB approval before initiating the study and re-
approval at least annually. 
Only an IRB/ERC approved version of the informed consent form will 
be used. 
10.2 Ethical Conduct of the Study 
This study will be conducted in accordance with the ethical principles that 
originated with the Declaration of Helsinki (Appendix 3). 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 41 of 65 
10.3 Subject Confidentiality 
All personal study subject data collected and processed for the purposes of 
this study should be maintained by the Investigator and his/her staff with 
adequate precautions as to ensure that the confidentiality of the data in 
accordance with local, state, and federal laws and regulations. 
Monitors, auditors and other authorized representatives of Ora, ReGenTree, 
the IRB/IEC approving this study, the FDA, the DHHS, other domestic 
government agencies, and other foreign regulatory agencies will be granted 
direct access to the study subject’s original medical and study records for 
verification of the data and/or clinical trial procedures.  Access to this 
information will be permitted to the aforementioned individuals to the extent 
permitted by law. 
A report of the results of this study may be published or sent to the 
appropriate health authorities in any country in which the investigational 
product may ultimately be marketed, but the subject’s identity will not be 
disclosed in these documents. 
10.4 Documentation 
Source documents may include a subject’s medical records, hospital charts, 
clinic charts, the Investigator’s study subject files, as well as the results of 
diagnostic tests such as X-rays, laboratory tests, and ECGs.  The 
Investigator’s copy of the Case Report Forms serves as the Investigator’s 
record of a subject’s study-related data. 
10.4.1 
Retention of Documentation 
All study related correspondence, patient records, consent forms, 
record of the distribution and use of all investigational products and 
copies of case report forms should be maintained on file for at least 
two years after the last approval of a marketing application in an ICH 
region and until there are no pending or contemplated marketing 
applications in an ICH region; or until at least two years have elapsed 
since the formal discontinuation of clinical development of the 
investigational product. These documents will be retained for a longer 
period if required by the applicable regulatory requirements or by an 
agreement with ReGenTree.  It is the responsibility of the ReGenTree 
or it’s representative to inform the Investigator/institution as to when 
these documents no longer need to be retained. 
If the responsible Investigator retires, relocates, or for other reasons 
withdraws from the responsibility of keeping study records, custody 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 42 of 65 
must be transferred to a person who will accept the responsibility.  
ReGenTree must be notified in writing of the name and address of the 
new custodian. 
10.5 Labeling, Packaging, Storage, Accountability, and Return or 
Disposal of Investigational Product 
10.5.1 
Labeling/Packaging 
This is a masked study and the subject numbers are assigned as described in 
Section 8.1.5. Pouches of five monodoses will be packed in clinical kits. Pouches 
and clinical kits will be labeled according to the applicable regulatory 
requirements. The labels will include instructions for use and storage.  
10.5.2 
Storage of Investigational Product 
The investigational product must be stored in a secure area, accessible 
only to the Investigator and his/her designees, refrigerated at 2-8º C, 
and protected from freezing and strong light.  The investigational 
product will be administered only to subjects entered into the clinical 
study, in accordance with the conditions specified in this protocol. 
Subjects will be provided with detailed instructions on drug storage 
and accountability. 
10.5.3 
Accountability of Investigational Product 
The investigational product is to only be prescribed by the principal 
Investigator or his/her named Sub Investigator(s), and is to only be 
used in accordance with this protocol.  The investigational product 
must only be distributed to subjects properly qualified under this 
protocol to receive investigational product. 
The Investigator must keep an accurate accounting of the 
investigational product received from the supplier. This includes the 
amount of investigational product dispensed to subjects, amount of 
investigational product returned to the Investigator by the subjects, and 
the amount returned or disposed upon the completion of the study.  A 
detailed inventory must be completed for the investigational product. 
10.5.4 
Return or Disposal of Investigational Product 
All investigational products will be returned to ReGenTree or their 
designee or destroyed at the study site.  The return or disposal of 
investigational product will be specified in writing. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 43 of 65 
10.6 Recording of Data on Source Documents and Electronic Case 
Reports Forms (eCRFs) 
The Investigator is responsible for ensuring that study data is completely and 
accurately recorded on each subject’s eCRF, source document, and all study-
related material.  All study data should also be attributable, legible, 
contemporaneous, and original.  Recorded datum should only be corrected in 
a manner that does not obliterate, destroy, or render illegible the previous 
entry (i.e., by drawing a single line through the incorrect entry and writing the 
revision next to the corrected data).  An individual who has corrected a data 
entry should make clear who made the correction and when, by adding to the 
correction his/her initials as well as the date of the correction. 
10.7 Handling of Biological Specimens 
Not applicable 
10.8 Publications 
Authorship and manuscript composition will reflect cooperation among all 
parties involved in the study.  Authorship will be established before writing 
the manuscript. The study Sponsor will have the final decision regarding the 
manuscript and publication. 
 
