Clinical trial NCT05470868
Conditions: Hyperemia Eye
Sponsor: Laboratorios Sophia S.A de C.V.
Start date: 2023-02-04; Completion date: 2023-05-22
Enrollment: 22 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Phase I Study to evaluate safety and tolerability of PRO-185 (naphazoline/hypromellose) ophthalmic solution through evaluation of vital signs, ocular signs such as intraocular pressure, hyperemia and mydriasis and adverse events.

Detailed description:
A total of 22 healthy volunteers will apply PRO-185 ocular solution on both eyes QID (four times per day) for 8 days. The safety variables will include intraocular pressure, heart rate, systolic and diastolic blood pressure. Tolerability variables will include: hyperemia, mydriasis, and expected and unexpected adverse events. The presence of any of these in ≤ 20% will deem PRO-185 as safe and tolerable.

Arms: arm 1: Healthy volunteers will apply one drop of PRO-185 ophthalmic solution (naphazoline / hypromellose) on both eyes, QID (four times per day) for 8 days.
Interventions: intervention 1: Naphazoline / Hypromellose Ophthalmic
Intervention descriptions: intervention 1: Naphazoline 0.03% / Hypromellose 0.2%

Protocol documents:
Prot_000.pdf: 
Study Protocol: 
SOPH185-0521/I 
Title: Phase I clinical study to evaluate the safety and tolerability of PRO-185 
ophthalmic solution applied in clinically healthy subjects
Sponsor: Laboratorios Sophia, S.A. de C.V. 
Version Date: July 27, 2022
Version: 2.0
This document was prepared in accordance with FDA, 
ICH, and GCP guidelines. 
 




Change History 
Sections, paragraphs and 
subsections 
Main modifications made 
Cover Page Headers and Footers 
Updated version number, version date, and page count. 
Change History 
Added Change History to describe the major changes between this version and the 
previous one. 
Content 
Section page numbers have been changed, and new subsections have been added. 
Index of tables, graphs and 
figures 
The page numbers of tables and figures were modified, and the table of contents of 
graphs was added. 
Study leaders 
The Study Director's affiliation was changed from “Medical Manager” to “Regional 
Clinical Research Manager.” 
List of abbreviations 
The abbreviation TRPL is being dropped. Tear film break-up time. 
1.1 Synopsis 
The protocol version number and version date have been changed. 
Hypothesis H 0 changed to: 
“PRO-185 ophthalmic solution is safe and tolerable for ophthalmic use, with any of 
the following adverse events occurring in 20% or less of subjects: increased 
intraocular pressure (IOP >5 mmHg), heart rate changes (>15 bpm), increased 
systemic blood pressure [>15 mmHg systolic (SBP) or >10 mmHg diastolic (DBP)], 
pharmacological mydriasis, or grade 3 and 4 conjunctival hyperemia.” The underlined 
words “any of” were added to highlight that only one of the adverse events described 
was necessary. It is described that comparisons will be between pre- and post-
treatment measurements at visits 1 and 2 and between measurements at the pre-PT 
baseline visit and measurements at the final visit. 
In Statistical Methodology, the Kolmogorov-Smirnov test and the Mann-Whitney U 
test were changed to the Wilcoxon signed-rank test; the Pearson X 2 test was also changed to 
the binomial X 2 , and the McNemar test was withdrawn . 
2.2.1 Naphazoline 
Typo fixed. 
2.2.3 Menthol 
Typographical and writing errors were corrected. 
Added information on menthol irritability and toxicity testing. 
Added information on the usefulness of using menthol. 
2.3 Background on the research 
product 
It was added that a preclinical safety and toxicity study of the PRO-185 ophthalmic 
solution has already been carried out. 
2.3.1 Preclinical safety and 
toxicity study of PRO-185 
solution 
New section describing the comparative preclinical safety and toxicity study of PRO-
185 ophthalmic solution versus a marketed product, Nazil® Contamination . The study 
involved administering the drugs four times daily for 30 days to New Zealand albino 
rabbits. The results show no relevant safety and toxicity data for any of the 
investigational products. There are also no clinical changes or manifestations 
demonstrating the unsafety or toxicity of the PRO-185 formulation. Therefore, it is 
concluded that the product is safe and nontoxic for ocular use in the selected animal 
model. 


2.3.2 Preclinical study of an 
ophthalmic solution of 
naphazoline 0.1% and 
hypromellose 0.5% (higher 
concentration of the main 
components of PRO-185 ) 
Information was added about another preclinical study different from the one 
already described. 
2.5.1 Known potential risks 
Typos were fixed. 
Information on the safety of menthol as an excipient is added to the concentration 
found in the PRO-185 solution. 
2.7 Justification of the study 
Information related to the preclinical study of PRO-185 is added, as well as the safety 
of menthol and its use at the concentration found in the PRO-185 solution. 
3.2 Hypothesis 
or "any of" are added to specify that only one of the adverse events described in the 
hypothesis must occur at the frequency also described. It is specified that 
comparisons will be made between pre- and post-treatment measurements from 
visits 1 and 2, and between measurements from the pre-PT baseline visit and 
measurements from the final visit. 
Table iii . Operational definition 
of variables in 7.4.4 Definition of 
variables, methods and scales 
for their measurement 
The Statistical Tests of all variables in the table are modified. 
7.4.5.7 SICCA ocular surface 
staining 
The statement is removed: After evaluating the TRPL, between 4 and 8 minutes after 
instillation of the dye. 
10.1.1 Calculation methodology 
Typo corrected. 
10.1.2 Size calculation 
Typographical errors are corrected. 
The equations for calculating the sample size are modified. 
Absolute values are added in parentheses to the description of the signs. 
10.2 Clinical data management 
Typo corrected. 
10.3 Statistical methodology 
The previous version specifies that the statistical analysis will be performed using 
SPSS version 19.0. The new version specifies that either SPSS or R software can be 
used, and that the available version will be used. 
10.3.2.1 Analysis for primary 
variables 
The Kolmogorov-Smirnov, Mann-Whitney U, and Pearson X2 tests are removed . The 
Wilcoxon signed-rank and Binomial X2 tests are added . 
It is specified that there will be a comparison between pre- and post-treatment 
measurements for visits 1 and 2; and another comparison between baseline (pre-PT) 
visit measurements and final visit measurements. 
Literature 
Bibliographies were added and the location of the bibliographic references in the list 
was modified, following the order of appearance in the protocol. 


Content 
Change History 
Contents 
Index of tables, graphs and figures 
Those responsible for study 
Signature Page 
From sponsor 
Researcher Agreement 
List of abbreviations 
1. Summary of protocol
1.1 Synopsis
1.2 Study diagram
1.3 Schedule of activities of subject
2. Introduction and background
2.1 Theoretical framework
2.1.1 The red eye 
2.1.2 Pathophysiology of red eye 
2.1.3 Ocular vasoconstrictors 
2.1.4 Mechanism of action of vasoconstrictors 
2.2 Background on the investigational product 
2.2.1 Naphazoline 
2.2.2 Hypromellose 


2.2.3 Menthol 
2.3 Background on the investigational product 
2.3.1 Preclinical safety and toxicity study of PRO-185 solution 
2.3.2 Preclinical study of an ophthalmic solution of naphazoline 0.1% and hypromellose 0.5% (higher 
concentration of the main components of PRO-185) 
2.3.3 Clinical study of an ophthalmic solution of naphazoline 0.1% and hypromellose 0.5% 
2.4 Background on the research 
2.4.1 From research question 
2.5 Risk-benefit assessment 
2.5.1 Known potential risks 
2.5.2 Known potential benefits 
2.6 Problem statement 
2.7 Justification of the study 
3. Objectives and hypotheses
3.1 Objectives
3.1.1 Main objective: 
3.1.2 Specific objectives: 
3.2 Hypothesis 
4. Study design
4.1 Overview of the study
4.2 Justification of the study design
4.3 Expected duration
5. Study population


5.1 Eligibility Criteria 
5.1.1 Inclusion criteria 
5.1.2 Exclusion criteria: 
5.2 Criteria for elimination and/or substitution of subjects 
5.2.1 Elimination criteria 
5.2.2 Substitution of subjects 
5.3 Scrutiny failures 
5.4 Recruitment and retention strategies 
5.5 Procedure in case of early discontinuation 
5.6 Identification of subject 
6. Investigational product and treatment
6.1 Managed Products
6.1.1 Investigational Product 
6.1.2 Dose of the investigational product 
6.1.3 Treatment with the investigational product 
6.2 Storage and handling of the investigational product at the study center 
6.3 Concomitant treatments and medications (permitted and prohibited) 
6.4 Procedure for monitoring and measuring adherence 
6.5 Strategies to improve adherence 
7. Methods and procedures of the study
7.1 Research Center
7.2 Clinical Study Registration


7.3 Treatment assignment 
 
7.4 Outcome variables 
 
7.4.1 Primary outcome variables 
 
7.4.2 Secondary outcome variables 
 
7.4.3 Exploratory variables 
 
7.4.4 Definition of variables, methods and scales for their measurement 
 
7.4.5 Description of the variables, methods and scales for their measurement 
 
7.5 Description of the procedures or assessments during the study 
 
7.5.1 Signature of informed consent 
 
7.5.2 Taking a clinical history (including ophthalmological and general clinical history) 
 
7.5.3 Eligibility criteria 
 
7.5.4 Subject code assignment 
 
7.5.5 Adverse events 
 
7.5.6 Measuring vital signs 
 
7.5.7 Urine Pregnancy Test 
 
7.5.8 Ophthalmological evaluation 
 
7.5.8.1 Best-corrected visual acuity 
 
7.5.9 Applying Medications During Visits 
 
7.5.10 Delivery of material for subject 
 
7.5.11 Delivery of study medication 
 
7.5.12 Evaluation of concomitant medications 
 
7.5.13 Assessment of treatment adherence 
 


7.5.14 Return of Study Drug 
7.5.15 Withdrawal from Subject Diary 
7.6 Diagram and schedule of activities for study 
7.6.1 Study diagram 
7.6.2 Study Activity Schedule 
7.7 Procedures to be performed per visit 
7.7.1 Baseline visit 
7.7.2 Visit 1 
7.7.3 Visit 2 
7.7.4 Final visit 
7.7.5 Unscheduled follow-up visits 
7.8 Data Collection 
7.8.1 Source documents 
7.8.2 Electronic forms of data collection 
7.8.3 File 
8. Evaluation and management of adverse events
8.1 Regulation and standards on adverse events
8.2 Definition of adverse event
8.3 Use of adverse events as a safety variable in the study
8.4 Definitions relevant to the classification of adverse events
8.5 Researcher Responsibilities
8.5.1 Recording of adverse events in the electronic case report form 


8.5.2 Monitoring of adverse events 
8.5.3 Procedures for a serious adverse event 
8.5.4 Causality Assessment 
8.6 Unanticipated Problems 
8.6.1 Reporting unanticipated problems 
9. Study monitoring
9.1 Monitoring of the study center
9.2 Audit and quality control
9.2.1 Pre-study audit 
9.2.2 Audit during the conduct of study 
10. Sample size calculation and statistical analysis
10.1 Sample size calculation
10.1.1 Calculation methodology 
10.1.2 Size Calculation 
10.2 Clinical data management 
10.3 Statistical Methodology 
10.3.1 Population analysis 
10.3.2 Safety and tolerability analysis 
10.3.3 Exploratory analysis 
10.4 Procedure for handling missing data 
11. Ethical considerations
11.1 Approval of committees


11.2 Amendments to Protocol 
11.3 Early termination of the study 
11.4 Informed consent 
11.4.1 Obtaining 
11.4.2 Special Considerations 
11.4.3 Modifications to informed consent 
11.5 Confidentiality 
11.6 Conflict of interest 
11.7 Access to information 
11.8 Ancillary and post-study care 
12. Biosecurity aspects
13. Publication Policy
13.1 Final Report
13.2 Communication of results
13.3 Publication of results
14. Financing and insurance
14.1 Compensation to Study Participants
14.2 Insurance for study participants
15. Bibliography
16. Annexes
16.1 Efron scale for conjunctival hyperemia
16.2 SICCA Ocular Staining Rating (modified from Whitcher et al, 2010)


16.3 Review of articles for sample size calculation 
Index of tables, graphs and figures 


Study leaders 
The administrative structure of the sponsoring party, corresponding to Laboratorios Sophia, SA de 
CV, is shown in Table 1. Study managers . 
Table i. Study leaders 
Function 
Name/Contact 
Membership ¥  
Medical Director of the 
study 
Medical Director 
Director of the study 
Regional Medical Affairs 
Manager 
Operations Manager 
Regional Clinical Research 
Manager 
Author of the Protocol 
Medical Editor 
Biostatistics 
Biostatistics Manager 


Signature page 
From the sponsor 
Name: 
Qualification: 
Signature 
Date 
Name: 
Qualification: 
Signature 
Date 
Name: 
Qualification: 
Signature 
Date 
Name: 
Qualification: 
Signature 
Date 


Researcher Agreement 
I agree to conduct this clinical study according to the design and guidelines of this protocol, adhering 
to its provisions. I declare that I will conduct the study in accordance with the standards of Good 
Clinical Practice and will report all information and data as outlined in the protocol, particularly any 
adverse events. I will also manage clinical supplies provided by the sponsor strictly in accordance 
with this protocol. I understand that the information that identifies me may be used by the sponsor. 
Because the information contained in this protocol and the Investigator's Manual is confidential, I 
understand that sharing it with any third party not involved in the approval, supervision, or conduct 
of the study is prohibited. I will ensure that necessary precautions are taken to protect the 
information from loss, inadvertent disclosure, or access by unauthorized third parties. 
Name: 
[Write the researcher's full 
name 
Qualification: 
Principal Investigator 
Name of the center: 
[ Write name of study center] 
Geographic location (city/state/country) 
[Write the geographic data of the center] 
Signature 
Date 


List of abbreviations 
Ad 
Adherence 
cAMP 
cyclic adenosine monophosphate 
AO 
Both eyes 
av 
Visual acuity 
AVMC 
Best corrected visual acuity 
BPC 
Good clinical practices 
CDM 
Clinical Data Management 
CEI 
Research Ethics Committee 
CI 
Investigation Committee 
COFEPRIS 
Federal Commission for the Protection against Sanitary Risks 
CTO 
Ocular staining grading 
OF 
Standard deviation 
IUD 
Intrauterine device 
EA 
Adverse event 
eCFR 
Electronic Case Form Report ) 
ECG 
Electrocardiogram 
EEP 
Punctate epithelial erosions 
USA 
United States of America 
FC 
Heart rate 
FCI 
Informed consent form 
FR 
Respiratory rate 
H 0 
Null hypothesis 
H 1 
Alternative hypothesis 
ICH 
International Council on Harmonization ( International Council for Harmonization ) 
ICMJE 
International Committee of Medical Journal Editors (ICME) of Medical Journal Editors 
) 
IP 
Principal Investigator 
IP 3 
Inositol triphosphate 
ITT 
By intention to treat (ITT ) to Treat ) 
bpm 
Beats per minute 
mmHg 
Millimeters of mercury 
NOM 
Mexican Official Standard 


WHO 
World Health Organization 
PAD
Diastolic blood pressure 
PI 
Research product 
PIO 
Intraocular pressure 
PNA 
Unanticipated problems 
Pre-TP 
Prior to applying TP Ofteno ® 
Post-Tx 
Post-treatment 
PP 
Population by protocol 
Post -TP 
After application of TP Ofteno ® 
Pre -Tx 
Pre-treatment 
RAM 
Adverse drug reaction 
RNEC 
National Registry of Clinical Trials 
SDV 
Source Document Verification Document Verification ) 
SICCA 
Sjögren 's International Collaborative Clinical Alliance ) 
SPSS 
Statistical Package for the Social Sciences ( Statistical Package for the Social Sciences) 
Package for the Social Sciences ) 
SRAM 
Suspected adverse drug reaction 
TEROS 40 
Tear evaporation rate at 40% ambient humidity ( Tear Evaporation Ocular Surface 
Rate ) 
TF 
Fluorescein staining 
TVL 
Lissamine green staining 
TRPM8 
Cation receptor potential channel subfamily 8 ( Transient Receptor Potential) Cation 
Channel Subfamily M Member 8 ), also known as the cold-sensing cation channel. 
UFTLS 
Pharmacovigilance and Technovigilance Unit of Sophia Laboratories 
VV 
Current version 
X 2 
Chi-square 
°C 
Degrees Celsius 




1. Summary of the protocol
1.1 Synopsis 
Title of the study: 
Phase I clinical study to evaluate the safety and tolerability of PRO-185 ophthalmic solution 
applied in clinically healthy subjects
Study number: 
SOPH185-0521/I 
Creation date: 
05/17/2021 
Protocol version: 
2.0 
Version date: 
27/07/2022 
Therapeutic indication: 
Ocular vasoconstrictor 
Use: 
Conjunctival hyperemia 
Estimated duration of the study (from the first 
visit of the first patient to the preparation of 
the final report) : 
5 months 
Clinical development phase: 
Yo 
Goals: 
Main objective: 
•
To evaluate the safety and tolerability of PRO-185 ophthalmic solution in
ophthalmologically and clinically healthy subjects.
Specific objectives: 
•
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of
subjects experiencing an increase of >5 mmHg in intraocular pressure 20 minutes after
application compared to the initial value.
•
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of
subjects experiencing a heart rate variation of >15 beats per minute 20 minutes after
application.
•
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of
subjects with an increase in systolic blood pressure of >15 mmHg or >10 mmHg in
diastolic blood pressure 20 minutes after application.