 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 44 of 65 
11 REFERENCES 
 
Dunn SP, Heidemann DG, Chow CYC, Crockford D, Turjman N, Angel J, Allan CB, Sosne 
G. Treatment of Chronic Nonhealing Neurotrophic Corneal Epithelial defects with 
Thymosin β4. Ann. N.Y. Acad. Sci. 2010a, 1194:199-206. 
Dunn SP, Heidemann DG, Chow CYC, Crockford D, Turjman N, Angel J, Allan CB, Sosne 
G. Treatment of Chronic Nonhealing Neurotrophic Corneal Epithelial defects with 
Thymosin β4. Archives of Ophthalmology 2010b, 128:636-638.   
Huff T, Müller CS, Otto AM, Netzker R, Hannappel E. Beta-Thymosins, small acidic 
peptides with multiple functions. Int Journal Biochem Cell Biol, 2001; 33(3):205-20. 
Huff T, Otto AM, Müller CS, Meier M, Hannappel E. Thymosin beta4 is released from 
human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and 
collagen. FASEB J, 2002; 16:691-6.Mackie IA: Neuroparalytic keratitis; in Roy FH, 
Meyer SM, Fraunfelder F (eds): Current Ocular Therapy. Philadelphia, WB Saunders, 
1995, pp 452-454. 
Malinda KM, Sidhu GS, Mani H, Banaudha K, Maheshwari RK, Goldstein AL, Kleinman 
HK.  
Thymosin beta4 accelerates wound healing. J Invest Dermatol, 1999; 113:364-8. 
Sosne G, Lihua Xu, Prach Lisa, et al. Thymosin beta 4 stimulates laminin-5 production 
independent of TGF-beta. Experimental Cell Research, 2004; 293:175-183. 
Sosne G, Qiu P, Christopherson PL, Kurpakus Wheater M. Thymosin beta 4 suppression 
of corneal NFkB: A potential anti-inflammatory pathway. Exp Eye Res, 2007, 84:663-669. 
 
 
 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 45 of 65
Appendix 1:
Schedule of Visits and Measurements 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 46 of 65
Appendix 2:
Examination Procedures, Tests, Equipment and 
Techniques 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 47 of 65
  
 
 
  
  
 
 
 
 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 48 of 65


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 49 of 65
   
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 50 of 65
 
 
  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 51 of 65
  
 
 
  
    
  
  
   


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 52 of 65
 
 
  
  
  


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 53 of 65


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 54 of 65
 
   


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 55 of 65
  
 
 
  
 
 
  
 
 
  