•
To assess tolerability by the incidence of grade 3 and 4 conjunctival hyperemia.
•
To assess tolerability by the incidence of pharmacological mydriasis.
•
Evaluate tolerability by the incidence of expected and unexpected adverse events.
Hypothesis : 
H 0 = PRO-185 ophthalmic solution is safe and tolerable for ophthalmic use, as it occurred in 20% 
or less of subjects with any of the following adverse events: increased intraocular pressure (IOP 
>5 mmHg), heart rate variation (>15 bpm), increased systemic blood pressure [>15 mmHg systolic
(SBP) or >10 mmHg diastolic (DBP)], pharmacological mydriasis, or grade 3 and 4 conjunctival
hyperemia.
𝐻0: 𝑝−𝑝0 ≤𝛿 
H 1 = PRO-185 ophthalmic solution is not safe or tolerable for ophthalmic use because more than 
20% of subjects experienced any of the following adverse events: increased IOP (>5 mmHg), heart 
rate variation (>15 bpm), increased systemic blood pressure [>15 mmHg systolic (SBP) or >10 
mmHg diastolic (DBP)], pharmacological mydriasis, or grade 3 and 4 conjunctival hyperemia. 
𝐻1:𝑝−𝑝0 > 𝛿 
Comparisons will be made between pre- and post-treatment measurements from visits 1 and 2, 
and between measurements from the baseline visit (before PT application) and measurements 
from the final visit. Only one of the adverse event values described in the hypothesis need be 
abnormal with a frequency >20% between the comparisons (pre-treatment with post-treatment; 
or pre-PT baseline with values from the final visit) for the null hypothesis to be rejected.
Study design: 
Phase I clinical study, controlled, non-comparative, open, single-center 
Number of subjects (planned and analyzed): 
Number of subjects planned: 22 subjects. 
Diagnosis and main inclusion criteria: 
-
Ophthalmologically and clinically healthy subjects.
Selection criteria: 
Inclusion criteria: 
-
Be clinically healthy.


-
Have the ability to voluntarily grant signed informed consent.
-
Be able and willing to comply with scheduled visits, treatment plan, and other study
procedures.
-
Be between 18 and 45 years old.
-
Women of childbearing potential must ensure continued use (started ≥ 30 days prior to
signing the ICF) of a hormonal contraceptive method or intrauterine device (IUD) during
the study period.
-
Have a best-corrected visual acuity of 20/30 or better in both eyes.
-
Have vital signs within normal parameters.
-
Have an intraocular pressure ≥10 and ≤ 21 mmHg.
Exclusion criteria: 
-
Be a user of topical ophthalmic products of any kind.
-
Being allergic to naphazoline or having a history of intolerance to nasal decongestants or
ocular vasoconstrictors.
-
Have a history of a suspected diagnosis of primary angle closure, primary angle closure,
or angle-closure glaucoma.
-
Having iridotomies or waiting for iridotomies.
-
Have conjunctival hyperemia grade 3 and 4 on the Efron scale.
-
Have ocular surface staining with a value equal to or greater than 3 on the SICCA scale in
either eye.
-
Being a user of medications or herbal products, by any other route of administration.
-
For women: be pregnant, breastfeeding, or planning to become pregnant during the
study period.
-
Having participated in clinical research studies 90 days prior to inclusion in this study.
-
Having previously participated in this same study.
-
Be a contact lens user who cannot discontinue use during the study.
-
Having a history of any chronic-degenerative disease, including diabetes and high blood
pressure.
-
Present inflammatory or infectious disease, active at the time of entering the study.
-
Present unresolved injuries or traumas at the time of entering the study.
-
Have a history of any type of eye surgery.
-
Having undergone non-ophthalmological surgical procedures in the last 3 months.
Research Product (RP): 
-
PRO-185. Naphazoline 0.03% and hypromellose 0.2%. Solución oftálmica. Laboratorios
Sophia, S.A. de C. V., Zapopan, Jalisco, Mexico.
-
Dosage : 1 drop 4 times a day in both eyes (AO).
-
Route of administration: Ophthalmic.
Duration of treatment: 
8 days 
Subject's duration in the study: 
10 days 


Evaluation criteria: 
Primary outcome variables 
−
Intraocular pressure
−
Heart rate
−
Systemic blood pressure
−
Pupillary diameter
−
Conjunctival hyperemia
Secondary outcome variables: 
−
Best corrected visual acuity
−
Changes in ocular surface staining
−
Incidence of chemosis
−
Incidence of expected adverse events
−
Incidence of unexpected adverse events
Statistical methodology 
Statistical analysis will be performed using specialized statistical software (SPSS statistical 
package, available version, or R software). Quantitative variables will be expressed as measures 
of central tendency: mean, median, standard deviation, and ranges. Qualitative variables will be 
presented as frequencies and percentages. Statistical analysis to rule out differences will be 
performed using nonparametric statistics, using the Wilcoxon signed-rank test for one sample, 
for quantitative variables. Analysis between qualitative variables will be performed using the 
binomial X 2 (Chi-square) test or Fisher's Exact test (when applicable). An alpha (α) ≤ 0.05 will be 
considered significant .


1.2 Study diagram 


1.3 Subject's activity schedule 
Procedures 
Baseline visit 
Day 1 
Visit 1 
Day 3 (+1) 
Visit 2 
Day 8 
Final visit 
Day 10 (+1) 
Pre-TP 
Post -
TP 
Pre -
Tx 
Post-
Tx 
Pre -
Tx 
Post-
Tx 
Without 
treatment 
FCI Signature 
X 
 
 
 
 
 
 
Medical record 
X 
 
 
 
 
 
 
Eligibility criteria 
X 
 
 
 
 
 
 
Subject code assignment 
X 
 
 
 
 
 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
Heart rate 
X 
X 
X 
X 
X 
X 
X 
Respiratory rate 
X 
 
X 
 
X 
 
X 
Blood pressure 
X 
X 
X 
X 
X 
X 
X 
Temperature (°C) 
X 
 
X 
 
X 
 
X 
Urine pregnancy test 
X 
 
 
 
 
 
X 
AVMC 
X 
 
X 
 
X 
 
X 
Anterior segment evaluation 
X 
X 
X 
X 
X 
X 
X 
Pupillary diameter 
measurement 
X 
X 
X 
X 
X 
X 
X 
Eye stains 
X 
X 
X 
X 
X 
X 
X 
Intraocular pressure 
X 
X 
X 
X 
X 
X 
X 
Gonioscopy 
X 
 
 
 
 
 
 
Pharmacological mydriasis 
(phenylephrine/tropicamide) 
X 
 
 
 
 
 
 
Evaluation of the posterior 
segment 
X 
 
X 
 
X 
 
X 
Application of PRO-185 
during a visit 
 
 
X 
 
X 
 
 
Delivery of material for the 
subject 
 
X 
 
 
 
 
 
Delivery of the study 
medication 
 
X 
 
 
 
 
 


Procedures 
Baseline visit 
Day 1 
Visit 1 
Day 3 (+1) 
Visit 2 
Day 8 
Final visit 
Day 10 (+1) 
Pre-TP 
Post -
TP 
Pre -
Tx 
Post-
Tx 
Pre -
Tx 
Post-
Tx 
Without 
treatment 
Evaluation of concomitant 
medications 
X 
 
X 
 
X 
 
X 
Adherence assessment 
 
 
X 
 
X 
 
 
Return of study medication 
 
 
 
 
 
X 
 
Withdrawal from the 
subject's diary 
 
 
 
 
 
X 
 
PRE-TP: prior to application of 0.8% tropicamide and 5% phenylephrine ophthalmic solution (TP 
Ofteno® , indicates that the checks will be performed prior to pharmacological mydriasis. Post-TP: 
post application of the TP Ofteno® solution, indicates that the checks will be performed with 
pharmacological mydriasis. Pre- Tx : pretreatment, indicates the checks to be performed before 
the application of the investigational medicinal product. Post-Tx : post-treatment, indicates the 
evaluations to be performed within 20-40 minutes after the application of the investigational 
medicinal product. ICF: informed consent form. BCVA: best corrected visual acuity. 


2. Introduction and background 
2.1 Theoretical framework 
2.1.1 The red eye  
Red eye is a cardinal sign of ocular inflammation, a very common presentation, and one of the most 
frequent ophthalmologic conditions in primary care. As a nonspecific sign of inflammation, its 
etiology is highly varied and can range from disorders that do not threaten vision (such as some 
infectious conjunctivitis, allergic and irritant conjunctivitis, and blepharitis, etc.) to diseases that are 
potentially harmful to the organ and its function (such as uveitis, acute angle-closure glaucoma, 
endophthalmitis, etc.). [1] 
Through the patient's medical history and examination, the etiology of the red eye can be diagnosed 
and the treatment to be followed determined accordingly. [1] [2] 
In self-limiting conditions, such as viral and irritant conjunctivitis, management is based on 
symptomatic measures focused on reducing conjunctival hyperemia and itching, relieving the 
patient's discomfort during the illness. This treatment may include cold compresses, decongestants, 
and lubricants. In some conditions, such as allergic and bacterial conjunctivitis, in addition to 
etiological treatment with antihistamines or antibiotics, respectively, symptomatic support 
measures are also recommended. [1] [2] [3] [4] 
Synthetic adrenergic agonists such as phenylephrine, naphazoline, oxymetazoline, and 
tetrahydrozoline are available as ocular decongestants. After topical application, they produce 
vasoconstriction at concentrations that do not cause mydriasis. These agents provide symptomatic 
therapy, as they have no effect on the conjunctival response to antigen. [5]  
Red eye is a common problem in the general population, accounting for approximately 15% of 
ophthalmology consultations and almost 6% of general practitioner visits. Symptoms and signs that 
may accompany red eye include irritation, itching, discharge, tearing, and eye pain. [6] 
The first thing to evaluate in patients with red eye is whether it is caused by a serious eye disease. 
To rule out a serious ophthalmologic disorder, it is important to inquire about the patient's absence 
of significant eye pain, blurred vision, and photophobia. [7] 
Ocular biomicroscopy is a useful tool that can diagnose the causes of more than 90% of conditions 
associated with red eye. During the examination, it is important to take an adequate history, 
detailing the onset of the red eye, the duration of its onset, whether the condition is unilateral or 
bilateral, associated symptoms, ocular and systemic diseases, use of topical and systemic 
medications, history of trauma, and contact lens use. [8] [9] 
The examination should include a visual inspection of the periocular region, adnexa, ocular surface, 
and anterior chamber. This allows for differentiation of hyperemia in conditions related to eyelid 


abnormalities, peripheral (predominantly in the fornix), perikeratic (greater intensity in the scleral-
corneal limbus ) , and localized conditions. Alterations in corneal transparency or elements in the 
anterior chamber, such as hyphema or hypopyon, should be sought. Fluorescein staining can 
identify de-epithelialized regions. [9]Table 2 summarizes the main causes of red eye, according to 
the predominant location of the conjunctival hyperemia. 
Table ii. Main causes of red eye 
Red eye of palpebral or periocular origin 
Diffuse superficial red eye 
• 
Blepharitis 
• 
Chalazion 
• 
Stye 
• 
Orbital cellulitis 
• 
Dacryocystitis 
• 
Infectious conjunctivitis 
• 
Allergic conjunctivitis 
• 
Dry eye syndrome 
Perikeratic red eye of intraocular origin 
Localized red eye 
• 
Keratitis, corneal erosion 
• 
Uveitis 
• 
Glaucoma 
• 
Subconjunctival hemorrhage 
• 
Pterygium 
• 
Episcleritis 
• 
Scleritis 
2. 1.2 Pathophysiology of red eye 
Red eye results from vasodilation of conjunctival blood vessels. Vascular tone in mammals is 
regulated by the activity of adrenergic receptors found in vascular smooth muscle, vascular 
endothelium, and nerve cell terminals. The contractile activity of smooth muscle cells alters blood 
vessel diameter. Stimulation of α1 and α2 adrenergic receptors causes vasoconstriction of vascular 
smooth muscle; meanwhile, stimulation of β adrenergic receptors causes vasodilation. Additionally, 
stimulation of α1 , α2 , and/or β adrenergic receptors on vascular endothelium will cause 
vasodilation, possibly by increasing nitric oxide. Basal vascular tone is governed by norepinephrine-
mediated vasoconstrictor and vasodilator activity, and red eye occurs when vasodilation 
predominates. [10] 
By analogy, the vasoactive effect of active ingredients with adrenergic agonist activity will depend 
on the balance between vasoconstrictor and vasodilator activity. Anatomical differences in the 


distribution of α1 and α2 adrenergic receptors are important , since α1 receptors are found in veins 
and arteries, while α2 receptors predominate in veins. Consequently, the effects of the active 
ingredients will depend on the receptors they activate. [10] 
2.1.3 Ocular vasoconstrictors 
Ocular vasoconstrictors available over-the-counter in the United States are all α adrenergic receptor 
agonists but vary in their affinity for the receptors. First-generation vasoconstrictors include 
phenylephrine, 
tetrahydrozoline, 
naphazoline, 
and 
oxymetazoline. 
Phenylephrine, 
a 
sympathomimetic amine with selective affinity for the α 1 receptor, and oxymetazoline, an 
imidazole derivative with approximately 5:1 affinity for α 2: α 1 receptors , are no longer 
commercially available in the United States. Tetrahydrozoline, an imidazole derivative available 
since the 1950s, is a selective α 1 receptor agonist  Naphazoline, another imidazole derivative, is an 
agonist with activity at alpha 1 and alpha 2 receptors, approved since 1974 as an ocular 
decongestant, and available over-the-counter in concentrations of 0.012-0.03% for the relief of 
ocular redness.[10] 
Brimonidine is an adrenergic agonist with a marked affinity for the α 2 receptor compared to the α 
1 receptor (1000:1). It was initially approved as an ocular hypotensive in 1996 at a concentration of 
0.2%; however, its off-label use in LASIK and pterygium surgery has demonstrated that brimonidine 
has effects in reducing subconjunctival hemorrhage and conjunctival hyperemia, which led to the 
development of a 0.025% solution to reduce ocular redness caused by minor irritations. [10] 
2.1.4 Mechanism of action of vasoconstrictors 
α1, mixed α1 and adrenergic agonists α 2, and α 2 selective vasoconstrictors produce constriction of 
conjunctival blood vessels through the activation of adrenergic receptors, this activation initiates a 
signal cascade of the G protein that culminates in the contraction of vascular smooth muscle. 
Specifically, the activation of the α 1 receptor, linked to the Gq protein, produces the contraction of 
vascular smooth muscle through the inositol triphosphate (IP 3 ) pathway, while the activation of the 
α 2 receptor , linked to the Gi protein, produces the contraction of the smooth muscle by the 
intracellular decrease of cyclic adenosine monophosphate (cAMP). Due to the differences in the 
distribution of α 1 and α 2 receptors , vasoconstrictors with activity in the α 1 receptor produce 
vasoconstriction of the conjunctival venules and arterioles; while those selective to the α 2 receptor 
act only on the venules.[10] 
2.2 Background on the investigational product 
The combination of naphazoline with hypromellose and menthol in ophthalmic solution is indicated 
to relieve eye redness caused by minor eye irritations. [11] [12] [13] 
These medications can reduce vascular congestion of the corneal and conjunctival surface in various 
disorders affecting the eyeball and/or ocular adnexa. [11] [12] [13] 


PRO-185 ophthalmic solution is a solution containing 0.03% naphazoline and 0.2% hypromellose. It 
also contains menthol among its excipients, distinguishing it from other products that are a 
combination of naphazoline and hypromellose. 
2.2.1 Naphazoline  
Naphazoline, classified by its chemical structure as an imidazole derivative, differs from other 
adrenergic agonists by the replacement of the benzene ring with an unsaturated ring. In general, 
naphazoline has a higher affinity for the α-adrenergic receptor than for the β-adrenergic receptor. 
After application to the ocular surface, naphazoline induces marked vasoconstriction by stimulating 
α-adrenergic receptors on vascular smooth muscle. Imidazole derivatives, such as naphazoline, have 
the advantage over phenylephrine in that they are less likely to induce rebound congestion and 
mydriasis. [12] [14] [5] 
In most cases of conjunctival hyperemia, naphazoline has an immediate and prolonged 
vasoconstrictive effect on the bulbar and eyelid capillaries of the conjunctiva. If patients have 
superficial corneal vessels, they may also be constricted. [14] 
Naphazoline can lower intraocular pressure by agonizing α-2 adrenergic receptors, thereby 
decreasing aqueous humor formation. The pressure reduction effect of naphazoline depends on the 
dose administered and the patient's sensitivity. [15] 
Systemic absorption has been reported after topical application of naphazoline solutions. Although 
not administered systemically, it can be absorbed through the gastrointestinal tract. The onset of 
naphazoline's action (conjunctival vasoconstriction) can be felt within 10 minutes and last up to 6 
hours. [16] 
Hurwitz and Thompson studied the effects of 0.1% naphazoline in over 100 subjects with congested 
and normal eyes. Slit-lamp evaluations revealed constriction of the conjunctival vessels, with no 
effects on the deep vessels. No accommodative effects were observed at this concentration. [5] 
The incidence of serious adverse reactions is low in patients receiving naphazoline ophthalmic 
solution at therapeutic doses. Excessive doses and/or prolonged use may irritate the conjunctiva 
and cause systemic adverse effects. [17] 
Ophthalmic naphazoline may cause blurred vision, mild transient itching, irritation, mydriasis, and 
increased or decreased intraocular pressure. Application of naphazoline to the conjunctiva may 
release pigment granules from the iris. Rebound congestion, characterized by reactive hyperemia, 
frequently occurs with prolonged use. [17] 
Systemic effects it can occasionally cause include headache, hypertension, cardiac irregularities, 
nervousness, nausea, dizziness, weakness, and sweating. Overdose can cause drowsiness and 
decreased body temperature, bradycardia, hypotension, and coma. [17] [18] 
Naphazoline poisoning is uncommon, although it can theoretically result in significant morbidity, 
especially in children, although it is very rare to see more than minimal effects. The toxic dose for 


oral ingestion is not established, but children have developed severe reactions after ingesting 1 or 
2 milliliters of ocular or nasal formulations containing imidazole derivative decongestants. [18] 
2.2.2 Hypromellose 
Hypromellose is a white, yellowish-white, or grayish-white powder that dissolves in water to form 
colloidal solutions. It is used in the pharmaceutical industry to make a film coating for tablets, as a 
tablet binder, emulsifier, matrix modifier, suspending agent, and stabilizer for gels and ointments. 
In the food industry, it is used as an emulsifier and stabilizer. In ophthalmic solutions, it is used to 
prolong the action of some medications (as an excipient) or as an ocular lubricant. It has also been 
used intraocularly in concentrations of 2% as a medical device for eye surgery. [16] 
Hypromellose is a chemically inert substance with a refractive index similar to that of the cornea, 
and has emollient and cohesive properties. It blends well with other polymers and substances 
present in artificial tears. It improves viscosity and prolongs the corneal retention time of solutions. 
[5] 
Hypromellose does not have the capacity for intraocular penetration or systemic absorption, and its 
pharmacokinetic properties have not been studied. [19] 
In recent years, ether-type cellulose substitutes, particularly hydroxymethylcellulose and 
hydroxypropylmethylcellulose, have been used more frequently. They are somewhat less viscous 
than methylcellulose, but possess cohesive and emollient properties equal to or superior to those 
of methylcellulose. Like methylcellulose, these ethers mix well with other polymers and substances 
present in artificial tears, and are compatible with many active ingredients and chemicals used in 
the eyes. Toda et al. found that 0.5% hydroxypropylmethylcellulose solution improved ocular 
symptoms in patients with dry eye associated with Sjögren's syndrome, and in those without it as 
well, although tear break-up time and rose bengal and fluorescein staining improved significantly 
only in the Sjögren's group. [5] 
Ocular surface damage in patients with Sjögren's syndrome is severe due to poor retention of tear 
components . Tear substitutes are ineffective due to their short retention time on the eye. However, 
hypromellose 0.5% was found to increase the tear evaporation rate at 40% ambient humidity 
(TEROS 40) for a longer period than sodium hyaluronate 0.1% or saline-based artificial tears. 
Hypromellose remains on the ocular surface longer and maintains its moisture. [20] 
Cellulose ethers, such as hypromellose, can absorb the tear film layer at the corneal-tear interface, 
stabilizing a thicker layer of fluid adjacent to the absorption site. The observation that these 
compounds prolong tear break-up time supports this assumption. [5] 
Hypromellose is a substance permitted for oral intake in humans, with an approved dose of 5 
mg/kg/day, and no effects have been demonstrated that influence carcinogenesis, teratogenesis, 
and alterations in fertility with its ingestion. [21] 


2.2.3 Menthol 
Menthol is a cyclic alcohol found as the main compound in the essential oils of Mentha canadensis 
and Mentha X piperita. Menthol is known for its cooling properties when inhaled, consumed, or 
applied to the skin, due to its ability to chemically activate the cold-sensitive cation channel, also 
known as transient receptor potential melastin-8 ( TRPM8). Studies have shown that menthol acts 
through the TRPM8 receptor by increasing the rate of intracellular calcium and mobilizing flow 
through the channels to induce a cold-like response at the site of application. Studies have shown 
that the cooling effect of menthol can last up to 70 minutes or more in 65% of humans. [22] 
Menthol has been used medicinally since ancient times and is frequently used in topical 
preparations for analgesic, antipruritic, and anti-inflammatory properties, as well as to enhance the 
penetration of other active ingredients. Menthol has been used in ophthalmic preparations to 
relieve visual fatigue and discomfort.[23] 
The cornea is innervated by three types of afferent neurons with free terminal endings. 
Mechanoreceptive neurons respond exclusively to mechanical stimuli from the corneal surface; 
polymodal neurons respond to mechanical, thermal, and chemical stimuli (including pH decreases); 
and cold-responsive neurons. Activation of mechanoreceptive and polymodal receptors evokes 
irritation, pain, and a tearing reflex; conversely, activation of cold-responsive neurons induces 
tearing without irritation or pain. [23] 
In addition to low temperatures, the activity of cold-sensitive cells is increased by the application of 
menthol, a TRPM8 receptor agonist, and by the use of hypertonic artificial tears. [23]Menthol 
application to the eye can increase tear production. [24] 
The irritancy and toxicity of menthol was evaluated by the Draize test in 30 rabbits divided into 5 
groups, each of which received solutions with concentrations of 0, 0.025, 0.05, 0.1, and 0.2% in a 
phosphate buffer at pH 7.4. Each application used 100 µl of the solution, and the dosage was 4 times 
a day for 7 days. The condition of the ocular tissue was observed at 1, 12, 24, 48, and 72 h after the 
last application. Corneal opacity and iris hyperemia were graded on a scale of 0-4, while conjunctival 
congestion, edema, and secretion were evaluated on a scale of 0-3, 0-4, and 0-3, respectively. For 
each solution, the average values of 4 eyes were calculated. According to the way the tests were 
evaluated, the concentrations were considered to be non-irritating at average values of 0-3.9, 
slightly irritating for values of 4-8.9, moderately irritating for values of 9-12.9 and seriously irritating 
for values of 13-16. [25] 
The mean values of the menthol irritation test were 0.0, 0.5 ± 0.5, 1.2 ± 0.8, and 4.2 ± 1 for the 0, 
0.025, 0.05, 0.1, and 0.2% solutions, respectively. The results showed that menthol was not irritating 
at concentrations ranging from 0.025% to 0.1%, and caused mild irritation at the 0.2% 
concentration. In this study, no visible damage was observed, nor were any abnormal clinical signs 
reported in the cornea, iris, or conjunctiva with the administration of any of the concentrations. All 
signs of mild irritation diminished and disappeared completely 48 h after the last application. [25] 


Menthol is an excipient in the PRO-185 solution. It is present at a concentration 80 times lower than 
the concentration that demonstrated mild irritation in the repeated-dose irritability test, and 10 
times lower than the lowest concentration that demonstrated no irritating effects. Therefore, no 
additional safety data are expected from its inclusion as an excipient in the PRO-185 formulation. 
[25] 
2.3 Background on the investigational product 
We do not have clinical studies of an ophthalmic solution containing naphazoline, hypromellose, 
and menthol. However, we have experience in clinical studies with naphazoline and hypromellose 
at higher concentrations than PRO-185, as well as in a preclinical safety and toxicity study of PRO-
185 ophthalmic solution. 
2.3.1 Preclinical safety and toxicity study of PRO-185 solution 
To determine the safety of PRO-185 (an ophthalmic solution containing naphazoline, hypromellose, 
and menthol), a preclinical study was conducted to compare its non-inferiority in terms of safety 
and toxicity with that of a currently marketed naphazoline ophthalmic solution (Nazil® 
Contamination ). In the study, the products were administered to the ocular surface of New Zealand 
albino rabbits (n=16 subjects). One drop was administered four times daily to the right eye for 30 
days, while the left eye served as the control eye. [26] 
In the study, 100% of all subjects in both groups and their respective control eyes presented corneal 
and conjunctival fluorescein staining between grades 0 and 2, values considered normal. At visits 2, 
3 and 5, 100% of cases in the PRO-185 and Nazil ® Contamination groups presented grade 0 (absent) 
fluorescein staining. Only at visit 1, two cases in each group presented grade 1 (mild), this finding 
not being statistically significant (E. Fisher, p=1.000). [26] 
Similarly, for conjunctival fluorescein staining, at visits 2, 3, 4, and 5, 100% of subjects in both groups 
had grade 0 (absent). While at visit 1, one case in the PRO-185 group was grade 2 (moderate) and 
one case in the Nazil ® Contamination group was mild, these findings not being statistically significant 
( X 2 , p = 0.368). In the subjects in the PRO-185 group, only at visit 1, two cases for the treated eyes 
presented grade 1 (mild) corneal staining with fluorescein, not being statistically significant with 
respect to the control eyes (E. Fisher, p = 1.000). For conjunctival fluorescein staining, at visit 1, both 
one treated eye and one control eye presented grade 1 staining (E. Fisher, p = 1.000). [26] 
At the initial, 3, 4 and 5 visits, 100% of the treated eyes in both groups showed grade 0 (normal) 
conjunctival hyperemia. At visit 1, two cases were mild and one was moderate in the PRO-185 group, 
compared to 3 mild cases in the Nazil ® Contamination group, although these findings were not 
statistically significant ( X 2 , p=0.549). However, in this case, one eye in the PRO-185 group presented 
grade 2 conjunctival hyperemia, compared to none in the Nazil ® Contamination group. At visit 2, 
only one case in the Nazil ® Contamination group presented mild hyperemia (E. Fisher, p=1.000). [26] 
In subjects treated with PRO-185, only at visit 1, two cases for the treated eyes presented grade 1 
(mild) conjunctival hyperemia and one case grade 2 (moderate), while in the control eyes only mild 


conjunctival hyperemia was reported in one eye. This difference was not statistically significant ( X 
2 , p=0.435). [26] 
In the eyes treated with Nazil® Contamination, at visit 1, three treated eyes, compared to one 
control eye, had mild hyperemia (E. Fisher, p=0.569). At visit 2, both one treated and one control 
eye had mild hyperemia (E. Fisher, p=1.000). [26] 
Only one case at visit 1 in the PRO-185-treated group showed minimal conjunctival edema; this 
finding was not statistically significant between treatments (E. Fisher, p=1.000). All treated and 
control eyes had normal conjunctival discharge during the study, as well as normal corneal opacity. 
[26] 
At the initial, 2, 3, 4 and 5 visits, 100% of the treated eyes in both groups presented normal tearing. 
At visit 1, five eyes in the PRO-185 group and five in the Nazil ® Contamination group presented grade 
1 tearing (increased tear level with concave meniscus), while one eye in each group presented grade 
2 tearing (increased tear meniscus level with convex meniscus). These findings were not statistically 
significant ( X 2 , p = 1.000). [26] 
In subjects treated with PRO-185, only in visit 1, five cases for the treated eyes (OD) presented grade 
1 and one case grade 2, compared to four control eyes grade 1 tearing, without these findings being 
statistically significant ( X 2 , p=0.411). [26] 
For subjects treated with Nazil ® Contamination, only in visit 1, five cases for the treated eyes (OD) 
presented grade 1 and one case grade 2, compared to three control eyes grade 1 tearing, without 
these findings being statistically significant ( X 2 , p=0.248). [26] 
No statistically significant differences were observed between groups in intraocular pressure during 
the execution of the study. The initial mean ± standard deviation (SD) value for PRO-185 was 8.88 ± 
0.64 mmHg vs 9.13 ± 1.55 mmHg for Nazil ® Contamination (Mann-Whitney, p = 0.824). While the 
final mean ± SD IOP was 9.38 ± 1.06 mmHg for PRO-185 vs 8.75 ± 1.28 mmHg for Nazil ® 
Contamination (Mann-Whitney, p = 0.354). Chart 1shows the IOP during the execution of the 
protocol. [26] 
No statistically significant differences were observed between treatment groups for the mean 
change in IOP during the execution of the protocol (Mann-Whitney, p>0.05). Chart 2shows the 
variation in IOP for both treatments. Only one subject (subject 12) treated with PRO-185 showed an 
increase of 5 mmHg at visit 2 compared to visit 1 (IOP visit 2 of 11 mmHg and IOP visit 1 of 0.6 
mmHg). Despite the significant difference between the intraocular pressure at visits 2 and 1 of 
subject 12, if the intraocular pressure at visit 2 is compared with the baseline visit, the difference is 
3 mmHg (baseline visit IOP of 9 mmHg and visit 2 IOP of 11 mmHg), this difference being insufficient 
to declare treatment-induced ocular hypertension in the study subject. [26] 


 
Chart 1Intraocular pressure (IOP) in mmHg during the study. Group A = PRO-185 Group B = Nazil® 
Contamination 
In Chart 1, the bars show the mean value ± 95% CI for the error bars. Mann-Whitney U p>0.05 in all 
comparisons. 


 
Chart 2Change in intraocular pressure (IOP) in mmHg during the study. Group A = PRO-185 Group B = 
Nazil® Contamination 
In the Chart 2, the bars show the mean value ± 95% CI for the error bars. Mann-Whitney U p>0.05 
in all comparisons. [26] 
For eyes treated with PRO-185, compared to their control eyes, no statistically significant 
differences in IOP were observed during the execution of the protocol ( Mann-Whitney, p>0.05 ), 
see Chart 3The IOP at visit 3 (8.38 ± 0.52 mmHg) was statistically lower than the IOP at visit 2 (9.63 
± 0.74 mmHg), for eyes treated with PRO-185 (Wilcoxon, p=0.026). [26] 
In eyes treated with Nazil ® Contamination, compared to their control eyes, no statistically significant 
differences in IOP were observed during the execution of the protocol (Mann-Whitney, p>0.05), see 
Chart 3The IOP at visit 2 (9.75 ± 0.87 mmHg) was statistically higher than the IOP at visit 1 (9.25 ± 
1.28 mmHg), (Wilcoxon, p=0.046). Meanwhile, the IOP at visit 3 (8.75 ± 0.89 mmHg) was statistically 
lower than its mean value at visit 2 (Wilcoxon, p=0.038). [26] 


 
Chart 3Intraocular pressure (IOP) in mmHg for eyes treated with PRO-185 (OD) (Group A) relative to their 
control eyes (OS) and for eyes treated with Nazil® Contamination (OD) (Group B) relative to their control 
eyes (OS) 
In Figure 3, the Wilcoxon signed-rank test for related samples was performed in PRO-185, for visit 2 
> visit 1 and in Nazil ® Contamination, for visit 2 > initial visit and visit 2 > visit 3; *p<0.05. [26] 
No adverse events occurred for either treatment during the protocol. Similarly, no alterations of any 
kind were observed for either treatment arm during the protocol. [26] 
In the histopathological results: there were no changes in the cornea, conjunctiva, ciliary body, 
retina, optic nerve for either treatment group. Regarding the goblet cells, the Subjects assigned to 
PRO-185 treatment had a mean ± SD value of 21.50 ± 2.33 cells ( AAPas , %) compared to 22.25 ± 
2.71 cells ( AAPas , %) in the Nazil® Contamination group , which was not statistically significant 
(Mann-Whitney, p = 0.546). Cell density in both the PRO-185 and Nazil ® Contamination treatments 
was not different in treated eyes compared to their controls (p values = 0.788 and 0.580 
respectively). [26] 
In general, the results of the variables studied do not show significant differences between the two 
products, nor do they show any unexpected abnormal results that could indicate the existence of 
any risk of these products in the research subjects. [26] 
Therefore, since there was no difference greater than 10% between the incidence of the main 
variables, we can accept the alternative hypothesis of the study, which states: The safety of the 
PRO-185 solution is not inferior to the safety of the Nazil ® Contamination solution within a non-
inferiority margin of 10% in abnormal fluorescein uptake and/or in the incidence of conjunctival 
hyperemia grade ≥ 2; given that there were no significant abnormal values in the secondary and 
toxicity variables with the administration of the product, and since it has been established by the 
main variables that the PRO-185 solution is not inferior in safety to Nazil ® Contamination, we can 
be certain that the product was safe to use with the animal model of the study (New Zealand albino 
rabbits). [26]  


2.3.2 Preclinical study of an ophthalmic solution of naphazoline 0.1% and hypromellose 
0.5% (higher concentration of the main components of PRO-185) 
Ten healthy New Zealand albino rabbits were administered a 0.1% naphazoline and 0.2% 
hypromellose solution four times daily for 10 days as part of a preclinical study to evaluate the 
product's safety and acute toxicity. The study results showed no changes to the ocular surface of 
the rabbits receiving the drug; only two rabbits exhibited minimal ocular secretion on the first day 
of the study. No dye uptake was observed in any of the rabbits. The study concluded that the 0.1% 
naphazoline and 0.2% hypromellose ophthalmic solution is safe for topical application to the ocular 
surface of healthy albino rabbits. [27] 
In another preclinical study, the safety and acute toxicity of naphazoline administered to the ocular 
surface of healthy New Zealand rabbits was evaluated. Two groups were evaluated with 
naphazoline; one group received Nazil Ofteno® , the other Naphacel Ofteno® ( naphazoline 0.1% and 
hypromellose 0.5%), and a third group received placebo. Each group consisted of 14 rabbits. The 
rabbits received the study product 4 times daily for a period of 30 days. [26] 
On days 15 and 30 of the study, the Nazil Ofteno ® group presented conjunctival hyperemia, 72% of 
the eyes evaluated, while 68% of those treated with Naphacel Ofteno ® had hyperemia, and 54% of 
those treated with placebo. [26] 
Comparing the intensity between both naphazoline groups, on day 5 the hyperemia was more 
intense for Naphacel Ofteno ® , and on day 15 for Nazil Ofteno ® . [26] 
There was no conjunctival discharge during the study in any of the groups, nor any changes in the 
posterior segment. There was also no corneal staining with fluorescein, lissamine green, or changes 
in intraocular pressure in the rabbits in all three groups. [26] 
This study concluded that Nazil Ofteno® and Naphacel Ofteno® have a similar safety and toxicity profile. 
[26] 
2.3.3 Clinical study of an ophthalmic solution of naphazoline 0.1% and hypromellose 0.5% 
An open-label, single-center, observational, prospective clinical study included thirty healthy 
volunteers. Volunteers were administered a 0.1% naphazoline and 0.5% hypromellose solution four 
times daily for ten days. Results showed no changes in conjunctival hyperemia, conjunctival 
discharge, ciliary injection, chemosis, and rose bengal and fluorescein staining. However, 86% of 
patients experienced burning, 70% of which was mild and the remainder moderate; 6.6% had mild 
red eyes; and 6.6% had mild ocular pain. The mean burning score was 0.6861, with a standard 
deviation of 0.42 and a standard error of 0.016. Student's t-test showed a p>0.05, leading to the 
conclusion that the burning score was not statistically significant. The study concludes that the 
solution of naphazoline 0.1% and hypromellose 0.5% is safe for application to the ocular surface of 
healthy volunteers. [28] 


2.4 Background on the research 
2.4.1 About the research question 
Is the application of PRO-185 ophthalmic solution safe and tolerable in clinically healthy subjects? 
The importance of the question preceding this paragraph is that there are no clinical studies with 
products identical to the PRO-185 solution, although there are studies with similar products 
(different concentration and excipients). 
2.5 Risk-benefit evaluation 
2.5.1 Known potential risks 
Formulations containing naphazoline and/or hypromellose at the same concentrations as PRO-185 
are available over-the-counter without the need for clinical or preclinical studies in high surveillance 
countries.[11] The risks associated with the use of this product in specific populations are described 
below; however, the use of this medication in the study will be in a population group without the 
alterations or characteristics described below. 
Patients with high blood pressure, cardiovascular abnormalities, hyperglycemia, hyperthyroidism, 
infections, and corneal wounds should use products containing naphazoline with caution due to the 
risk of aggravation of these conditions.[29] 
Patients with angle-closure glaucoma or closed angles may experience significant increases in 
intraocular pressure and should not use products similar to PRO-185.[29] [30] [31] 
Overuse of this product may cause rebound conjunctival hyperemia.[29] [11] 
Some patients, when using this product as recommended, may experience pharmacological 
mydriasis, with the resulting decreased vision.[11] 
Ophthalmic vasoconstrictors, such as this product, should not be used in children under 12 years of 
age, and serious reactions requiring hospitalization have been reported with accidental ingestion of 
naphazoline products by children.[29] [32] 
Administration of this product during pregnancy and lactation is prohibited.[29] [32] 
Although menthol is present as one of the components, it is an excipient and is present at a 
concentration 80 times lower than the concentration that demonstrated mild irritation in repeated-
dose irritability tests, and 10 times lower than the lowest concentration that demonstrated no 
irritating effects. Therefore, no additional safety data are expected from incorporating it as an 
excipient in the PRO-185 formulation. Furthermore, there is already experience in other markets 
with the use of eye drops with menthol as an excipient, and a lubricant and vasoconstrictor.[25] 