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 56 of 65 
Appendix 3:  World Medical Association Declaration of Helsinki 
(2000) 
Ethical principles for medical research involving human subjects. 
Adopted by the 18th WMA General Assembly Helsinki, Finland, June 1964 and amended 
by the 29th WMA General Assembly, Tokyo, Japan, October 1975. 
35th WMA General Assembly, Venice, Italy, October 1983. 
41st WMA General Assembly, Hong Kong, September, 1989. 
48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 
and the 52nd WMA General Assembly, Edinburgh, Scotland, October 2000. 
Note of Clarification on Paragraph 29 added by the WMA General Assembly, 
Washington 2002. 
A. 
Introduction 
1. The World Medical Association has developed the Declaration of Helsinki as a 
statement of ethical principles to provide guidance to physicians and other 
participants in medical research involving human subjects. Medical research 
involving human subjects includes research on identifiable human material or 
identifiable data. 
2. It is the duty of the physician to promote and safeguard the health of the people. 
The physician's knowledge and conscience are dedicated to the fulfilment of this 
duty. 
3. The Declaration of Geneva of the World Medical Association binds the physician 
with the words, ‘The health of my patient will be my first consideration’, and the 
International Code of Medical Ethics declares that ‘A physician shall act only in 
the patient's interest when providing medical care which might have the effect of 
weakening the physical and mental condition of the patient’. 
4. Medical progress is based on research which ultimately must rest in part on 
experimentation involving human subjects. 
5. In medical research on human subjects, considerations related to the well-being of 
the human subject should take preference over the interests of science and society. 
6. The primary purpose of medical research involving human subjects is to improve 
prophylactic, diagnostic and therapeutic procedures and the understanding of the 
aetiology and pathogenesis of disease. Even the best proven prophylactic, 
diagnostic, and therapeutic methods must continuously be challenged through 
research for their effectiveness, efficiency, accessibility and quality. 
7. In current medical practice and in medical research, most prophylactic, diagnostic 
and therapeutic procedures involve risks and burdens. 
8. Medical research is subject to ethical standards that promote respect for all human 
beings and protect their health and rights. Some research populations are 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 57 of 65 
vulnerable and need special protection. The particular needs of the economically 
and medically disadvantaged must be recognized. Special attention is also 
required for those who cannot give or refuse consent for themselves, for those 
who may be subject to giving consent under duress, for those who will not benefit 
personally from the research, and for those for whom the research is combined 
with care. 
9. Research investigators should be aware of the ethical, legal and regulatory 
requirements for research on human subjects in their own countries as well as 
applicable international requirements. No national ethical, legal or regulatory 
requirement should be allowed to reduce or eliminate any of the protections for 
human subjects set forth in this document. 
B. 
Basic principles for all medical research 
1. It is the duty of the physician in medical research to protect the life, health, 
privacy, and dignity of the human subject. 
2. Medical research involving human subjects must conform to generally accepted 
scientific principles, be based on a thorough knowledge of the scientific literature, 
other relevant sources of information, and on adequate laboratory and, where 
appropriate, animal experimentation. 
3. Appropriate caution must be exercised in the conduct of research which may 
affect the environment, and the welfare of animals used for research must be 
respected. 
4. The design and performance of each experimental procedure involving human 
subjects should be clearly formulated in an experimental protocol. This protocol 
should be submitted for consideration, comment, guidance, and where 
appropriate, approval to a specially appointed ethical review committee, which 
must be independent of the investigator, the sponsor or any other kind of undue 
influence. This independent committee should be in conformity with the laws and 
regulations of the country in which the research experiment is performed. The 
committee has the right to monitor ongoing trials. The researcher has the 
obligation to provide monitoring information to the committee, especially any 
serious adverse events. The researcher should also submit to the committee, for 
review, information regarding funding, sponsors, institutional affiliations, other 
potential conflicts of interest and incentives for subjects. 
5. The research protocol should always contain a statement of the ethical 
considerations involved and should indicate that there is compliance with the 
principles enunciated in this Declaration. 
6. Medical research involving human subjects should be conducted only by 
scientifically qualified persons and under the supervision of a clinically competent 
medical person. The responsibility for the human subject must always rest with a 
medically qualified person and never rest on the subject of the research, even 
though the subject has given consent. 
7. Every medical research project involving human subjects should be preceded by 
careful assessment of predictable risks and burdens in comparison with 
foreseeable benefits to the subject or to others. This does not preclude the 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 58 of 65 
participation of healthy volunteers in medical research. The design of all studies 
should be publicly available. 
8. Physicians should abstain from engaging in research projects involving human 
subjects unless they are confident that the risks involved have been adequately 
assessed and can be satisfactorily managed. Physicians should cease any 
investigation if the risks are found to outweigh the potential benefits or if there is 
conclusive proof of positive and beneficial results. 
9. Medical research involving human subjects should only be conducted if the 
importance of the objective outweighs the inherent risks and burdens to the 
subject. This is especially important when the human subjects are healthy 
volunteers. 
10. Medical research is only justified if there is a reasonable likelihood that the 
populations in which the research is carried out stand to benefit from the results of 
the research. 
11. The subjects must be volunteers and informed participants in the research project. 
12. The right of research subjects to safeguard their integrity must always be 
respected. Every precaution should be taken to respect the privacy of the subject, 
the confidentiality of the patient's information and to minimize the impact of the 
study on the subject's physical and mental integrity and on the personality of the 
subject. 
13. In any research on human beings, each potential subject must be adequately 
informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and 
potential risks of the study and the discomfort it may entail. The subject should be 
informed of the right to abstain from participation in the study or to withdraw 
consent to participate at any time without reprisal. After ensuring that the subject 
has understood the information, the physician should then obtain the subject's 
freely given informed consent, preferably in writing. If the consent cannot be 
obtained in writing, the nonwritten consent must be formally documented and 
witnessed. 
14. When obtaining informed consent for the research project the physician should be 
particularly cautious if the subject is in a dependent relationship with the 
physician or may consent under duress. In that case the informed consent should 
be obtained by a well-informed physician who is not engaged in the investigation 
and who is completely independent of this relationship. 
15. For a research subject who is legally incompetent, physically or mentally 
incapable of giving consent or is a legally incompetent minor, the investigator 
must obtain informed consent from the legally authorized representative in 
accordance with applicable law. These groups should not be included in research 
unless the research is necessary to promote the health of the population 
represented and this research cannot instead be performed on legally competent 
persons. 
16. When a subject deemed legally incompetent, such as a minor child, is able to give 
assent to decisions about participation in research, the investigator must obtain 
that assent in addition to the consent of the legally authorized representative. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 59 of 65 
17. Research on individuals from whom it is not possible to obtain consent, including 
proxy or advance consent, should be done only if the physical/mental condition 
that prevents obtaining informed consent is a necessary characteristic of the 
research population. The specific reasons for involving research subjects with a 
condition that renders them unable to give informed consent should be stated in 
the experimental protocol for consideration and approval of the review 
committee. The protocol should state that consent to remain in the research should 
be obtained as soon as possible from the individual or a legally authorized 
surrogate. 
18. Both authors and publishers have ethical obligations. In publication of the results 
of research, the investigators are obliged to preserve the accuracy of the results. 
Negative as well as positive results should be published or otherwise publicly 
available. Sources of funding, institutional affiliations and any possible conflicts 
of interest should be declared in the publication. Reports of experimentation not 
in accordance with the principles laid down in this Declaration should not be 
accepted for publication. 
C. 
Additional principles for medical research combined with medical care 
1. The physician may combine medical research with medical care, only to the 
extent that the research is justified by its potential prophylactic, diagnostic or 
therapeutic value. When medical research is combined with medical care, 
additional standards apply to protect the patients who are research subjects. 
2. The benefits, risks, burdens and effectiveness of a new method should be tested 
against those of the best current prophylactic, diagnostic, and therapeutic 
methods. This does not exclude the use of placebo, or no treatment, in studies 
where no proven prophylactic, diagnostic or therapeutic method exists. 
To clarify further the WMA position on the use of placebo-controlled trials, the WMA 
Council issued, during October 2001, a note of clarification on article 29.  
1. At the conclusion of the study, every patient entered into the study should be 
assured of access to the best proven prophylactic, diagnostic and therapeutic 
methods identified by the study. 
2. The physician should fully inform the patient which aspects of the care are related 
to the research. The refusal of a patient to participate in a study must never 
interfere with the patient–physician relationship. 
3. In the treatment of a patient, where proven prophylactic, diagnostic and 
therapeutic methods do not exist or have been ineffective, the physician, with 
informed consent from the patient, must be free to use unproven or new 
prophylactic, diagnostic and therapeutic measures, if in the physician's judgement 
it offers hope of saving life, re-establishing health or alleviating suffering. Where 
possible, these measures should be made the object of research, designed to 
evaluate their safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, published. The other relevant guidelines of this 
Declaration should be followed. 


RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 60 of 65 
D. 
Note of clarification on paragraph 29 of the WMA Declaration of Helsinki 
The WMA is concerned that paragraph 29 of the revised Declaration of Helsinki 
(October 2000) has led to diverse interpretations and possible confusion. It hereby 
reaffirms its position that extreme care must be taken in making use of a placebo-
controlled trial and that in general this methodology should only be used in the absence 
of existing proven therapy. However, a placebo-controlled trial may be ethically 
acceptable, even if proven therapy is available, under the following circumstances:  
1. Where for compelling and scientifically sound methodological reasons it is 
necessary to determine the efficacy or safety of a prophylactic, diagnostic or 
therapeutic method; or 
2. Where a prophylactic, diagnostic or therapeutic method is being investigated for a 
minor condition and the patients who receive placebo will not be subject to any 
additional risk of serious or irreversible harm. 
All other provisions of the Declaration of Helsinki must be adhered to, especially the 
need for appropriate ethical and scientific review. 
1Note of clarification on paragraph 29 of the WMA Declaration of Helsinki. The WMA is 
concerned that paragraph 29 of the revised Declaration of Helsinki (October 2000) has 
led to diverse interpretations and possible confusion. It hereby reaffirms its position that 
extreme care must be taken in making use of a placebo-controlled trial and that in general 
this methodology should only be used in the absence of existing proven therapy. 
However, a placebo-controlled trial may be ethically acceptable, even if proven therapy is 
available, under the following circumstances:  
1. Where for compelling and scientifically sound methodological reasons it is 
necessary to determine the efficacy or safety of a prophylactic, diagnostic or 
therapeutic method; or 
2. Where a prophylactic, diagnostic or therapeutic method is being investigated for a 
minor condition and the patients who receive placebo will not be subject to any 
additional risk of serious or irreversible harm. 
All other provisions of the Declaration of Helsinki must be adhered to, especially the 
need for appropriate ethical and scientific review. 
 
 
 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 61 of 65
Appendix 4:
Amendment Summary of Changes 


RGN-259
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 62 of 65






RGN-259 
ReGenTree, LLC 
Amendment 4 Protocol: RGN-NK-301/15-110-0006  
Final V5.0 04MAY2017 
Confidential 
Page 65 of 65 
Appendix 6:  Investigator’s Signature 
Protocol Title: 
A Phase 3, Multi-Center, Randomized, Double Masked, 
Placebo Controlled Clinical Study to Assess the Safety and 
Efficacy of 0.1% RGN-259 Ophthalmic Solution for the 
Treatment of Neurotrophic Keratopathy 
Protocol Number: 
RGN-NK-301/15-110-0006 
Amendment 4 
Date: 
04 May 2017 
I agree to implement and conduct the study diligently and in strict compliance with the 
protocol, good clinical practices and all applicable laws and regulations.  I agree to 
maintain all information supplied by Ora and ReGenTree, LLC. in confidence and, when 
this information is submitted to an Institutional Review Board (IRB), Ethical Review 
Committee (ERC) or another group, it will be submitted with a designation that the 
material is confidential. 
I have read this protocol in its entirety, including the above statement, and I agree to all 
aspects. 
Signed: 
 
 
Date:  
 

SAP_001.pdf: 
 
 
 
 
 
STATISTICAL ANALYSIS PLAN  
 
 
A Phase 3, Multi-Center, Randomized, Double Masked, Placebo Controlled 
Clinical Study to Assess the Safety and Efficacy of 0.1% RGN-259 Ophthalmic 
Solution for the Treatment of Neurotrophic Keratopathy 
 
 
 
Sponsor:   
 
ReGenTree, LLC. 
116 Village Boulevard, Suite 200 
Princeton, NJ 08540 
USA  
 
 
 
 
 
 
Protocol Number: 
RGN-NK-301/15-110-0006 
 
 
Author:   
 
  
 
 
 
 
 
 
Date:    
 
10Sep2019 
Version:  
 
1.0 
 




Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 3 of 27 
 
 
Table of Contents 
1. 
Introduction .............................................................................................................................. 6 
2. 
Study Objectives ...................................................................................................................... 6 
3. 
Study Variables ........................................................................................................................ 6 
3.1 Primary Variables ..................................................................................................................... 6 
3.2 Secondary Variables ................................................................................................................ 6 
3.3 Safety Variables ....................................................................................................................... 7 
3.4 Statistical Hypotheses .............................................................................................................. 7 
4. 
Study Design and Procedures ................................................................................................. 7 
4.1 General Study Design .............................................................................................................. 7 
4.2 Schedule of Visits and Assessments ....................................................................................... 8 
4.3 Early Termination of the Study................................................................................................. 8 
5. 
Study Treatments ..................................................................................................................... 9 
5.1 Method of Assigning Subjects to Treatment Groups ............................................................... 9 
5.2 Masking and Unmasking .......................................................................................................... 9 
6. 
Sample Size and Power Considerations ................................................................................. 9 
6.1 Revised Power Calculations .................................................................................................. 10 
7. 
Data Preparation .................................................................................................................... 10 
8. 
Analysis Populations .............................................................................................................. 11 
8.1 Intent-to-Treat ........................................................................................................................ 11 
8.2 Per Protocol ........................................................................................................................... 11 
8.3 Safety ..................................................................................................................................... 11 
9. 
General Statistical Considerations ......................................................................................... 11 
9.1 Unit of Analysis ...................................................................................................................... 11 
9.2 Missing or Inconclusive Data Handling .................................................................................. 11 
9.3 Definition of Baseline ............................................................................................................. 12 
9.4 Data Analysis Conventions .................................................................................................... 12 
9.5 Adjustments for Multiplicity .................................................................................................... 12 
10. 
Disposition of Subjects ........................................................................................................... 13 
11. 
Demographic and Pretreatment Variables ............................................................................. 13 
11.1 Demographic Variables .......................................................................................................... 13 
11.2 Pretreatment Variables and Baseline Disease Characteristics ............................................. 14 
12. 
Medical History, Prior and Concomitant Medications ............................................................ 14 
12.1 Medical History ...................................................................................................................... 14 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 4 of 27 
 