2.5.2 Known potential benefits 
The benefit of the drug in the target population is a reduction in hyperemia or ocular redness. [11] 
[12] [13]However, as this is a Phase I study, the study population will be a healthy population. 
Therefore, no direct benefit is expected for each individual, and the benefit of the research will be 
to understand the formulation's behavior and to have more data on its safety in order to prevent or 
identify adverse effects in users or problems with tolerance. 
2.6 Problem statement 
There are many formulations on the market containing naphazoline or hypromellose, some of them 
a combination of both. However, although there is experience with these products in the post-
marketing stages, there are no published clinical studies that have examined the behavior of these 
drugs. 
Since these products can be approved for marketing directly, without the need for clinical trials, 
there is little information available in the medical literature on these products. Furthermore, when 
they are marketed as over-the-counter products, the information included in the instructions or 
prescribing information is summarized and, in most cases, lacks a description of the experience of 
the drug's post-marketing behavior. 
2.7 Justification of the study 
Laboratorios Sophia, SA de CV, has conducted a preclinical and phase I clinical study of a similar 
formulation, but with a higher concentration of components. The reduction in the concentration of 
the main components (naphazoline and hypromellose) in the PRO-185 solution compared to this 
formulation studied could mean greater safety of PRO-185, but not necessarily greater tolerance, 
since the excipients may be involved in clinical manifestations unrelated to the main components. 
Menthol has been used in ophthalmic preparations to relieve visual fatigue and ocular discomfort. 
[23]The inclusion of menthol among the excipients in PRO-185, as well as the lack of clinical 
evaluation of this formulation, necessitates clinical evaluation of the product. This would provide a 
more complete safety and tolerability profile. 
In a preclinical safety and toxicity study of PRO-185 ophthalmic solution in New Zealand albino 
rabbits, the drug was shown to be non-toxic and safe to administer. Information also indicates that, 
at the menthol concentration used in Draize tests, there were no irritating or toxic effects. 
Because there is preclinical evidence of safety and toxicity with the PRO-185 formulation, as well as 
the absence of irritation at the concentration used; and because there are ophthalmic products with 
menthol as an excipient in similar combinations on other markets, it was decided to advance to 
clinical research to test the safety and tolerability of PRO-185 in a controlled environment with 
healthy subjects. 


3. Objectives and hypotheses 
3.1 Objectives 
3.1.1 Main objective:  
• 
To evaluate the safety and tolerability of PRO-185 ophthalmic solution in 
ophthalmologically healthy subjects. 
3.1.2 Specific objectives: 
• 
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of 
subjects who present an increase of > 5 mmHg in intraocular pressure after 20 minutes of 
application compared to the initial value. 
• 
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of 
subjects experiencing a heart rate variation of >15 beats per minute 20 minutes after 
application. 
• 
To evaluate the safety of PRO-185 ophthalmic solution by measuring the incidence of 
subjects with an increase in systolic blood pressure of >15 mmHg or >10 mmHg in diastolic 
blood pressure 20 minutes after application. 
• 
To assess tolerability by the incidence of grade 3 and 4 conjunctival hyperemia. 
• 
To assess tolerability by the incidence of pharmacological mydriasis. 
• 
Evaluate tolerability by the incidence of expected and unexpected adverse events. 
3.2 Hypothesis 
H 0 = PRO-185 ophthalmic solution is safe and tolerable for ophthalmic use, as it occurred in 20% or 
less of subjects with any of the following adverse events: increased intraocular pressure (IOP >5 
mmHg), heart rate variation (>15 bpm), increased systemic blood pressure [>15 mmHg systolic (SBP) 
or >10 mmHg diastolic (DBP)], pharmacological mydriasis, or grade 3 and 4 conjunctival hyperemia. 
𝐻0: 𝑝−𝑝0 ≤𝛿 
H 1 = PRO-185 ophthalmic solution is not safe or tolerable for ophthalmic application because more 
than 20% of subjects experienced any of the following adverse events: increased IOP (>5 mmHg), 
heart rate variation (>15 bpm), increased systemic blood pressure [>15 mmHg systolic (SBP) or >10 
mmHg diastolic (DBP)], pharmacological mydriasis, or grade 3 and 4 conjunctival hyperemia. 
𝐻1: 𝑝−𝑝0 > 𝛿
Comparisons will be made between pre- and post-treatment measurements from visits 1 and 2, and 
between measurements from the baseline visit (before PT application) and measurements from the 


final visit. Only one of the adverse event values described in the hypothesis need be abnormal with 
a frequency >20% between the comparisons (pre-treatment with post-treatment; or pre-PT baseline 
with values from the final visit) for the null hypothesis to be rejected. 
 


4. Study design 
4.1 General description of the study 
Phase I clinical study, controlled, non-comparative, open, single-center 
4.2 Justification of the study design  
Study design (clinical trial) is considered the highest standard of data quality when exploring the 
effect of an intervention. The drug development phase (Phase I) corresponds to the study's 
objective, which is to evaluate safety and tolerability. Therefore, the intervention time is short and 
the required sample size is smaller than that of an efficacy clinical trial. Blinding was not considered 
in the study because there will only be one treatment group. 
4.3 Expected duration  
The total duration of the study, from the first patient visit to the final report, is estimated to be 5 
months, with a 4-month recruitment period. 
The approximate duration of each subject in the study is 10 days. 
 


5. Study population  
5.1 Eligibility criteria 
5.1.1 Inclusion criteria 
• 
Be clinically healthy 
• 
Have the ability to voluntarily grant signed informed consent 
• 
Be able and willing to comply with scheduled visits, treatment plan, and other study 
procedures 
• 
Be between 18 and 45 years old. 
• 
Women of childbearing potential must ensure continued use (started ≥ 30 days prior to 
signing the ICF) of a hormonal contraceptive method or intrauterine device (IUD) during the 
study period. 
• 
Have a best-corrected visual acuity of 20/30 or better in both eyes. 
• 
Have vital signs within normal parameters. 
• 
Have an intraocular pressure ≥10 and ≤ 21 mmHg. 
 
5.1.2 Exclusion criteria: 
• 
Be a user of topical ophthalmic products of any kind. 
• 
Being allergic to naphazoline or having a history of intolerance to nasal decongestants or 
ocular vasoconstrictors. 
• 
Have a history of a suspected diagnosis of primary angle closure, primary angle closure, or 
angle-closure glaucoma. 
• 
Having iridotomies or waiting for iridotomies. 
• 
Have conjunctival hyperemia grade 3 and 4 on the Efron scale. 
• 
Have ocular surface staining with a value equal to or greater than 3 on the SICCA scale in 
either eye. 
• 
Being a user of medications or herbal products, by any other route of administration. 
• 
For women: be pregnant, breastfeeding, or planning to become pregnant during the study 
period. 
• 
Having participated in clinical research studies 90 days prior to inclusion in this study. 
• 
Having previously participated in this same study. 
• 
Be a contact lens user who cannot discontinue use during the study. 
• 
Having a history of any chronic-degenerative disease, including diabetes and high blood 
pressure. 
• 
Present inflammatory or infectious disease, active at the time of entering the study. 
• 
Present unresolved injuries or traumas at the time of entering the study. 
• 
Have a history of any type of eye surgery 
• 
Having undergone non-ophthalmological surgical procedures in the last 3 months. 


5.2 Criteria for elimination and/or substitution of subjects 
5.2.1 Elimination criteria 
▪ 
Withdrawal of informed consent. 
▪ 
Major deviation from the protocol that could impact the integrity of the results. 
▪ 
Presentation of an adverse event, whether or not related to the investigational product, 
which, in the opinion of the PI and/or the sponsor, could affect the subject's ability to safely 
continue the study procedures. 
▪ 
Non-tolerability or hypersensitivity to any of the compounds used during the tests. 
▪ 
Non-tolerability or hypersensitivity to the investigational product. 
5.2.2 Subject substitution 
The sponsor, with prior authorization from the research ethics committees, may decide to replace 
subjects who withdraw their FCI or those who are lost to follow-up, if it is necessary to balance the 
study groups so that they are evaluable. 
5.3 Scrutiny failures 
A screening failure is defined as a participant who agrees to participate in the study, giving their 
consent, but who is not assigned to a treatment group; that is, they do not enter the study. The 
following information regarding screening failures must be reported, at a minimum: 
- 
Demographic data. 
- 
Details of the counting failure (specify whether due to eligibility criteria, which one, or some 
other reason for the failure). 
- 
Presence of serious adverse events during the scrutiny. 
The above is necessary to comply with the CONSORT guidelines ( Consolidated Standards of 
Reporting Trials ) for the publication of results or to respond to potential questions from regulatory 
authorities. 
5.4 Recruitment and retention strategies  
The subject's participation in the study lasts approximately 10 days, during which they will attend 
four visits in total, corresponding to the baseline, first, second, and final visits. Strategies to improve 
subject retention include, but are not limited to: 
• 
Clearly report the objectives of the study. 
• 
Make calls or send text messages to remind yourself of appointments or activities to 
do. 
• 
Provide a printed calendar and ID card to remind you of upcoming appointments and 
activities, as well as their estimated duration. 


• 
Systematic organization of the study procedures, so that the subject does not stay 
longer than necessary during his visit. 
• 
Minimize subject wait times. 
All materials to be delivered to the subject or recruitment strategies implemented by the center will 
be submitted for approval by the corresponding committees. 
5.5 Procedure in case of early discontinuation  
For this protocol, early discontinuation is defined as those subjects who were assigned the PI, who 
at some point were active subjects in the study, but their final evaluation could not be completed. 
If the subject does not complete their participation due to withdrawal of consent or a major 
deviation, the last visit at which their withdrawal was determined will be considered their final visit. 
Subjects withdrawn due to the presence of AEs will continue the follow-up as determined until their 
AE is resolved. 
In cases where the participating subject does not attend their appointment, the research site will 
call to determine the reason and will attempt to schedule a new appointment within the established 
window or an unscheduled appointment. If an appointment cannot be scheduled, the subject will 
be considered lost to follow-up, and the presence of adverse events and the reason for 
discontinuing the study will be asked as minimum data. 
5.6 Subject identification  
Study subjects will be identified by a number and the initials of their name. 
The initials of the subject of study will be obtained starting with the first letter of the name, followed 
by the first letter of the first surname and the first letter of the second surname, obtaining a 
maximum of three letters. In case the person has two names or a compound surname, the first 
letter will always be used. 
Example: 
 
TO. A rieh Daniel M ercado C arrizalez B. J uan D e la Torre O rozco 
 
to. Initials: AMC  
b. Initials: JDO 
During the counting stage, you will be assigned a participant number consecutively, using 3 
consecutive digits. 
Once the subject has been selected, they will be assigned a number that will identify them 
throughout the study. This code will consist of eight numbers in the following order from left to 
right: 
• 
three digits of the molecule under study according to the name given by the sponsor. 
• 
two digits corresponding to the research center number. 
• 
three digits of the consecutive number assigned to its inclusion in the research center. 


Example of assigned number: 
 
 
149 - 01 - 001   
Molecule Code 
Inclusion number 
  
Center number 
  


6. Investigational product and treatment 
6.1 Managed Products 
6.1.1 Investigational Product  
• 
PRO-185. Naphazoline 0.03% and hypromellose 0.2%. Solución oftálmica. Laboratorios 
Sophia, S.A. de C. V., Zapopan, Jalisco, Mexico. 
• 
Route of administration: Ophthalmic. 
6.1.2 Dose of the investigational product 
• 
Dosage: 1 drop 4 times a day in both eyes (AO). 
6.1.3 Treatment with the investigational product 
The investigational product will be administered at the conclusion of the baseline visit, and the 
subject will administer the drops approximately every 2-4 hours between drops, applying them a 
total of 4 times per day to each eye. 
Baseline visits, 1, and 2, should be completed in the morning, before 11 a.m., to allow the patient 
more time to apply PRO-185 throughout the day. During visits 1 and 2, the patient will come to the 
clinic without having previously applied the investigational product. The first application will be 
considered the drop applied by the researcher during the visit. For visit 1, the patient will be required 
to apply the product three times after the visit has concluded. For visit 2, the patient will not need 
to apply the product again because the investigational product will have been returned to the 
researcher. 
6.2 Storage and handling of the investigational product at the study center  
Delivery will be made via a courier service contracted by the sponsor, specifically selected for this 
purpose, to the address of the research center in accordance with the study plan. 
Reception will be carried out by the assigned research team staff. They must verify the condition of 
the primary packaging (box). If it shows alterations or defects in its integrity that, in their judgment, 
could have damaged the contents, they must report this to the sponsor. If the package shows no 
significant defects, they will proceed to open it. 
Inside the shipment, you must locate the receipt and temperature data logger . You must verify that 
the recorded temperature meets the specifications for its transport and storage. You will verify the 
contents (PI) with what is reported on the document. If the document matches the contents, you 
will sign the receipt and send it to the sponsor. If not, you will notify the sponsor. 
Storage and safeguarding are the responsibility of the research center. The medication must be kept 
in a secure area with restricted access. 
Storage temperature should be 2° to 30°C. 


Upon receipt at the center and until the PI is out of stock, the research center is required to review 
the PI storage conditions daily and manually record the temperature recorded by the data logger 
(current, minimum, and maximum temperatures) in the designated format . These data will be 
reviewed by the clinical monitor during their monitoring visits, based on the records stored in the 
data logger's memory .  
In the event of material loss, this must be documented in the input and output log along with a clear 
description of the mechanism by which the loss occurred. 
Upon completion of the protocol, all study materials will be retrieved by the sponsor as part of the 
closing visit. The final return of materials will be made by the principal investigator or the person 
designated by the principal investigator to return materials at the end of the study. 
The sponsor reserves the right to initiate civil and criminal action against the principal investigator 
in the event of undocumented material missing at the end of the study. 
6.3 Concomitant treatments and medications (permitted and prohibited)  
Any medication used, in addition to appearing in the clinical note, must be recorded in the 
concomitant medications section of the eCRF . 
Permitted medications:  
- 
Ophthalmic: 
All permitted medications administered ophthally during the study must wait a minimum of 10 
minutes after the last application of the study or reference treatments. This is to avoid treatment 
interactions with the tear film, based on the physiological tear flow rate and volume. [24] 
o Tetracaine 0.5% 
o Tropicamide 0.8% / Phenylephrine 5% 
o Hypromellose 2% 
o Fluorescein 
o Lissamine green 
- 
Systemic or other routes of administration other than ophthalmic: 
o Any hormonal contraceptive will be included, including tablets, injections, and 
drug-releasing devices. 
 
Prohibited medications: 
- 
Any medication with ophthalmic application that is not on the list of permitted medications 
- 
Any systemic medication that is not on the list of permitted medications. 


6.4 Procedure for monitoring and measuring adherence  
For over four decades, numerous investigations have been conducted on the appropriate way to 
measure and quantify medication adherence, however, none has reached a consensus to establish 
itself as the gold standard, both in cross-sectional and longitudinal studies. [25, 26, 27, 28, 29, 30, 
31, 32] 
There are different procedures for measuring adherence to pharmacological interventions. The 
most common procedure involves self-reports, which include patient interviews, questionnaires, 
and self-monitoring diaries. Their strengths are speed, flexibility, low cost, and ease of 
implementation; they have a high degree of specificity for nonadherence; however, their sensitivity 
and reliability for adherence are low. [32, 33] 
Biochemical measurement of the drug, or its metabolite, is one of the methods that best confirms 
drug use. However, in addition to being costly and impractical, it is of little use in ophthalmic 
applications, as peripheral concentrations may be undetectable; and samples from other tissues 
require more invasive methods that would not be advisable. [32] 
Medication counting is another way to measure adherence. Classically referred to as "pill counting," 
in ophthalmology it is translated as the weight of the bottle. This is a simple, inexpensive, and 
noninvasive method. The main disadvantages of this method are: 1. It cannot confirm the 
application of the medication (it could have been intentionally dropped or instilled outside the eye), 
and 2. It depends on the subject bringing the medication back. [32, 33] 
Adhesion assessment will be based on the bottle's weight and will be performed taking into account 
the following information: drop weight, initial container weight, final container weight, and the total 
number of applications. The following simplified formula will be used: 
(𝑃 𝑖 − 𝑃 𝑓 )100
𝐴𝑑 =
𝑃 𝑇
Where: 
Ad = adhesion 
𝑃 𝑖 = weight of the container delivered to the subject at the beginning 
𝑃 𝑓 = weight of the container returned by the subject 
𝑃 𝑇 = weight of the dosage indicated for the investigational products 
𝑃 𝑇 = ( 𝑃 𝑔 ) 𝐺  
  
Where: 
𝑃 𝑔 = weight of one drop of the medicine, determined by the research and development 
department 


G = number of applications indicated for the investigational products 
Containers that do not maintain their physical integrity will not be considered for the adherence 
calculation. Subjects who do not return the investigational product container by Visit 2 will be 
considered non-compliant. 
There is no standardized parameter to define adequate adherence; this must be defined and 
outlined by the objectives of the particular research. [32] 
For this study, a minimum adherence of 80% (by weight and diary) will be considered necessary to 
meet the research objectives. Therefore, subjects with less than 80% adherence will be included in 
the intention-to-treat population. 
6.5 Strategies to improve adherence  
1. The PI will educate the subject on the importance of correctly administering 
concomitant treatment to achieve the study objectives. 
2. Direct questioning by the IP regarding the application of concomitant treatment. 
3. Delivery of a printed calendar specifying the date of the visit and its activities. 
If deemed necessary, text messages may be sent as reminders. The content of these messages must 
be approved in advance by the IEC. 
 


7. Methods and procedures of the study  
7.1 Research center  
This study will be conducted at a research center previously evaluated by the sponsor. The center 
will be an institution or facility that conducts health research and complies with current regulations. 
The research center will be responsible for forming a multidisciplinary research team to execute the 
clinical study according to the protocol. It is its prerogative to design the organization and select the 
personnel who will perform these functions. However, the sponsor requires that the PI and sub-
investigator be physicians specializing in ophthalmology. 
Any person assigned, under the PI's responsibility, to a part of the study monitoring (sub-
investigator, nurse, etc.) or a specific role in the study (pharmacist, administrative assistant, study 
coordinator, etc.) must be listed in the "Delegation of Responsibilities." 
The competency and training of all individuals directly involved in study activities must be verified 
prior to the conduct of any protocol-related activities. This must be recorded, and documents 
constituting evidence of this competency and/or training must be retained in the study master file. 
The competency and training of personnel involved in the study, both at the central level and at the 
study site, are the responsibility of the sponsor. 
The sponsor must ensure that all study site personnel participating in the study are adequately 
trained in the study (research protocol, investigator's manual, amendments, standard operating 
procedures, etc.) and in ICH Good Clinical Practices prior to the start of their participation in the 
study. Training must be documented in writing and filed in the study master file. 
7.2 Clinical study registration  
This clinical study will be registered by the sponsor in public clinical trial registries prior to its 
initiation (inclusion of the first subject): the National Registry of Clinical Trials (RNEC) of the Federal 
Commission for the Protection against Sanitary Risks (COFEPRIS) and on a WHO primary registry 
platform. WHO primary registries meet specific criteria regarding content, quality and validity, 
accessibility, unique identification, technical capacity, and administration. WHO primary registries 
meet the requirements of the International Committee of Medical Journal Editors (ICMJE). 
7.3 Treatment assignment 
- 
This is an open-label, comparative study. All patients will receive the same treatment. 
7.4 Outcome variables  
7.4.1 Primary outcome variables  
− 
Intraocular pressure. 


− 
Heart rate. 
− 
Systemic blood pressure. 
− 
Pupillary diameter. 
− 
Conjunctival hyperemia. 
7.4.2 Secondary outcome variables  
− 
Best corrected visual acuity (BCVA). 
− 
Changes in ocular surface staining. 
− 
Incidence of chemosis. 
− 
Incidence of expected adverse events. 
− 
Incidence of unexpected adverse events. 
7.4.3 Exploratory variables 
− 
Respiratory rate. 
− 
Body temperature. 
7.4.4 Definition of variables, methods and scales for their measurement 
Table iii. Operational definition of variables 
Variable 
Conceptual 
Definition 
Operational 
Definition 
Type of 
measurement 
Reference 
value 
Statistical 
test 
Intraocular 
Pressure 
Tonometry is the 
objective 
measurement of IOP, 
based on the force 
required to flatten the 
cornea or the degree 
of corneal indentation 
produced by a fixed 
force. 
By means of 
Goldmann tonometry 
based on the Imberk 
-Fick principle. 
Discrete 
quantitative 
10 – 21 mmHg 
Wilcoxon 
one-sample 
signed-rank 
test 
 
Heart rate 
Pulse is a 
measurement of HR, 
that is, the number of 
times the heart beats 
per minute. 
With each heartbeat, 
the left ventricle 
contracts and expels 
blood into the aorta. 
This forceful 
expulsion of blood 
creates a wave that is 
transmitted to the 
periphery of the body 
through the arteries. 
Discrete 
quantitative 
60 – 100 bpm 
Wilcoxon 
one-sample 
signed-rank 
test 
 


Variable 
Conceptual 
Definition 
Operational 
Definition 
Type of 
measurement 
Reference 
value 
Statistical 
test 
It is measured with 
an electronic blood 
pressure monitor or 
manually. 
Systemic 
blood 
pressure 
It is the force exerted 
by blood against the 
walls of the arteries. 
SBP is the pressure 
inside the artery 
when the heart 
contracts and pumps 
blood through the 
body; while DBP is the 
pressure inside the 
artery when the heart 
is resting and filling 
with blood. 
Measured with a 
blood pressure 
monitor and a 
stethoscope. 
Discrete 
quantitative 
SBP: 120 – 
139 mmHg 
DBP: 80 – 89 
mmHg 
Wilcoxon 
one-sample 
signed-rank 
test 
 
Pupillary 
diameter 
The natural pupil of 
the human eye is 
usually approximately 
circular, for a given 
subject, the pattern of 
aberrations, 
diffraction, depth of 
field and retinal 
illumination depend 
on the pupil diameter, 
which in turn varies 
depending on the 
ambient lighting. 
The pupillary 
diameter results 
from the balance 
between the 
pupillary sphincter 
muscle and the radial 
fibers of the iris, 
which have only 
autonomic 
innervation  [33]. 
Continuous 
quantitative 
2 – 6 mm 
Wilcoxon 
one-sample 
signed-rank 
test 
 
Conjunctival 
hyperemia 
It is defined as the 
simplest reaction of 
the conjunctiva to a 
stimulus; a red 
Direct observation. 
Classification using 
Qualitative 
ordinal 
Degrees: 
0= Normal 
1= Very mild 
2= Mild 
Binomial X 2   


Variable 
Conceptual 
Definition 
Operational 
Definition 
Type of 
measurement 
Reference 
value 
Statistical 
test 
appearance is 
observed secondary 
to vasodilation of the 
vessels of the 
conjunctiva of 
variable intensity. 
the Efron scale (see 
appendix 16.1) 
3= Moderate 
4= Severe 
Changes in 
the AVMC 
Spatial visual acuity is 
the ability to 
distinguish separate 
elements of an object 
and identify them as a 
whole. It is quantified 
as the minimum angle 
of separation (located 
at the nodal point of 
the eye) between two 
objects that allows 
them to be perceived 
as separate objects. 
Snellen chart 
Continuous 
quantitative 
0.6 to 2.0 
Wilcoxon 
one-sample 
signed-rank 
test 
Changes in 
ocular 
surface 
staining 
Detection of epithelial 
defects in the cornea 
and conjunctiva. 
Direct observation 
with a slit lamp will 
be graded according 
to the SICCA (see 
annex 16.2)  [34]. 
Discrete 
quantitative 
0 to 12 
Wilcoxon 
one-sample 
signed-rank 
test 
 
Incidence of 
Chemosis 
It is conjunctival 
edema, the result of 
an inflammatory 
reaction. It is 
classified as present 
or absent. 
 
The evaluator will 
use a narrow beam 
of light at 60° and 
measure whether the 
conjunctiva separates 
by ≥1/3 of the entire 
eyelid opening or if it 
extends beyond the 
gray line. 
Nominal 
qualitative 
Present / 
Absent 
Binomial 
 
 


Variable 
Conceptual 
Definition 
Operational 
Definition 
Type of 
measurement 
Reference 
value 
Statistical 
test 
Incidence of 
AE 
Any adverse medical 
event that occurs in a 
patient or clinical 
research subject who 
has been 
administered a 
pharmaceutical 
product and that does 
not necessarily have a 
causal relationship 
with this treatment  
[35]. 
All AEs will be 
reported (according 
to the protocol) and 
not only those in 
which the PI suspects 
a causal relationship 
with the treatments. 
• Discrete 
quantitative 
• Nominal 
qualitative 
• Incidence 
• Seriousness 
(gravity) 
• Causality 
 
Binomial 
X 2  
 
 
Respiratory 
rate 
The process involves 
inspiration and 
expiration, diffusion 
of oxygen from the 
pulmonary alveoli to 
the blood and carbon 
dioxide from the 
blood to the alveoli, 
and the transport of 
oxygen to body 
tissues and organs. 
RR is the number of 
breaths a person 
takes per minute at 
rest. 
Discrete 
quantitative 
12 – 16 
rpm 
Wilcoxon 
one-sample 
signed-rank 
test 
 
Body 
temperature 
A clinical parameter 
that reflects the 
physiological state of 
the body. It is the 
balance between heat 
production and heat 
loss. 
Degree of heat or 
cold, expressed in 
terms of a specific 
scale, using a 
thermometer. 
Continuous 
quantitative 
36.7 – 
37.4°C 
Wilcoxon 
one-sample 
signed-rank 
test 
Abbreviations: BCVA, best-corrected visual acuity; AE, adverse event; HR, heart rate; RR, respiratory rate; PI, principal 
investigator; bpm, beats per minute; DBP, diastolic blood pressure; SBP, systolic blood pressure; IOP, intraocular 
pressure; rpm, breaths per minute; χ 2 , Chi-square. 


7.4.5 Description of the variables, methods and scales for their measurement 
7.4.5.1 Intraocular pressure 
Tonometry is the objective measure of IOP, based primarily on the force required to flatten the 
cornea, or the degree of corneal depression produced by a fixed force. Goldman tonometry is based 
on the Imbert-Fick principle. [34] 
Tonometry will be performed after instillation of topical anesthetic with fluorescein and the use of 
a cobalt blue filter (after assessing surface staining). Two readings will be taken, and the average 
will be recorded in the clinical record. The average will be recorded in the eCRF . 
Management as AEs: IOP peaks ≥24 mmHg should be reported as AEs, as well as increases in 
intraocular pressure greater than 5 mmHg between evaluations before and after application of the 
investigational product (visits 1 and 2). 
7.4.5.2 Heart rate 
Heart rate is the number of times the heart's ventricles contract per unit of time, usually per minute.  
[36]Heart rate measurement will be performed by direct auscultation of the chest with a 
stethoscope. In the case of women, wrist pulse measurement may be permitted. However, the 
method chosen for the patient will be the same one used at all visits. 
For heart rate measurement, the patient must be calm and at rest. They will be asked to rest for at 
least 20 minutes upon arrival at the clinic before the measurement, and when the measurement is 
to be performed after medication administration, they must also rest for 20 minutes. 
For protocol purposes, rest is defined as a state in which the patient is not agitated due to 
unnecessary physical exertion. The patient should be seated for most of the rest period, but may 
get up to walk to the bathroom. 
An increase or decrease in heart rate greater than 15 bpm between the pre- and post-treatment 
check-ups of visits 1 and 2 will be considered abnormal. 
Management as AE: Patients with heart rate greater than 100 bpm or less than 60 bpm. 
7.4.5.3 Systemic blood pressure 
It is the force exerted by blood flow on the walls of the arteries. Blood pressure is measured with 
two measurements: systolic (measured when the heart beats, when the pressure is at its highest) 
and diastolic (measured between heartbeats, when the pressure is at its lowest). When reporting, 
the systolic is written first, followed by the diastolic.[37] 
A calibrated aerobic or mercury sphygmomanometer and a stethoscope will be used for 
measurement. The measurement should be taken with the left arm and should be done while 
resting. 


An increase or decrease in systolic blood pressure greater than 15 mmHg and in diastolic blood 
pressure greater than 10 mmHg will be considered abnormal, between the evaluations before and 
after the application of the investigational product at visits 1 and 2. 
Management as AE: Patients with systolic blood pressure of 140 mmHg or more and diastolic blood 
pressure of 90 mmHg or more. 
7.4.5.4 Pupillary diameter 
It is the opening in the middle of the iris through which light enters the eyeball. Its diameter is 
determined by the balance between the pupillary sphincter muscle and the radial fibers of the iris 
(pupil dilator). Normal values are 2–6 mm. [33] 
Since the research product is not a mydriatic, we will consider significant changes in the pupil of the 
research subjects of > 2 mm (pharmacological mydriasis), whether this change is in photopic or 
mesopic vision. The measurement will be performed using any of the following tools: OPD III, 
autorefractors , or topographers that include these measurements. We will only require that the 
tool used be the same for all patients at the research center and for all their visits. The measurement 
must always be performed in the same office, under the same artificial lighting conditions, and that 
the patient be in the room for at least 5 minutes before the measurement. 
If there is a change greater than 2 mm between the baseline visit and any of the other 
measurements, the product will be considered to have produced pharmacological mydriasis, also if 
there is this change between the examination before and after the application of the investigational 
product at visits 1 and 2. This change will also be considered an adverse event. 
7.4.5.5 Conjunctival hyperemia 
It is defined as the simplest reaction of the conjunctiva to a stimulus. A red appearance is observed 
secondary to vasodilation of the conjunctival vessels of varying intensity. It is graded using the Efron 
scale. [38] 
 
 
Management as AE: Grade 3 and 4 at visits 1, 2 and final will be considered as aggravation and 
adverse event. 
7.4.5.6 Best-corrected visual acuity 
Visual acuity (VA) is a test of visual function. Spatial visual acuity is the ability to distinguish separate 
elements of an object and identify them as a whole. It is quantified as the minimum angle of 
Figura 1. Escala de Efron para hiperemia conjuntival 
Figura 1. Escala de Efron para hiperemia conjuntival 


separation (located at the nodal point of the eye) between two objects that allows them to be 
perceived as separate objects. 
Snellen notation is described as the distance at which the test is performed divided by the distance 
at which the letter is vertically equivalent to 5 minutes of arc. Thus, at 6 meters a letter 6/6 (20/20) 
is equivalent to 5 minutes of arc, a letter 6/12 (20/40) is equivalent to 10 minutes, and a letter 6/60 
(20/200) is equivalent to 50 minutes. The Snellen fraction can also be expressed as a decimal (i.e. 
20/20 = 1 and 20/40 = 0.5).[34] 
VA will be assessed at baseline, without refractive correction, using the Snellen chart. This chart will 
be placed in a location with adequate natural or artificial lighting and at a distance of 3 m from the 
subject being assessed. Visual acuity will be measured in the study eye, asking the subject to keep 
both eyes open and using an occluder to cover the contralateral eye. The subject will read aloud the 
lines indicated by the evaluator. The line with the smallest letters visible will be recorded by the 
evaluator as a fraction of the OD VA in the clinical record. 
The subject's best refractive correction will then be performed, and the examination will be 
repeated using the obtained refraction. This result will be reported as best-corrected visual acuity, 
recorded as a fraction and decimal in both the clinical record and the eCRF . 
Management as AE: A decrease of 2 or more lines of vision in BCVA compared to that obtained at 
the baseline visit will be considered an adverse event. 
7.4.5.7 SICCA ocular surface staining 
The sequence of ocular surface staining is important for reproducibility and accuracy. Fluorescein 
should be applied before lissamine green. 
Fluorescein staining (TF): A drop of topical anesthetic is instilled in the conjunctival fornix. A second 
drop is then applied to the tip of the fluorescein strip, allowing it to sit on the strip for 5 seconds to 
elute the dye, shaking off the excess at the end. A small contact of the strip with the conjunctiva is 
made in the temporal fornix while the patient looks upward, avoiding damaging the conjunctiva. 
Corneal fluorescein staining is assessed using a slit lamp with a cobalt blue filter. It is graded 
according to the Sjögren's Clinical Collaboration Alliance (SICCA) Ocular Staining Grading (OSG). [39]  
According to the CTO, grade 0 corresponds to the absence of punctate epithelial erosions (PEEs); 
grade 1 is defined as the presence of 1-5 PEEs; grade 2 corresponds to 6-30 PEEs; and >30 PEEs will 
be classified as grade 3. An additional grading point will be added if: 1) PEEs with a diameter of 4 
mm are present in the central portion of the cornea; 2) filaments are observed; and 3) confluent 
staining patches, including linear staining, are observed.  [39]See Figure 2. 
the grades awarded for corneal staining for OD and OS, respectively, in the file and on the eCFR . 
The maximum grade per eye is 6. 
Lissamine green staining (LGS): After the fluorescein examination, a drop of saline solution is applied 
to the tip of the lissamine green strip, allowing it to sit on the strip for 5 seconds to elute the dye. A 


drop is instilled from the strip into the temporal fornix while the patient looks up, avoiding damaging 
the conjunctiva. The patient may be asked to blink repeatedly to avoid accumulation in the 
conjunctival folds. The examination should be performed between 1 and 4 minutes after instillation 
through a neutral density filter or a red-free filter. It will be graded according to the SICCA CTO. [39] 
In CTO, grade 0 is defined as the presence of 0 to 9 lissamine green staining points in the 
interpalpebral bulbar conjunctiva (with the temporal and nasal portions scored separately); grade 1 
is defined as the presence of 10 to 32 points; grade 2 by 33 to 100; and grade 3 by >100 points. 
Because of the difficulty in counting individual points in a moving eye, any area ≥4 mm 2 of confluent 
points is considered >100 points  [39]. See Figure 2. 
The PI will record, in the file and in the eCFR , the sum of the grades awarded to the temporal and 
nasal portions for OD and OS, respectively. The maximum grade per eye is 6. 
Figure 2SICCA ocular staining grading (Modified from Whitcher et al, 2010)[34] 
Ocular surface staining using SICCA is determined by the sum of corneal fluorescein staining and 
conjunctival lissamine green staining. This is shown in Figure 2. The values that can be obtained for 
Lissamine green 
(conjunctiva only)
Fluorescein 
(cornea only) 
Grade      Score 
Grade      Score 
0-9 
1 
10-32 
2 
33-100 
3 
>100 
0 
0 
1 
1-5 
2 
6-30 
3 
>30 
Score by staining pattern 
Additional points for fluorescein 
staining only: 
 