12.2 Concomitant Medications ....................................................................................................... 14 
13. 
Dosing Compliance and Treatment Exposure ....................................................................... 15 
13.1 Dosing Compliance ................................................................................................................ 15 
13.2 Treatment Exposure .............................................................................................................. 15 
14. 
Efficacy Analyses ................................................................................................................... 15 
14.1 Primary Analysis .................................................................................................................... 15 
14.2 Sensitivity Analyses ............................................................................................................... 16 
14.3 Secondary Analyses .............................................................................................................. 18 
15. 
Exploratory Analyses ............................................................................................................. 20 
16. 
Safety Analyses ..................................................................................................................... 20 
16.1 Adverse Events ...................................................................................................................... 20 
16.2 Visual Acuity........................................................................................................................... 23 
16.3 Corneal Sensitivity (Cochet-Bonnet) ...................................................................................... 23 
16.4 Slit-Lamp Biomicroscopy ....................................................................................................... 24 
16.5 Dilated Fundoscopy ............................................................................................................... 24 
16.6 Intraocular Pressure (IOP) ..................................................................................................... 24 
17. 
Interim Analyses .................................................................................................................... 24 
18. 
Deviations from Protocol-Stated Analyses ............................................................................ 24 
19. 
References ............................................................................................................................. 25 
20. 
Tables .................................................................................................................................... 25 
21. 
Listings ................................................................................................................................... 26 
22. 
Figures ................................................................................................................................... 27 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 5 of 27 
 
List of Abbreviations 
Abbreviation 
Definition 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
ATC 
Anatomical Therapeutic Chemical 
BCVA 
Best Corrected Visual Acuity 
CI 
Confidence Interval 
CRF 
Case Report Form 
CS 
Clinically Significant 
eCRF 
Electronic Case Report Form 
ETDRS 
Early Treatment of Diabetic Retinopathy Study 
HIPAA 
Health Information Portability and Accountability Act 
ICH 
International Conference on Harmonization 
IOP 
Intraocular Pressure 
ITT 
Intent-to-Treat 
LOCF 
Last Observation Carried Forward 
logMAR 
Logarithm of the Minimum Angle of Resolution 
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
Multiple Imputation 
NCS 
Not clinically significant 
NK 
Neurotrophic Keratopathy 
PDF 
Portable Document Format 
PP 
Per Protocol 
PT 
Preferred Term 
RTF 
Rich Text Format 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SDC 
Statistics and Data Corporation, Incorporated 
SOC 
System Organ Class 
TEAE 
Treatment-Emergent Adverse Event 
TFBUT 
Tear Film Break-up Time 
VA 
Visual Acuity 
WHODrug 
World Health Organization Drug Dictionary 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 6 of 27 
 
1. Introduction 
The purpose of this statistical analysis plan (SAP) is to describe the planned analyses and reporting for 
protocol RGN-NK-301/15-110-0006, version 5.0 dated 04May2017.   
This SAP is being written with due consideration of the recommendations outlined in the most recent 
International Conference on Harmonization (ICH) E9 Guideline entitled Guidance for Industry: Statistical 
Principles for Clinical Trials and the most recent ICH E3 Guideline, entitled Guidance for Industry: 
Structure and Content of Clinical Study Reports. 
This SAP describes the data that will be analyzed and the subject characteristics, efficacy, and safety 
assessments that will be evaluated.  This SAP provides details of the specific statistical methods that 
will be used.  The statistical analysis methods presented in this document will supersede the statistical 
analysis methods described in the clinical protocol.  If additional analyses are required to supplement 
the planned analyses described in this SAP, they may be completed and will be identified in the CSR. 
2. Study Objectives 
The objective of this study is to compare the safety and efficacy of RGN-259 Ophthalmic Solution to 
placebo for the treatment of neurotrophic keratopathy (NK). 
3. Study Variables 
3.1 
Primary Variables 
The primary efficacy variable is the following: 
• 
 
   
3.2 
Secondary Variables 
The secondary efficacy variables include the following: 
• 
 
 
• 
   
• 
 
• 
 
 
• 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 7 of 27 
 
3.3 
Safety Variables 
The safety variables include the following: 
• 
VA assessed by Early Treatment of Diabetic Retinopathy Study (ETDRS) scale; 
• 
Slit-Lamp Biomicroscopy; 
• 
Cochet-Bonnet; 
• 
Adverse Event (AE) Query; 
• 
Dilated Fundoscopy; 
• 
Intraocular Pressure (IOP). 
3.4 
Statistical Hypotheses 
H0: 
There is no difference between study eyes treated with RGN-259 and study eyes treated 
with placebo, RGN-259 – placebo, in the percentage of study eyes achieving complete 
healing of epithelial defect at Day 29 (Visit 5). 
HA: 
There is a difference between study eyes treated with RGN-259 and study eyes treated 
with placebo, RGN-259 – placebo, in the percentage of study eyes achieving complete 
healing of epithelial defect at Day 29 (Visit 5). 
 
 
4. Study Design and Procedures 
4.1 
General Study Design 
This is a Phase 3, multicenter, randomized, double-masked, placebo controlled safety and efficacy 
study.  It consists of two treatment arms, a 0.1% concentration of RGN-259 ophthalmic solution and 
placebo ophthalmic solution, 
.  There are seven scheduled visits 
over approximately 6 weeks (43 days), with approximately 100 subjects being screened 
 
.   Visit 1 (Day 1) is the screening 
visit, as well as the baseline visit for subjects who are eligible for the study.  Eligible subjects will be 
randomized to receive one of the two study treatments.  Randomized subjects will then begin using 
study drug in the affected eye(s) 5 times per day for 28 days.   
Study visits will be referred to in all tables and listings as the expected study day corresponding to the 
visit to enable reviewers to understand the assessment timing without referring to the protocol visit 
schedule. The following table shows the scheduled study visits, their planned study day (Day 1 
corresponds to the day of randomization), and the acceptable visit window for each study visit: 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 8 of 27 
 