+1 if there are patches of confluent 
staining 
 
+1 if there is staining in the pupillary 
area 
 
+1 if there are filaments 


each eye range from 0 to 12, with staining considered abnormal when a value of 3 or higher is 
reached. 
Management as AE: It will be considered an adverse event when the rating is greater than or equal 
to 3 points. 
7.4.5.8 Chemosis 
It is defined as conjunctival edema resulting from an inflammatory reaction. It is graded as present 
or absent. The evaluator will use a narrow 60° beam of light and measure whether the conjunctiva 
separates from the sclera by ≥ 1/3 of the entire eyelid opening or whether it extends beyond the 
gray line. [40]  
Management as AE: The presence of chemosis will be considered an adverse event. 
7.4.5.9 Adverse events 
As described in subsection 8.2 Definition of Adverse Event , an adverse event is defined as Any 
adverse medical occurrence in a subject to whom an investigational product is administered, 
regardless of causal attribution. 
The management of adverse events will be carried out in accordance with the provisions of Section 
8. Evaluation and management of adverse events and incidents. 
The Principal Investigator will record any adverse events that may occur in the study subjects in the 
corresponding section of the eCRF and will also report them in the clinical record. 
For an adequate assessment of adverse events, in addition to the targeted questioning, a 
Comprehensive Ophthalmologic Evaluation must be performed at each visit . This evaluation 
consists of: ophthalmologic examination of the eyelids and adnexa; anterior and posterior 
segments, which are performed during a routine ophthalmologic examination, procedures not 
specifically included in the study variables. The posterior pole evaluation may be performed with 
direct or indirect ophthalmoscopy, with or without pharmacological mydriasis, at the discretion of 
the PI. The fundus will be assessed for abnormalities that could alter the study results. IOP will be 
measured during this evaluation, using the PI's chosen instrument, and should be measured after 
the stain evaluation. The results of the evaluation will be recorded in the clinical record. Only those 
findings that the Principal Investigator considers to be adverse events will be reported in the eCRF 
. 
Adverse events that may occur with PRO-185 are: ocular burning, ocular hyperemia, foreign body 
sensation, itching, red eye, eye irritation, tearing, little relief of discomfort, blurred vision, burning, 
superficial punctate keratitis, stinging, mydriasis, increase or decrease in intraocular pressure, 
release of pigment granules, rebound hyperemia, headache, vertigo, hypertension, hypotension, 
cardiac irregularities (including tachycardia, bradycardia, among other types of cardiac 
irregularities), nervousness or excitability, nausea, dizziness, weakness, drowsiness and 
diaphoresis. [29]  


7.4.5.10 Respiratory rate 
This is the measurement of the number of breaths per minute taken by research subjects. It is 
assessed discreetly by observing, without the subject's awareness, the movements of the rib cage, 
counting each rise/rise of the subject's rib cage as one breath. A range of 12 to 24 breaths per minute 
is considered normal. 
Management as an AE: Any abnormal value will be considered an adverse event. In some cases, the 
research subject may need to be considered a possible case of COVID-19, and the investigator may 
need to refer the patient for medical care and withdraw the subject from the research. 
7.4.5.11 Body temperature 
It is the temperature level of a body. Normal values range from 35°C to 37.4°C. It can be measured 
using a contact or non-contact thermometer on different parts of the body. Any thermometer 
calibrated at the research center may be used in the study, as long as the same one is used for all 
patients and for all their visits. 
Management as an AE: Any abnormal value will be considered an adverse event. In some cases, 
the research subject may need to be considered a possible case of COVID-19, and the investigator 
may need to refer the patient for medical care and withdraw the subject from the research. 
7.5 Description of the procedures or assessments during the study 
The various procedures that will be performed during the study are described below. The list may 
not be in order and could be arranged in the most optimal way according to the needs of the 
research center. 
7.5.1 Signature of informed consent 
Procedure that ensures that the research subject has voluntarily expressed his or her intention to 
participate in this research, after having understood the information given to him or her about the 
objectives of this research, benefits, discomforts, and possible risks. 
7.5.2 Taking a medical history (including ophthalmological and general medical history) 
This includes questions about medical history, symptoms, or diagnosed conditions, as well as any 
medications currently being used, regardless of their route of administration. It includes 
measurements of body weight and height (somatometry), and a complete ophthalmological 
evaluation. 
7.5.3 Eligibility criteria 
It is the evaluation that the patient meets all the inclusion criteria and none of the exclusion or 
elimination criteria. 
7.5.4 Subject code assignment 
It is the granting of a code that represents the subject in the study. This code is assigned when the 
research subject is included in the study. 


7.5.5 Adverse events 
Described in section 7.4.5.9 Adverse events. 
7.5.6 Measuring vital signs 
This involves measuring heart rate, respiratory rate, blood pressure, and body temperature. These 
measurements can be performed with a stethoscope, sphygmomanometer, and mercury or digital 
thermometer. 
Vital signs are described in sections 7.4.5.2, 7.4.5.3, 7.4.5.10 and 7.4.5.11. 
7.5.7 Urine pregnancy test 
Pregnancy testing will be performed at the baseline and final visits. The researcher must provide the 
research subject with a female, reproductive-age subject (no natural or induced menopause, 
defined as 12 consecutive months of amenorrhea)  [41]. The patient will be allowed to go to the 
bathroom and have privacy for the test. After the test is performed, the researcher must verify the 
result by observing the medical device. 
7.5.8 Ophthalmological evaluation 
Evaluation of the eyeball, eyelids, eyelashes, and other ocular structures through inspection, slit 
lamp (biomicroscopy), and palpation (touch). This evaluation includes best-corrected visual acuity, 
assessment of the integrity of the ocular surface, anterior segment, intraocular pressure, and 
gonioscopy and posterior segment (fundoscopy). 
7.5.8.1 Best-corrected visual acuity 
Described in section 7.4.5.6 Best corrected visual acuity. 
7.5.8.2 Integrity of the ocular surface 
This will be performed using biomicroscopy using the research center's slit lamp. The cornea, 
conjunctiva, and tear film will be inspected, and ocular staining will be performed with lissamine 
green and fluorescein ( the stains are described in section 7.4.5.7). 
7.5.8.3 Anterior segment 
This is the evaluation of the anterior segment structures (cornea, conjunctiva, anterior chamber, 
iris, pupil (including measurement of pupillary diameter), lens, aqueous humor). The evaluation will 
be performed using a slit lamp. 
7.5.8.4 Intraocular pressure 
Described in section 7.4.5.1 Intraocular pressure. 
7.5.8.5 Gonioscopy 
This is the assessment of the iridotrabecular angle by attaching a gonioscopy lens to the patient's 
cornea. In some cases, the lens will be filled with a gel to improve its fit with the patient's cornea. 
The Shaffer system will be used for angle classification. 
 


Table iv. Shaffer classification 
Degree 
Angular 
opening 
Description 
Risk of 
occlusion 
4 
45°-35° 
Open 
Impossible 
3 
35°-20° 
Open 
Impossible 
2 
20° 
Narrow 
Possible 
1 
≤ 10° 
Extremely 
narrow 
Likely 
0 
0° 
Closed 
Occluded 
 
Angle closure or suspected angle closure will be considered if Grade 2 or less is present in more than 
180° of the angular circumference. 
7.5.8.2 Posterior Segment (fundoscopy) 
Also called ophthalmoscopy, this is an examination performed with a light and magnifying glass to 
observe the fundus (optic nerve and retina) through the pupil. Sometimes, the pupil will need to be 
dilated to allow a better evaluation of the fundus. 
7.5.9 Applying medication during visits 
During patient visits, various medications or medical devices may be applied to the ocular surface 
for ophthalmological examination. 
Examples: 
• 
Tetracaine 0.5% ophthalmic solution, used to anesthetize the patient and facilitate 
measurement of intraocular pressure, application of dyes for staining, and placement of the 
gonioscopy lens. 
• 
Ophthalmic solution of 0.8% tropicamide and 5% phenylephrine, used to dilate the pupil 
and better assess the posterior segment. 
• 
PRO-185 ophthalmic solution, an investigational drug, applied at visits 1 and 2. 
• 
Hypromellose 2% ophthalmic solution may be used by the Researcher for gonioscopy 
assessment. 
7.5.10 Delivery of material for the subject 
This refers to the provision of the subject's ID card and the subject's diary. The ID card will serve as 
identification with the treatment assignment number; this card can also serve as an appointment 
card. The Subject's Diary is used to record the number of times the treatment is administered. 


7.5.11 Delivery of the study medication 
Once the patient is in the study, the investigational product will be given to them, and the dosage 
will be explained (one drop four times a day in both eyes).
7.5.12 Evaluation of concomitant medications 
This question asks about any medications you are currently taking regularly or have used in the past 
month. If you have required medication therapy injected into the eye, you should be asked about 
any medications you have injected in the past 6 months. 
7.5.13 Assessment of treatment adherence 
This refers to indirectly assessing the number of applications during the period between visits. To 
assess the approximate number of drops, the medication dropper bottle (concomitant or 
investigational treatment) can be weighed. The Subject Diary is also reviewed to determine the 
recorded applications. 
7.5.14 Return of study medication 
It refers to the return of the product under investigation by the research subject. 
7.5.15 Withdrawal from the subject's journal 
It refers to the delivery of the subject's diary by the research subject.
7.6 Diagram and schedule of study activities 
7.6.1 Study diagram 
Figure 3Study diagram 


7.6.2 Study activity schedule 
Table v. Study schedule 
Procedures 
Baseline visit 
Day 1 
Visit 1 
Day 3 (+1) 
Visit 2 
Day 8 
Final visit 
Day 10 (+1) 
Pre-TP 
Post -
TP 
Pre -
Tx 
Post-
Tx 
Pre -
Tx 
Post-
Tx 
Without 
treatment 
FCI Signature 
X 
 
 
 
 
 
 
Medical record 
X 
 
 
 
 
 
 
Eligibility criteria 
X 
 
 
 
 
 
 
Subject code assignment 
X 
 
 
 
 
 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
Heart rate 
X 
X 
X 
X 
X 
X 
X 
Respiratory rate 
X 
 
X 
 
X 
 
X 
Blood pressure 
X 
X 
X 
X 
X 
X 
X 
Temperature (°C) 
X 
 
X 
 
X 
 
X 
Urine pregnancy test 
X 
 
 
 
 
 
X 
AVMC 
X 
 
X 
 
X 
 
X 
Anterior segment evaluation 
X 
X 
X 
X 
X 
X 
X 
Pupillary diameter 
measurement 
X 
X 
X 
X 
X 
X 
X 
Eye stains 
X 
X 
X 
X 
X 
X 
X 
Intraocular pressure 
X 
X 
X 
X 
X 
X 
X 
Gonioscopy 
X 
 
 
 
 
 
 
Pharmacological mydriasis 
(phenylephrine/tropicamide) 
X 
 
 
 
 
 
 
Evaluation of the posterior 
segment 
X 
 
X 
 
X 
 
X 
Application of PRO-185 
during a visit 
 
 
X 
 
X 
 
 
Delivery of material for the 
subject 
 
X 
 
 
 
 
 
Delivery of the study 
medication 
 
X 
 
 
 
 
 


Procedures 
Baseline visit 
Day 1 
Visit 1 
Day 3 (+1) 
Visit 2 
Day 8 
Final visit 
Day 10 (+1) 
Pre-TP 
Post -
TP 
Pre -
Tx 
Post-
Tx 
Pre -
Tx 
Post-
Tx 
Without 
treatment 
Evaluation of concomitant 
medications 
X 
 
X 
 
X 
 
X 
Adherence assessment 
 
 
X 
 
X 
 
 
Return of study medication 
 
 
 
 
 
X 
 
Withdrawal from the 
subject's diary 
 
 
 
 
 
X 
 
PRE-TP: prior to application of 0.8% tropicamide and 5% phenylephrine ophthalmic solution (TP 
Ofteno® , indicates that the checks will be performed prior to pharmacological mydriasis. Post-TP: 
post application of the TP Ofteno®  solution , indicates that the checks will be performed with 
pharmacological mydriasis. Pre- Tx : pretreatment, indicates the checks to be performed before 
the application of the investigational medicinal product. Post-Tx : post-treatment, indicates the 
evaluations to be performed within 20-40 minutes after the application of the investigational 
medicinal product. ICF: informed consent form. BCVA: best corrected visual acuity. 
7.7 Procedures to be performed per visit 
The procedures to be performed at each visit are listed below; these may not be in the optimal order 
for the Research Center. The researcher should organize them according to their needs, and the 
needs of the study and sponsor. 
In assessing treatment adherence, the subject's diary may be reviewed and/or the dispensed 
dropper bottles may be weighed. 
7.7.1 Baseline visit  
- 
Consent signature informed 
- 
General and ophthalmological clinical history taking 
- 
Measurement of vital signs (temperature, blood pressure, heart rate, respiratory rate) 
- 
Drug evaluation concomitants 
- 
Measurement of best-corrected visual acuity 
- 
Ophthalmological evaluation (before and after pupil dilation) 
- 
Evaluation of pupil diameter 
- 
Ocular staining with fluorescein and lissamine green 
- 
Measurement of intraocular pressure 
- 
Gonioscopy 
- 
Review of eligibility criteria 
- 
Event evaluation adverse 
- 
Pupil dilation 
- 
Delivery of the investigational product 


7.7.2 Visit 1 
- 
Pre-application procedures for the investigational product 
o Measurement of vital signs (temperature, blood pressure, heart rate, respiratory 
rate) 
o Drug evaluation concomitants 
o Measurement of best-corrected visual acuity 
o Assessment ophthalmological 
o Evaluation of pupil diameter 
o Ocular staining with fluorescein and lissamine green 
o Measurement of intraocular pressure 
o Review of continuation criteria 
o Event evaluation adverse 
 
- 
Post-application procedures for the investigational product 
o Measurement of vital signs (blood pressure and heart rate) 
o Measurement of best-corrected visual acuity 
o Assessment ophthalmological 
o Evaluation of pupil diameter 
o Ocular staining with fluorescein and lissamine green 
o Measurement of intraocular pressure 
o Review of continuation criteria 
o Event evaluation adverse 
7.7.3 Visit 2 
- 
Pre-application procedures for the investigational product 
o Measurement of vital signs (temperature, blood pressure, heart rate, respiratory 
rate) 
o Drug evaluation concomitants 
o Measurement of best-corrected visual acuity 
o Assessment ophthalmological 
o Evaluation of pupil diameter 
o Ocular staining with fluorescein and lissamine green 
o Measurement of intraocular pressure 
o Review of continuation criteria 
o Event evaluation adverse 
 
- 
Post-application procedures for the investigational product 
o Measurement of vital signs (blood pressure and heart rate) 
o Measurement of best-corrected visual acuity 
o Assessment ophthalmological 
o Evaluation of pupil diameter 
o Ocular staining with fluorescein and lissamine green 
o Measurement of intraocular pressure 
o Review of continuation criteria 
o Event evaluation adverse 
o Return of the research product 


o Return of the subject's diary 
 
7.7.4 Final visit 
- 
Measurement of vital signs (temperature, blood pressure, heart rate, respiratory rate) 
- 
Drug evaluation concomitants 
- 
Measurement of best-corrected visual acuity 
- 
Assessment ophthalmological 
- 
Evaluation of pupil diameter 
- 
Ocular staining with fluorescein and lissamine green 
- 
Measurement of intraocular pressure 
- 
Event evaluation adverse 
- 
Urine pregnancy test 
7.7.5 Unscheduled follow-up visits 
At the request of the subject or study personnel, unscheduled follow-up visits may be conducted to 
report adverse events or any other situation that warrants it. During these visits, all relevant data 
on reported adverse events must be collected, and an appropriate management plan must be 
established, if applicable. 
7.8 Data collection 
7.8.1 Source documents 
Source documents are all written or printed records derived from automated processes (e.g., 
printouts of laboratory results issued by automated analytical equipment) where information is first 
recorded and which become part of the subject's permanent medical record. Examples of source 
documents include medical records, clinical progress notes, laboratory reports, office study reports, 
nursing notes, follow-up notes, surgical records, etc. 
The PI is obligated to accept monitoring of study-related information, audits, review by ethics and 
research committees, and inspections by the health authority. This obligation implies direct access 
to source documents. 
7.8.2 Electronic forms of data collection 
All protocol-related data will be captured via an electronic case report form ( eCRF ) by research 
team staff. Protocol-related data should NOT be captured directly into the eCRF , but rather 
transcribed from the corresponding source document. This procedure allows for monitoring to 
verify the information captured in the eCRF . It is the researcher's responsibility to ensure that the 
information is transcribed into the eCRF correctly, completely, and in a timely manner. It is 
understood that all data captured and submitted via the eCRF for data analysis have been approved 
by the researcher. 
7.8.3 File 
The data collected in this database are anonymous (only the subject number is stored along with 
other relevant information). The software used for data capture and storage meets the traceability 


requirements necessary for conducting clinical studies. The collected data will be stored by the 
sponsor or designated clinical research organization for a period of 10 years. Records of subject 
number assignment will remain at the participating institutions under the care of the PI or their 
team and must be maintained for at least 5 years. 
 