Scheduled Visit 
Planned Study Day 
Visit Window 
Visit 1 
Day 1 
n/a 
Visit 2 
Day 8 
± 2 Days 
Visit 3 
Day 15 
± 2 Days 
Visit 4 
Day 22 
± 2 Days 
Visit 5 
Day 29 
± 2 Days 
Visit 6 
Day 36 
± 3 Days 
Visit 7 
Day 43 
± 3 Days 
4.2 
Schedule of Visits and Assessments 
The schedule of visits and assessments is provided below. 
4.3 
Early Termination of the Study 
Due to the rarity of the disease and the roughly 5 year enrollment period at multiple sites the Sponsor 
has decided to end this phase 3 trial based on the lack of enrollment. Even with the extended enrollment 
period, only 18 subjects were enrolled and subsequently completed the study. Analyses will be 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 9 of 27 
 
conducted as originally intended; however, the study is no longer powered to detect statistical 
differences. See the revised power calculations in section 6.1 of this SAP.  
As with other studies of rare diseases this study will focus on clinically meaningful difference. Clinically 
meaningful results will be assessed by the Sponsor’s medical and/or regulatory teams. 
5. Study Treatments 
The study treatments are 0.1% RGN-259 Ophthalmic Solution and Placebo Ophthalmic Solution 
(Vehicle). 
5.1 
Method of Assigning Subjects to Treatment Groups 
At Visit 1, subjects who provide written informed consent will be assigned a unique 5-digit screening 
number, which includes the 2-digit site number plus a unique 3-digit screening number beginning with 
001 (e.g., 11001, 11002, 11003, etc.). Screening numbers must be assigned in ascending consecutive 
order. 
If all inclusion and exclusion criteria are met at Visits 1, each qualifying subject will then be assigned a 
4-digit randomization number at the end of Visit 1. All randomization numbers will be assigned in strict 
numerical sequence and no numbers will be skipped or omitted. Assignment of qualified subjects to a 
treatment group will be accomplished by using a randomization code generated by an independent 
biostatistician. Investigators and study staff who will conduct the assignment procedure will be masked 
to the randomization code.  
5.2 
Masking and Unmasking 
This is a double-masked study. Subjects, Investigators and site staff, sponsor and Ora/Statistics & Data 
Corporation (SDC) study staff are masked to the treatment group assignments for the duration of the 
study.  The randomization schedule was created by an unmasked statistician who is not otherwise 
involved in the conduct of the study.   
Under normal circumstances, the mask should not be broken.  When medically necessary, the 
investigator may need to determine what treatment arm has been assigned to a subject. When possible 
(i.e., in non-emergent situations), Ora and/or the study sponsor should be notified before unmasking 
study drug. 
6. Sample Size and Power Considerations 
 
 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 10 of 27 
 
 
 
 
 
 
 
6.1 
Revised Power Calculations 
Due to the rarity of the disease and the Sponsor’s early termination of the study the proposed sample 
size of 42 will not be attained. 
 
 
 
 
 
 
7. Data Preparation 
All reported study data will be recorded on the electronic case report forms (eCRFs) supplied by SDC 
using iMedNet™.  Only the Principal Investigator and authorized study staff according to the Delegation 
of Responsibilities log are entitled to make entries in the eCRF. 
After data are entered into the clinical study database, electronic edit checks and data review will be 
performed.  All data validation specifications and procedures are detailed in the Data Validation Manual 
as a separate document.  When the database has been declared to be complete and accurate, the 
database will be locked.  Any changes to the database after data have been locked can only be made 
with the approval of the Sponsor and Ora in consultation with SDC.  
All analyses outlined in this document will be carried out after the following have occurred: 
• 
All data management requirements are met according to SDC standard operating procedures, 
including data entry, performance of edit and validation checks, documentation and resolution 
of data queries, and database lock with written authorization provided by appropriate SDC and 
Sponsor personnel; 
• 
Protocol deviations have been identified and status defined (major/minor deviations);  
• 
Analysis populations have been determined; and 
• 
Randomized treatment codes have been unmasked. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 11 of 27 
 
8. Analysis Populations 
8.1 
 
 
   
8.2 
 
 
 
 
 
   
8.3 
Safety 
The safety population includes all randomized subjects who have received at least one dose of the 
investigational medicinal product. The safety population will be analyzed for all safety assessments 
according to the treatment actually received. 
The statistical analysis of safety data will be performed for the safety population.  The analysis of 
baseline and efficacy data will be performed for the ITT population.  The primary efficacy analysis will 
also be performed on the PP population as sensitivity analyses. 
9. General Statistical Considerations 
9.1 
Unit of Analysis 
The unit of analysis in this study will be the study eye for all efficacy summaries and any treated eye for 
ocular safety summaries. In general, NK is unilateral.  If a patient presents with bilateral epithelial defects 
that both meet the entry criteria, the study eye will be defined as the affected eye with the largest affected 
area, and the other eye will be considered a qualified fellow eye.  If both affected eyes have the same 
affected area, the right eye will be the study eye and the left eye will be considered a qualified fellow 
eye.  
Ocular AEs will be summarized for all eyes and for any treated eye (if at least one subject has bilateral 
NK in which both eyes are treated), and ocular safety assessments will be analyzed by study eye and 
both treated eyes (again, if at least one subject has bilateral NK in which both eyes are treated), where 
the worst safety outcome over both eyes is summarized.  Medical history and non-ocular AEs and safety 
assessments will be presented at the subject level. 
9.2 
Missing or Inconclusive Data Handling 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 12 of 27 
 
 
 
 
 
     
 
9.3 
Definition of Baseline 
Baseline measures are defined as the last measure prior to the initiation of study treatment, usually at 
Visit 1 (Day 1).   
9.4 
Data Analysis Conventions 
All data analysis will be performed by SDC after the study is completed and the database has been 
locked and released for unmasking.  Statistical programming and analyses will be performed using 
 
  Output will be provided in rich text format (RTF) format for tables and portable 
document file (PDF) format for tables, listings, and figures using landscape orientation.  All study data 
will be listed by subject, treatment, visit (as applicable), and time point (as applicable) based on all 
randomized subjects unless otherwise specified. 
 
 
 
 
 
 
 
  
 
 
 
 
 
9.5 
Adjustments for Multiplicity 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 13 of 27 
 
10. Disposition of Subjects 
Subject disposition will be presented in terms of the numbers and percentages 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
11. Demographic and Pretreatment Variables 
11.1 
Demographic Variables 
The demographic variables collected in this study include age, sex, race, ethnicity, and iris color.  
Subjects who record more than one race will be grouped into a single category denoted as multi-racial. 
Demographic variables will be summarized for the ITT population and Safety populations, separately. 
Age (years) will be summarized, overall and by treatment, using continuous descriptive statistics.  Age 
will also be categorized as follows:  
 
 
 
The number and percentage of subjects will be presented, overall and by treatment, for age category, 
sex, race, ethnicity and iris color (by study eye).   
No statistical inference testing will be performed for the demographic variables.  A subject listing that 
includes all demographic variables will be provided. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 14 of 27 
 
11.2 
Pretreatment Variables and Baseline Disease Characteristics 
 
 
 
 
12. Medical History, Prior and Concomitant Medications 
12.1 
Medical History 
Medical history will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) Version 
22.0. 
Non-ocular medical history will be summarized using discrete summary statistics and presented by 
treatment group at the subject and event level by System Organ Class (SOC) and Preferred Term (PT) 
using the Safety population.  Ocular medical history will be similarly summarized at the subject level.  If 
a subject reports the same PT multiple times within the same SOC, that PT will only be reported once 
within that SOC.  As with the PT, if a subject reports multiple conditions within the same SOC, that SOC 
will only be reported once.   
Listings of medical history will be generated separately for ocular and non-ocular data. 
12.2 
Concomitant Medications 
Prior and concomitant medications will be coded using the World Health Organization Drug Dictionary 
(WHODrug Global, B3, March 2019) and summarized to the therapeutic drug class (Anatomical 
Therapeutic Chemical (ATC) 4 classification) and preferred name (drug name). 
Concomitant medications are defined as those medications listed as having been taken 1) prior to 
initiation of study drug administration and continuing for any period of time following the first 
administration of study drug or 2) at any time following the first administration of study drug.  Prior 
medications are defined as those medications listed as having been taken prior to initiation of study drug 
administration, but not taken after initiation of study drug administration. 
Both ocular and non-ocular concomitant medications will be summarized using the Safety population.  
Medications will be tabulated for each treatment group using frequencies and percentages.  Subjects 
may have more than 1 medication per ATC 4 classification.  At each level of subject summarization, a 
subject will be counted once if he/she reports 1 or more medications.  Percentages will be based on the 
number of subjects in each treatment group.   
Listings of prior and concomitant medications will be generated separately for ocular and non-ocular 
data. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 15 of 27 
 
13. Dosing Compliance and Treatment Exposure 
13.1 
Dosing Compliance 
 
 
 
 
 
 
 
 
 
 
   
 
13.2 
Treatment Exposure 
Treatment exposure will be measured at the subject level; 
 
 
 
 
 
 
 
 
 
 
14. Efficacy Analyses 
14.1 
Primary Analysis 
The primary analysis variables will be presented in subject listings by visit and time point. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 16 of 27 
 
 
 
 
    
 
 
 
 
 
 
 
 
14.2 
Sensitivity Analyses 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 17 of 27 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 18 of 27 
 
 
 
  
 
  
 
 
 
 
 
 
 
14.3 
Secondary Analyses 
The continuous and ordinal secondary efficacy variables collected at each visit will be summarized 
descriptively
 
 
 
 
 
 
14.3.1 Percentage of Subjects with Complete Healing 
The percentage of subjects achieving complete healing of the epithelial defect at Visits 
 
 
 
 
 
 
14.3.2 Epithelial Defect Measurement 
The epithelial defect will be measured 
 
 
  
 
 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 19 of 27 
 
 
 
 
14.3.3 Epithelial Defect Classification 
  
 
 
 
 
 
 
14.3.4 Tear Film Break-Up Time 
 
 
 
  
 
 
 
 
   


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 20 of 27 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
14.3.6 Visual Acuity (ETDRS) 
 
 
 
 
  
 
 
 
 
 
 
15. Exploratory Analyses 
There are no exploratory analyses in this study. 
16. Safety Analyses 
All safety analyses will be conducted using the Safety Population. 
16.1 
Adverse Events 
An adverse event (AE) is defined as any untoward medical occurrence associated with the use of an 
investigational medicinal product (IMP) in humans, whether or not considered IMP-related. An AE can 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 21 of 27 
 