8. Evaluation and management of adverse events 
8.1 Regulation and standards on adverse events 
The registration and reporting of adverse events and incidents will be carried out in accordance with 
the guidelines established in NOM-220-SSA1-2016 and the international ICH E6 guidelines. [35] [42] 
[43] [44] 
8.2 Definition of adverse event 
According to the International Conference on Harmonization (ICH), an adverse event (AE) is any 
unfavorable medical occurrence in a clinical research subject administered a pharmaceutical 
product, regardless of causal attribution.[42] [43] [44] 
Therefore, an AE may be any of the following: any unfavorable, unintended disease, symptom, or 
sign (including an abnormal laboratory finding) that is temporally related to the use of a medical 
product, whether or not considered to be related to that product; any new disease or exacerbation 
of an existing disease (worsening of the nature, frequency, or severity of a known condition); relapse 
of an intermittent medical condition (e.g., headache) not present at baseline; any deterioration in 
a laboratory value or other clinical test (e.g., electrocardiogram [ECG], x-ray) that is related to the 
symptoms or that results in a change in study or concomitant treatment or discontinuation of study 
drug.[42] [43] [44] 
As defined in the previous paragraph, an adverse event is defined as any event that occurs during 
treatment with a drug or device. However, the definition can also apply to any undesirable event 
that occurs during a clinical trial, including behavioral disturbances.[44] 
8.3 Use of adverse events as a study safety variable 
Measuring the safety of PRO-185 use is paramount to the study, so reporting any undesirable 
symptoms or illnesses that occur during the course of the study is important, regardless of whether 
the symptoms are considered related to the investigational treatment.[44] 
8.4 Definitions relevant to the classification of adverse events  
Severity (serious/non-serious), also called seriousness (serious/non-serious). A serious event is 
defined as any event that: results in death, threatens life, requires hospitalization or prolongs 
hospitalization, causes permanent or significant disability or incapacity, causes abnormalities or 
malformations in the newborn, or other medically significant conditions. 
Severity (mild, moderate, or severe). Mild conditions present with minimal symptoms and do not 
require treatment or discontinuation of the medication; moderate conditions interfere with normal 
activities without threatening the patient's life, require treatment, and may or may not require 
discontinuation of the medication; severe conditions interfere with normal activities and require 
pharmacological treatment and discontinuation of the medication.[35] [42] [43] 
Causality. The relationship assigned between the pharmaceutical product and the adverse event: 
certainly caused by the pharmaceutical product, there is clear evidence of causality, i.e. the adverse 
event recurs with the administration of the pharmaceutical product; probably caused by the 


pharmaceutical product, there is a high suspicion of causality but direct evidence is lacking or it is 
considered unnecessary or dangerous, i.e. the reaction disappears upon discontinuation of the 
pharmaceutical product; possibly caused by the pharmaceutical product, there is additional 
information suggesting that the cause may be due to another pharmaceutical product or disease; 
unlikely to be caused by the pharmaceutical product, there is a clear explanation for the origin due 
to the underlying disease or the use of another pharmaceutical product; conditional, there is a lack 
of data to establish a clear causality; unclassifiable, those for which, once all possible information 
on the adverse event has been obtained, it remains unclassifiable.[35] [42] [43] [44] 
8.5 Researcher Responsibilities  
The investigator is responsible for verifying the AE through a history, a relevant physical 
examination, assessment of progress, and appropriate medical and pharmacological management. 
The investigator is also responsible for monitoring the AE until it is resolved or resolved, and the 
patient is discharged, following the definitions established in national and international 
regulations.[35] [42] [43] 
In the event of an AE or any event that puts the health and well-being of the subjects at risk, 
appropriate medical care will be provided, either at the research center or by referring the subject 
to the highest-resolution hospital with which the research center has a medical care agreement. 
The PI will notify the sponsor's clinical monitor, in accordance with the timeframes established in 
national and international regulations. In the case of serious adverse events, the PI will notify the 
sponsor and record the corresponding information in the eCRF , and in turn, will inform the IEC and 
the IC. 
The attention of the AE will be carried out according to the event attention diagram (see Figure 4. 
Adverse event attention). 
The sponsor's final report will include adverse event reporting in compliance with current national 
and international regulations.[35] [42] 
If the research subject develops a chronic adverse event during their participation in the study, such 
as diabetes or arterial hypertension, they will be referred to a healthcare professional for chronic 
treatment. Follow-up and termination of participation will be in accordance with ICH guidelines. 
8.5.1 Recording of adverse events in the electronic case report form  
The adverse event registry considers: 
 
Subject identification information such as: subject number, age, sex, and if 
applicable, specify the eye. 
 
Information about the causality of the adverse event, its relationship to the 
investigational products, or to another study-related drug, as appropriate. 
 
Information on important dates: 
o Date on which the adverse event occurs. 
o Date on which the Principal Investigator is informed of the same. 
o Date of resolution or outcome, as applicable. 
 
Information on diagnosis and clinical management. 


 
Establish the outcome or resolution of the event: 
o 
Recovered/resolved without sequelae 
o 
Recovered/resolved with sequelae 
o 
Not recovered/Not resolved 
o 
Subject who died due to the adverse event 
o 
Subject who presented death and it is judged that the research product may 
have contributed 
o 
Subject who died and this was not related to the product under 
investigation, 
o 
A stranger 
 
Information about the investigational product or the product associated with the 
adverse event, incident, adverse event, ADR, or SRAM must be recorded. The essential 
information to be recorded is the generic name, distinctive name, or code of the 
investigational product or the product associated with the undesirable clinical 
manifestation. It will also be necessary to record data concerning the lot number, 
manufacturing laboratory, expiration date, dose, route of administration, start and end 
dates of administration and/or consumption, and reason for the prescription, depending 
on whether it is an investigational product or medication (a protocol in which the subject is 
currently participating) or a medication that the research subject is taking for the treatment 
of underlying concomitant diseases or for the management of any transient signs or 
symptoms that do not correspond to the natural history of the pathology that motivated 
their entry into the research protocol. 
 
Indicate whether the adverse event disappears upon withdrawal of the suspected 
product (which caused the event). Also indicate whether a dose adjustment is made, 
whether the event changes in intensity or severity, and whether the reaction persists. It is 
important to indicate whether the AE reappears in subjects who are re-exposed to the 
product after having been previously discontinued. 
 
Information regarding concomitant pharmacotherapy. Indicate the generic name, 
dose, route of administration, start and end dates, and the reason for the prescription, 
regardless of whether it is in accordance with the prescribing information or the data sheet 
or if it is used outside of the regulations or as authorized by the local, national, or 
international regulatory body. 
 
Information on relevant clinical history. The analysis of the AE considers the 
information previously described. However, the clinical context in which the adverse event 
occurs in the participants in the clinical research protocol is of particular interest. Therefore, 
information about previous conditions, hypersensitivity or allergy symptoms, previous 
surgical procedures, laboratory tests or imaging examinations the participant has 
undergone, etc., that the researcher deems appropriate may be mentioned. 
8.5.2 Monitoring of adverse events  
The Principal Investigator will provide care and follow-up of the adverse event presented by the 
participant until its outcome, in accordance with the provisions of the following section. 


8.5.3 Procedures for a serious adverse event  
The adverse event handling process considers the following stages: 
Figure 4Adverse event care 
During the development and conduct of this study, undesirable harmful events or adverse 
reactions/incidents with medical implications may occur in the research subject, which are not 
necessarily causally related to the investigational products. These harmful phenomena may occur 
during the use of investigational pharmaceutical products at doses authorized for human use by a 
local, national, or international regulatory body. However, it may be suspected that the 
investigational product may cause some unwanted clinical manifestation. AEs, Incidents, Adverse 
Incidents, ADRs, or SRAMs related to one or more pharmaceutical products may occur during the 
systematic evaluation of participants (on the days on which the clinical review is scheduled, 
according to the schedule of activities) or suddenly, in such a way that: 
1. The investigator should be the first person to whom the subject notifies that he or 
she has developed or experienced any clinically harmful phenomena during his or her 
participation in this study. 
2. Based on their clinical judgment, the principal investigator will determine the 
appropriate course of treatment for the adverse event/reaction based on the relevant 
Document 
Appropriate care 
and treatment 
Categorization 
Medical note preparation 
Recording and reporting in eCRF 
At the research site 
At a 1st-3rd level hospital (if 
required) 
  
By outcome: 
Serious/ Not Serious 
By severity: 
Mild, moderate, or severe 
Physical examination 
and evaluation of 
the EA 
EA not serious 
Serious EA 
Follow up the EA 
until its outcome 
END 
+ Inform the sponsor within 24 
hours of becoming aware of it 
+ Follow up on the AE until its 
resolution 
+ Inform the IEC 
Notify: 
+ Study Director 
+ Protocol Author 
+ Pharmacovigilance Unit 
END 


physical examination, history, etc., as well as the analysis of information available in the 
medical literature and the information provided in the investigator's manual, 
prescribing information, or the comparator drug's data sheet. 
3. This care may be provided at the research center or at the hospital with the highest 
capacity for treatment. Thus, if the subject is referred by the PI to a hospital, they will 
be provided care through a referral system. The referral may be through a card 
identifying the subject as a study participant and linking them to the pre-established 
agreement with the institution, or through a referral medical note issued by the 
Principal Investigator. Laboratorios Sophia, SA de CV, will pay the costs for the 
participating subject's medical care when the adverse event is associated with or 
related to the investigational product. 
4. Taking into account the clinical information collected, either during the care 
provided at the research center or provided by the treating physician(s) at the hospital, 
the IP will record the AE in his/her clinical note, stating the seriousness, intensity (mild, 
moderate, or severe), and relationship to the investigational product. 
5. The PI must migrate the relevant data to the eCRF and its respective adverse event 
section. Serious adverse events must be reported to the study's clinical monitor within 
24 hours of becoming aware of them, so that they can then inform the Clinical Team 
and the UTFLS, and subsequently notify the IEC/CI. Non-serious adverse events will be 
recorded and appropriately addressed, and the corresponding regulatory body will be 
informed about the safety profile of the PI or investigational drug in the final clinical 
trial report. 
The recording of the outcome of the AE depends substantially on the Principal Investigator's follow-
up of the subject, since most adverse events (see subsection 2.5 Risk-Benefit Assessment and the 
Investigator's Manual) are expected to be ophthalmic in nature; however, systemic alterations may 
exist. Therefore, at the investigator's discretion, the participant's withdrawal or continuation will be 
considered. 
8.5.4 Assessment of causality  
Causality assessment is the methodology used to estimate the probability of attributing an observed 
adverse event to a pharmaceutical product. It considers probabilistic categories according to the 
available evidence and the quality of the information, based on national pharmacovigilance and 
technovigilance regulations.[35] [45] 
An adverse event may or may not be related to the clinical study. A causal relationship means that 
the intervention caused (or is reasonably likely to have caused) the adverse event. This usually 
involves a relationship between the timing of the intervention and the adverse event (for example, 
the adverse event occurred shortly after the research subject received the intervention).[44] 
For all adverse events, the Principal Investigator is responsible for examining and evaluating the 
patient to determine the association of the event with the clinical study and intervention, whether 
related to experimental treatment, concomitant treatment, surgical procedure, or diagnostic 
procedures performed during the study.[44] 
Accepting that the adverse event is related to the clinical study requires a plausible mechanism of 
action—that is, a logical sequence between the event and the intervention that caused it. In some 


cases, it is helpful to know the opinions of other physicians directly or indirectly involved in the 
study, as well as whether the patient believes there is a relationship.[44] 
The Pharmacovigilance and Technovigilance Unit of Sophia Laboratories (UFTLS) may use the 
causality categories described by The Uppsala Monitoring Centre to categorize the probability of the 
adverse event to the investigational product or to concomitant or used treatments during the 
visits:[35] [45] 
• 
Definite (certain): A clinical event, including laboratory test abnormalities, that occurs with 
a plausible time sequence related to drug administration and that cannot be explained by 
concurrent disease or other drugs or substances. The response to drug withdrawal must be 
clinically plausible. The event must be definitive from a pharmacological or 
phenomenological standpoint, using, if necessary, a conclusive rechallenge procedure.[35] 
[45] 
• 
Probable: A clinical event, including laboratory test abnormalities, that occurs in a 
reasonable time sequence related to drug administration, is unlikely to be attributed to the 
concurrent disease or other drugs or substances, and to which withdrawal of the drug 
produces a clinically reasonable response. Rechallenge information is not required for this 
definition.[35] [45] 
• 
Possible: A clinical event, including laboratory test abnormalities, that manifests within a 
reasonable timeframe related to medication administration, but which can also be 
explained by concurrent illness, or by other drugs or substances. Information regarding 
medication discontinuation may be missing or unclear.[35] [45] 
• 
Unlikely: A clinical event, including laboratory test abnormalities, that occurs in a time 
sequence that is unlikely to occur in relation to the administration of the drug and that can 
be more plausibly explained by concurrent disease, or by other drugs or substances.[35] 
[45] 
• 
Conditional/Unclassified: A clinical event, including laboratory test abnormalities, reported 
as an adverse reaction, for which further data are essential for proper evaluation, or 
additional data are under review.[35] [45] 
• 
Not Evaluable/Unclassifiable: A report that suggests an adverse reaction, but which cannot 
be judged due to insufficient or contradictory information, and which cannot be verified or 
completed in its data .[35] [45] 
Thus, the degree of certainty to establish the research product as the causal agent of the harmful 
phenomenon that occurs in the subject of the clinical study can be indicated directly by the Principal 
Investigator based on his or her clinical experience or through the application of the causality 
categories described by The Uppsala Monitoring Centre . It is important that the researcher and the 
UFTLS take into account the following arguments in favor of the causal relationship: 
a) 
Strength of association, which refers to the number of cases in relation to those 
exposed. 
b) 
The consistency of the data, that is, the presence of a common characteristic or 
pattern. 
c) 
The exposure-effect pattern, which determines the relationship with the site of 
onset, time, dose and reversibility after suppression. 


d) 
Biological plausibility, which refers to the possible pharmacological or 
pathophysiological mechanisms involved in the development or presentation of the adverse 
event. 
e) 
Experimental findings, for example, the appearance of anomalous metabolites or 
high levels of drug or its biotransformation product. 
f) 
Analogy, which refers to the experience acquired with other related drugs, adverse 
reactions frequently produced by the same family of pharmacological agents. 
g) 
Nature and characteristics of the data, i.e. objectivity, accuracy and validity of the 
relevant documentation.[46] 
8.6 Unanticipated problems  
Unanticipated problems (ANP) are considered situations that pose risks to the participating 
subjects, generally any incident, experience or result that meets all of the following criteria: 
• 
Unexpected in terms of its nature, severity, or frequency in relation to: 1) study-related 
documents such as the investigator's manual, study protocol, and informed consent form; and 
2) the characteristics of the study population. 
• 
Related or possibly related to your participation in the study (possibly related means that 
there is a reasonable possibility that the incident or results were caused by study procedures). 
• 
Indication that the research places participants at greater risk of harm (including physical, 
psychological, economic, or social) than previously recognized. 
8.6.1 Reporting unanticipated problems 
The PI will be responsible for reporting PNAs to the sponsor, the IC, and the IEC. The report should 
contain the following information: 
• 
Study identification: protocol title and number, name of the PI and, where applicable, the 
center. 
• 
Detailed description of the event, incident, experience or outcome. 
• 
Explanation, justification of the reasons why the incident represents a PNA. 
• 
Description of changes to the protocol or corrective actions taken or proposed in response 
to the NAP. 
PNAs that are EAS must be reported to the IEC/ CI and the sponsor within the first 24 hours of the 
IP becoming aware of it. 
Any other PNA will be reported to the IEC/CI and the sponsor within the first 5 business days after 
the IP becomes aware of it. 
 


9. Study monitoring  
The study sponsor is responsible for monitoring the study. Monitoring activities include, but are not 
limited to: general safety monitoring, general study quality monitoring, study site monitoring, 
adverse event detection monitoring, reporting and follow-up, monitoring to resolve data entry 
discrepancies, etc. 
Responsibility for monitoring activities and ultimate responsibility for monitoring rests with the 
sponsor. 
The details of the monitoring activities are specified in a separate document from this protocol in a 
Monitoring Plan. 
9.1 Study center monitoring  
Monitoring will be conducted at the research center participating in the study. At least one initial 
visit and one closing visit must be conducted for each center, although one or more follow-up visits 
may be required between these two mandatory visits. 
The initial visit must be conducted before the first participant is enrolled at that center. During this 
visit, the monitor will verify that the materials to be used during the study have been received and 
that the personnel involved in study activities have been trained in the study. The monitor will also 
verify compliance with applicable regulatory requirements and standard operating procedures. 
At the follow-up visit(s), the monitor will review the study documents to confirm that the research 
protocol and applicable standard operating procedures are being followed, that data entry is 
complete and timely, and that adverse event reporting is being conducted appropriately. At each 
visit, the monitor will discuss the findings with the investigator and determine the appropriate 
actions to be taken. 
The closing visit will take place at the end of the study, once the last participant at the site has been 
discharged from follow-up. During this visit, the monitor will verify that the site has all necessary 
documentation for archiving, that all biological samples have been analyzed, that all PI (used and 
unused) has been returned to the sponsor, and that all unused materials have been recovered. 
Details of monitoring are set out in the relevant plan. 
9.2 Audit and quality control  
To ensure compliance with GCPs and all applicable regulatory requirements, Laboratorios Sophia, 
SA de CV may conduct quality assurance audits. Regulatory agencies may also conduct a regulatory 
inspection of this study. 
Details of the audit process are set out separately in an Audit Plan. 
9.2.1 Pre-study audit  
The research center included in the study will be subject to a feasibility visit prior to center selection, 
where it will be verified that it meets the minimum requirements indicated by the sponsor. 


9.2.2 Audit during the conduct of the study  
They may take place at any time before, during, or after the conclusion of the study. If an audit or 
inspection is conducted, the investigator and the institution must agree to allow the 
auditor/inspector direct access to all relevant documents and must allocate their time and staff time 
to the auditor/inspector to discuss the findings and any pertinent issues. If the audit has not been 
scheduled by the sponsor, the center must notify Laboratorios Sophia, SA de CV immediately. 