be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of an investigational product, without any judgment about causality. 
An AE can arise from any use of the investigational product (e.g. off-label use, use in combination with 
another drug or medical device) and from any route of administration, formulation, or dose, including an 
overdose. All AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) Version 
22.0. 
Documentation regarding the AE should be made as to the nature, date of onset, end date, severity, 
relationship to study drug, action(s) taken, seriousness, and outcome of any sign or symptom observed 
by the physician or reported by the subject upon indirect questioning. 
Treatment-emergent AEs (TEAEs) are defined as any event that occurs or worsens on or after the day 
that randomized study treatment is initiated.  As treatment is initiated at Visit 1 in this study, all AEs will 
be considered TEAEs.  
Serious adverse events (SAE) are defined as any event that results in death, is life-threatening, requires 
inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant 
disability/incapability, is a congenital anomaly/birth defect, or is medically significant. 
An overall summary will be presented that includes the number of AEs and the number and percentage 
of subjects who experienced at least one AE, by treatment group.  This summary will also include 
breakdowns of AEs further categorized as ocular (study eye, treated fellow eye, and untreated fellow 
eye separately) or non-ocular, SAEs, AEs by maximum severity, AEs by maximum relationship, and 
AEs leading to subject withdrawal. 
Additional summaries of AEs will be provided showing the number and percentage of subjects who 
experienced at least one AE.  These summaries will be presented by SOC and PT.  Non-ocular AEs will 
be summarized using discrete summary statistics and presented by treatment group at the subject and 
event level by SOC and PT.  Ocular AEs will be similarly summarized at the subject level for all eyes 
and treated eyes separately.  If a subject reports the same PT multiple times within the same SOC, that 
PT will only be reported once within that SOC.  As with the PT, if a subject reports multiple conditions 
within the same SOC, that SOC will only be reported once.  In the summary, SOC will be listed in 
ascending alphabetical order; PTs will be listed in order of descending frequency for all subjects within 
each SOC.   
Severity of an AE is defined as a qualitative assessment of the degree of intensity of an AE as 
determined by the Investigator or reported to him/her by the patient/subject.  The assessment of severity 
is made irrespective of relationship to investigational product or seriousness of the event and should be 
evaluated according to the following scale: 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 22 of 27 
 
• Mild: Event is noticeable to the subject, but is easily tolerated and does not interfere with the 
subject’s daily activities. 
• Moderate: Event is bothersome, possibly requiring additional therapy, and may interfere with the 
subject’s daily activities. 
• Severe: Event is intolerable, necessitates additional therapy or alteration of therapy, and 
interferes with the subject’s daily activities. 
Summaries of AEs by maximal severity will be presented for ocular AEs and non-ocular AEs separately.  
The number of subjects with any AEs (along with percentages) will be tabulated by SOC and PT within 
each SOC by treatment group.  To count the number of subjects with any AEs, if a subject has multiple 
AEs coded to the same PT within the same SOC, the subject will be counted once under the maximal 
severity. 
The relationship of each AE to the study drug should be determined by the investigator using these 
explanations: 
• Definite: When there are good reason and sufficient documentation to demonstrate a direct 
causal relationship between investigational product and AE; 
• Probable: When there are good reasons and sufficient documentation to assume a causal 
relationship in the sense of plausible, conceivable, likely but not necessarily highly probable. 
• Possible: When there is sufficient information to accept the possibility of a causal relationship in 
the sense of not impossible and not unlikely, although the connection is uncertain or doubtful, 
for example; due to missing data or insufficient evidence. 
• None: When there is sufficient information to accept a lack of a causal relationship, in the sense 
of impossible and improbable. 
• Unclassified: When the causal relationship is not assessable for whatever reason due to 
insufficient evidence, conflicting data or poor documentation. 
Only AEs with a relationship of definite, probable, or possible, or with a missing relationship, will be 
considered as treatment-related AEs. 
The expectedness of an AE should be determined based upon existing safety information about the 
study drug using these explanations: 
• Unexpected: An AE that is not listed in the Investigator’s brochure or is not listed at the specificity 
or severity that has been observed. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 23 of 27 
 
• Expected: An AE that is listed in the Investigator’s brochure at the specificity and severity that 
has been observed. 
• Not Applicable: Any AE that is unrelated to the study drug. 
AEs that are mentioned in the Investigator’s brochure 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 are to be considered unexpected.  AEs that are reported as suspected 
with respect to relationship to study drug, and are also categorized as unexpected are to be reported 
directly to Ora, the sponsor, and other authorities as appropriate.  Expectedness of the AE will be 
included in the subject level listings. 
As described above, separate summaries will be provided for the following categories of AEs by SOC 
and PT:  
• 
Ocular AEs in any eye 
• 
Ocular AEs in treated eyes 
• 
Non-ocular AEs  
• 
Treatment-related ocular AEs 
• 
Treatment-related non-ocular AEs 
• 
SAEs 
All AEs will be presented in a subject listing.  The AEs leading to study treatment discontinuation will be 
listed separately.  In addition, all SAEs will be presented in a separate listing. 
16.2 
Visual Acuity 
 
 
 
 
   
16.3 
Corneal Sensitivity (Cochet-Bonnet) 
 
 
 
 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 24 of 27 
 
16.4 
Slit-Lamp Biomicroscopy 
 
  
 
  
 
 
 
 
 
 
16.5 
Dilated Fundoscopy 
Dilated fundoscopy examinations will be conducted on both eyes 
 
 
 
 
 
 
 
 
 
16.6 
Intraocular Pressure (IOP) 
IOP will be measured for both eyes 
 
 
 
 
  
 
17. Interim Analyses 
No interim analyses are planned for this study. 
18. Deviations from Protocol-Stated Analyses 
No deviations from the protocol specified analyses are currently planned. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 25 of 27 
 
19. References 
Dunn SP, Heidemann DG, Chow CYC, Crockford D, Turjman N, Angel J, Allan CB, Sosne 
G. Treatment of Chronic Nonhealing Neurotrophic Corneal Epithelial defects with Thymosin β4. 
Archives of Ophthalmology 2010b, 128:636-638. 
Ratitch, B., Lipkovich, I., and M. O’Kelley.  “Combining Analysis Results from Multiply Imputed 
Categorical Data.”  PharmaSUG 2013 - Paper SP03.  
20. Tables 
Tables that will be included in the topline delivery are shown in boldface font. 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 26 of 27 
 


Protocol RGN-NK-301/15-110-0006, SAP Version 1.0 
10SEP2019 
Confidential & Proprietary 
Page 27 of 27