10. Sample size calculation and statistical analysis 
10.1 Sample size calculation 
The sample size calculation was based on the study's primary objective: to evaluate the safety and 
tolerability of PRO-185 ophthalmic solution (naphazoline hydrochloride 0.03% and hypromellose 
0.2%) in clinically healthy subjects. To meet the study's primary objective, an estimated 22 subjects 
will contribute OA to the study. 
10.1.1 Calculation methodology 
A search was performed on the open access search engines PubMed (pubmed.ncbi.nlm.nih.gov) and 
Web of Science (webofscience.com), finding until May 2021, 102 results in PubMed and 24 in Web 
of Science that matched the search keywords: “ Naphazoline Ophthalmic ”. The extraction of this 
search was generated and the integrated database was reviewed, to eliminate duplicate matches 
and filter those articles that were in accordance with the objective of the study. After reviewing the 
abstracts (available only in 89 articles), 7 articles were chosen for their complete review (full text), 
of these, 5 articles were selected to make the proposal for the sample size [47, 48, 49, 50], one of 
them is a review article [51], this was used only as a reference. See appendix 16.3. 
The mean ± standard deviation (SD) sample size used in the selected studies was 24.4 ± 21.24 
subjects (range, 6–60). Considering a 20% incidence rate for AEs, these values were entered into an 
online tool [52]to estimate sample size and power, following specific equations for the calculation 
[53]. Finally, the calculated value was increased by 20% to account for potential losses. 
10.1.2 Size calculation 
PRO-185 ophthalmic solution is expected to present in less than 20% of subjects any of the following 
AEs: increased IOP (>5 mmHg), heart rate changes (>15 bpm), increased systemic blood pressure 
(>15 mmHg in SBP or >10 mmHg in DBP), pharmacological mydriasis or grade 3 and 4 conjunctival 
hyperemia, 20 minutes after its application with respect to its initial value. 
The sample size was calculated using the equation for a non-superiority proportion [53]. This 
equation is useful for non-inferiority/superiority tests, if we want to test whether a proportion (p) 
is not superior to a reference value, p 0 (20% in this case). The idea is that statistically significant 
differences between the proportion and the reference value cannot be of interest unless the 
difference is greater than a threshold δ (10% in this case). This type of calculation is useful in clinical 
studies where the value of δ is chosen based on clinical judgment and knowledge of the subject. 
The hypothesis test is: 
𝐻0: 𝑝−𝑝0 ≤𝛿 
𝐻1: 𝑝−𝑝0 > 𝛿 


Equations 
The calculation to estimate the sample size and power was performed using an online tool and 
following the equations [53]: 
18 = 𝑝(1 −0.2) ( 1.96 + 𝑧1−𝛽
𝑝−0.2 −0.1)
2
 
 
1 −𝛽= 𝛷(𝑧−1.96) + 𝛷(−𝑧−1.96) , 𝑧= 𝑝−0.2 −0.1
√𝑝(1 −0.2)
18
 
 
Where: 
n is the sample size (18) 
p 0 is the reference value (0.20) 
𝛷 is the function of the standard normal distribution 
𝛷 -1 is the function of the standard normal distribution 
α is the Type I error (1.96) 
β is the Type II error, which means that, 1-β (1-0.8) is the power 
δ is the test margin (0.10) 
According to the previous calculation, 18 subjects are estimated, increasing the calculation by 20% 
considering possible losses, 22 subjects are estimated to contribute OA to the study. This calculation 
is in accordance with the average number of subjects used in the clinical studies reviewed in the 
search performed in PubMed and Web of Science (24 subjects), where the incidence of adverse 
events was similar to that expected in this study. 
10.2 Clinical data management 
Clinical data management (CDM ) enables the generation of high-quality, reliable, and statistically 
valuable data. CDM is the process of collecting, cleaning, and managing subject information in a 
study in compliance with regulatory standards (21 CFR Part 11, ICH, and GCP guidelines). It covers 
eCFR design , eCFR commenting , database design, data entry , validation ( Source Document 
Verification ( SDV), discrepancy handling ( queries ), medical coding ( medical coding ), extraction ( 
soft lock ) and closing the database ( hard lock ).[54] 
In accordance with roles and responsibilities, multiple users can be created, whose access types to 
the eCRF can be limited to data entry (principal investigator, PI), medical coding, database design, 
or quality control . check ) [54, 55]Discrepancy handling will be based on the flow in Figure 5. 


The CDM team will include the following roles: 
• 
Data Manager 
• 
Database Designer/Programmer 
• 
Clinical coder ( Medical coder ) 
• 
Clinical Data Coordinator 
• 
Quality control 
• 
Data entry associate ) 
Figure 5Discrepancy management (DCF, medical note; CRA, clinical monitor; SEC, self-evident) correction ) 
[54] 
10.3 Statistical methodology 
The statistical analysis plan was developed considering the evaluation criteria described in the study 
protocol. 
Statistical analysis will be performed by personnel from Laboratorios Sophia, SA de CV. Specialized 
statistical software will be used (SPSS statistical package, available version, or R software). Coding 
will be performed using consecutive numbers. Data will be collected and organized in an Excel 
spreadsheet (Microsoft® Office). The data will then be exported to the specialized statistical 
software. Variables will be categorized according to their nature (see Table 3 ). The statistical tests 
used will follow the corresponding assumptions for nonparametric statistics. 
10.3.1 Population analysis 
Statistical analysis will be presented to provide a general overview of the subjects enrolled in the 
study and an overview of the safety and tolerability of the results. Data provided by the research 
 


site will be summarized for this purpose, according to their nature. The Shapiro-Wilk test will be 
performed to test whether the quantitative data are normally distributed. 
The results of the quantitative variables will be presented as measures of central tendency: mean, 
standard deviation, maximum, and minimum. Changes in pupil diameter, BCVA, and body 
temperature will be expressed as continuous variables. Changes in IOP, HR, systemic blood pressure, 
TVL, and TF will be expressed as discrete variables. 
The results of nominal and ordinal qualitative variables will be presented as frequencies, 
proportions, and/or percentages. For these, 2 x 2 frequency tables will be constructed. All 
percentages will be presented with one decimal place. 
The level of difference considered significant will be an alpha (α) of 0.05 or less. The triangulation 
between the type of variable and the measurements is shown in Table 6 . 
Table vi. Triangulation of concepts 
Variable 
type 
Variable 
A1 
A2 
B1 
B2 
C1 
C2 
C3 
C4 
D1 
D2 
D3 
D4 
E1 
E2 
Selection 
A1 
Demographics 
DT 
 
 
 
 
 
 
 
 
 
 
 
 
 
A2 
Medical 
history/Selection 
criteria 
 
DT 
 
 
 
 
 
 
 
 
 
 
 
 
Basal 
B1 
Vital signs 
 
B 
B 
 
B 
 
B 
T 
 
 
 
TM 
 
 
B2 
Comprehensive 
ophthalmological 
evaluation 
 
DTB 
 
TB 
B 
 
 
 
 
 
 
 
 
 
Security 
C1 
PIO 
 
 
 
B 
B 
 
 
 
 
 
 
TM 
 
 
C2 
HR, SBP, DBP 
 
 
B 
 
 
B 
 
 
 
 
 
TM 
 
 
C3 
Pupillary diameter 
 
 
 
B 
 
 
B 
 
 
 
 
TM 
 
 
C4 
Conjunctival 
hyperemia 
 
 
 
T 
 
 
 
T 
 
 
 
T 
 
 
Secondary Safety Outcome 
D1 
AVMC 
 
 
 
B 
 
 
 
 
B 
 
 
TM 
 
 
D2 
Corneal and 
conjunctival stains 
 
 
 
B 
 
 
 
 
 
B 
 
TM 
 
 
D3 
Chemosis 
 
 
 
T 
 
 
 
 
 
 
T 
T 
 
 
D4 
Incidence of AE 
 
 
TM 
TM 
TM 
TM 
TM 
T 
TM 
TM 
T 
TM 
TM 
TM 
Exploratory 
E1 
FR 
 
 
B 
 
 
 
 
 
 
 
 
TM 
B 
 
E2 
Body temperature 
 
 
B 
 
 
 
 
 
 
 
 
TM 
 
B 
Abbreviations: BCVA, best-corrected visual acuity; B, bivariate analysis; D, descriptive statistics; AE, adverse event; HR, heart 
rate; RR, respiratory rate; M, multivariate analysis; DBP, diastolic blood pressure; SBP, systolic blood pressure; IOP, intraocular 
pressure; T, 2x2 contingency table. 


10.3.2 Safety and tolerability analysis 
10.3.2.1 Analysis for primary variables 
The analysis of safety and tolerability outcomes will be conducted in the safety population, defined 
as all subjects who received at least one dose of the investigational product (PRO-185), regardless 
of their adherence to the protocol (intention-to-treat, ITT, population). 
Statistical analysis for the primary quantitative variables will be estimated using the Wilcoxon 
signed-rank test for a sample for the difference in measurements before and after treatment for 
visits 1 and 2, while the baseline visit (pre-PT) will be compared against the final visit, once the values 
have been adjusted with respect to their baseline within each individual. 
For conjunctival hyperemia, the X2 binomial test or Fisher's exact test will be used for expected 
values less than 5. 
10.3.2.2 Analysis for secondary variables 
For the analysis of these variables, the same primary analysis will be performed as long as the 
necessary measurements are available. 
10.3.3 Exploratory analysis 
The analysis of exploratory variables, FR and body temperature, will be performed on those subjects 
who complete their participation without deviations from the study protocol (per-protocol 
population, PP). 
10.4 Procedure for handling missing data 
An imputation procedure for missing data is not contemplated. 
 


11. Ethical considerations 
11.1 Approval of committees 
This study will be conducted in accordance with the standards of the Declaration of Helsinki, World 
Medical Association 2013. Nuremberg Code ; Nuremberg Judgment by the International Tribunal at 
Nuremberg , 1947. Belmont Report, National Commission for the Protection of Subjects of 
Biomedical and Conduct Research, 1979. It will be conducted in accordance with the scientific and 
technical requirements necessary for the registration of medicines for human use of the 
International Conference on Harmonization ( The International Council for Harmonisation (ICH) 
Guideline for Good Clinical Practice. International Ethical Guidelines for Biomedical Research 
Involving Human Subjects of the Council for International Organizations of Medical Sciences of 
Medical Sciences , CIOMS, 2002). International Ethical Guidelines for Epidemiological Studies of the 
Council for International Organizations of Medical Sciences of Medical Sciences , CIOMS, 2008). The 
Research Ethics Committee and the Research Committee will evaluate the protocol before 
conducting the study and will issue their approval or any possible modifications for its 
implementation. These Committees must be notified of any significant changes to the protocol. In 
addition to the above, the current regulations of the regulatory authority must also be complied 
with. 
The sponsor's authorized personnel will submit the essential documentation of the research project 
for evaluation by the Research Ethics Committees, Research Committees, and when applicable, to 
the Biosafety Committee: research protocol, informed consent form, researcher's manual, subject 
material, as well as other additionally requested documents, in accordance with the local, national 
or international requirements applicable by regulatory entities. 
The study will not be initiated at the research center without the confidentiality agreements and 
financial proposals from each of the principal investigators, duly signed, and without having 
previously obtained the favorable opinion and/or approval of the corresponding Research Ethics 
Committees, Research Committees, and, where applicable, the Biosafety Committee. 
The study will not begin without meeting the relevant local, national, or international regulatory 
requirements and obtaining the appropriate health authorization. 
The study is considered to be research with greater than minimum risk, in accordance with the 
Regulations of the General Health Law on Health Research, Title Two, Chapter I, Article 17, Section 
III, published in the Official Gazette on January 6, 1987. 
11.2 Amendments to the protocol  
The amendment process will be relevant when there is a need to make any changes to a document 
that is part of the research project or protocol, due to changes in the methodological structure, 
replacement of the principal investigator, or the identification of risks to the research subjects. 


Documents that may be amended include: the protocol, informed consent letter, researcher's 
manual, subject documents, measurement scales, and activity schedule. 
Any amendment must be approved by the sponsor and/or the principal investigator. The amended 
document(s), once reviewed and approved by the Research Ethics Committee and the Research 
Committee or, when applicable, by the Biosafety Committee (entities that issued the initial 
favorable opinion for the conduct of the research), will be sent for authorization by the regulatory 
entity. 
Amendments that substantially modify the protocol or impose additional or different risks to 
research subjects must be approved by the aforementioned Committees. It is the investigator's 
responsibility to take measures in situations requiring immediate action to prevent unnecessary 
harm to study participants. 
The principal investigator is responsible for communicating to the Research Ethics Committee any 
amendments to the protocol that could affect the rights, safety, or well-being of the research 
participants. They must also report any situation or new knowledge that indicates an increased risk 
to the participants, the premature termination or suspension of the study, the reasons for this, and 
the results obtained to that point. They must also report the conclusion of the study upon 
completion of the research protocol. 
11.3 Early termination of the study  
The study may be temporarily suspended or terminated prematurely if there is sufficiently 
reasonable cause. Written notification documenting the reason for the suspension or early 
termination must be provided by the party executing the suspension. The PI must promptly inform 
the study participants, the IC, and the IRB, providing the reasons. 
Situations in which suspension or early termination of the study will be considered include, but are 
not limited to: 
1. The presence of serious adverse events in more than 10% of participants in a study 
group. 
2. The regulatory authority considers it to be a security alert. 
3. The Sponsor determines it for its convenience or eventualities such as: financial 
support, manufacturing errors, etc. 
4. The determination of unexpected risks to participants that are significant or 
unacceptable. 
5. Obtaining new relevant safety information. 
6. Insufficient adherence to protocol requirements. 
7. The data obtained are not evaluable or are not sufficiently complete. 
8. The determination that the primary objective has been achieved. 
9. The determination of futility. 


In the event of suspension, the study may be resumed once the situations that led to the suspension 
have been resolved, provided this justification is sufficient for the sponsor, IC, IEC, and regulatory 
authorities. 
11.4 Informed consent  
The FCI contains complete and understandable information about the study and the investigational 
product, in accordance with current applicable regulations and Good Clinical Practices. 
The FCI will be considered a source document and will be filed as such. The site's principal 
investigator is responsible for ensuring that all new versions of the informed consent form undergo 
the appropriate approvals (the same ones that the original informed consent form underwent) and 
that the most current approved version is presented to the study subjects. 
11.4.1 Obtaining  
Informed consent must be obtained before the subject undergoes any procedure indicated in the 
protocol. For this purpose, the informed consent form must be signed. 
Written consent documents will incorporate the elements of informed consent described in the 
Declaration of Helsinki and the ICH Guidelines for Good Clinical Practice and will be in compliance 
with all applicable laws and regulations. 
The PI, or the study staff delegated by him or her, will provide the potential participant with all 
information regarding the characteristics of the study, its potential benefits, risks, objectives, and 
procedures. 
This information will be provided in a language understandable to the subject. The subject will be 
explained that they have the right to discontinue their participation in the study at any stage, 
without affecting their relationship with the researcher and/or their future participation. Informed 
consent will be presented to the potential participant; they must have sufficient time to review each 
and every aspect mentioned above. Any questions they may have will be clarified by the person 
responsible for obtaining informed consent. 
Once the participant agrees to participate in the study, he or she must sign and date the informed 
consent letter in the presence of two witnesses, whether or not related to the study subject. These 
witnesses will participate in the informed consent process and sign, confirming that the process was 
carried out prior to any study procedure, that the study information was clearly explained, and that 
any questions were clarified. 
In the event that a subject is illiterate, acceptance will be with his or her fingerprint, and in the event 
that the subject is not capable of providing adequate written informed consent, a “legally 
authorized” representative of the subject may provide such consent for the subject in accordance 
with applicable laws and regulations. 
Likewise, the PI, or the study staff delegated by him, must sign and date this consent. 


The FCI must be signed in duplicate by all involved; one copy will be filed in the researcher's folder 
and the other will be given to the participant. The PI or designated staff member must document 
the process of obtaining Informed Consent through a detailed, accurate, and contemporaneous 
medical note, specifying the signed version, the date the document was signed, and how the process 
was carried out. 
11.4.2 Special considerations  
The procedures that will be performed during the conduct of the study do not pose any additional 
risk that should be considered apart from the procedures listed in the informed consent. 
11.4.3 Modifications to informed consent  
Any changes to the FCI constitute an amendment to this document and must be submitted for 
approval to the regulatory committees and authorities. 
Such amendments may be implemented only after obtaining written approval from the Research 
Ethics Committee and the Regulatory Body (as applicable), except for an amendment that is 
required to eliminate an immediate danger to the study subjects. 
re-consent process must be conducted for each subject affected by the amendment under the same 
conditions as those described above, in order to promptly communicate the new information 
contained in the document. The subject will be given a signed original of the amendment, and the 
researcher will retain the second original. 
11.5 Confidentiality  
All documents and information provided to the research center by the sponsor are strictly 
confidential. The PI expressly agrees that the data regarding his or her professional and clinical 
experience, provided to the sponsor in paper form and stored electronically, are solely for use in 
connection with his or her activities with the clinical study sponsor, in accordance with Good Clinical 
Practice. 
The PI agrees that he and his team members will use the information only within the scope of this 
study, to carry out the protocol. This agreement is binding as long as the confidential information 
has not been publicly disclosed by the sponsor. 
The clinical study protocol provided to the PI may be used by the PI and his or her team to obtain 
informed consent from the subjects for the study. The clinical study protocol, as well as any 
information derived from it, must not be disclosed to other parties without the sponsor's written 
authorization. 
The PI will not disclose any information without the prior written consent of Laboratorios Sophia, 
SA de CV, except to representatives of the Competent Authorities, and only at their request. In the 
latter case, the researcher is obligated to inform Laboratorios Sophia, SA de CV before disclosing 
the information to these authorities. 


The PI will complete and maintain a subject selection log, as well as the identification and 
enrollment list of each subject participating in the study. The researcher agrees to grant on-site 
access to the auditor and/or representatives of the Competent Authorities. The information will be 
treated in compliance with professional secrecy. 
In the eCRF and all communications related to study subjects, they will be identified only by their 
study subject identification number, either the screening number or the allocation number. The 
information collected in this study will be exchanged between the sponsor and the research site 
and must be treated confidentially. The Health Authority, the IRB, the IC, the sponsor, the 
monitors/auditors, and third-party auditors will be the only bodies authorized to review the study 
documentation. If publications arise from this research project, under no circumstances will they 
contain information about the identification of the study subjects. If the study results are published, 
no personal information about the study subjects will be revealed. 
The protection of personal data will be in accordance with the corresponding current regulations. 
11.6 Conflict of interest  
The independence of the study's conduct and results from any actual or perceived external 
influences is critical. Therefore, any current conflict of interest of any person playing a role in the 
design, conduct, analysis, publication, or any other aspect of this study will be declared. 
Furthermore, those with a perceived conflict of interest will be asked to manage it in a manner 
appropriate to their participation in the study. 
11.7 Access to information  
The final study database will be the property of Laboratorios Sophia, SA de CV, and access to it will 
be restricted. The PI will not have access to it except with prior written authorization from the 
sponsor. 
Any information obtained that is relevant to the safety of the subjects participating in the study 
must be immediately shared with the research center, so that the study subjects can be notified. 
11.8 Auxiliary and post-study care  
Once the study is completed and adverse events are closed in accordance with section 8, the 
sponsor will not extend care to the research subject. 
 


12. Biosecurity aspects 
NO BIOSECURITY IMPLICATIONS 
This protocol, entitled: “Phase I clinical study to evaluate the safety and tolerability of PRO-185 
ophthalmic solution applied in clinically healthy subjects,” and number: SOPH185-0521/I, HAS NO 
BIOSAFETY IMPLICATIONS, since no infectious biological material will be used; pathogenic strains of 
bacteria or parasites; viruses of any type; radioactive material of any type; genetically modified 
animals and/or cells and/or plants; toxic, hazardous, or explosive substances; or any other material 
that puts the health or physical integrity of the research center staff or research subjects at risk, or 
affects the environment. It is also declared that no cell, tissue, or organ transplant procedures, or 
cell therapy, will be carried out in this project, nor will laboratory, farm, or wildlife animals be used. 


13. Publication Policy 
13.1 Final report  
Once the statistical analysis is completed, the final report will be written with the results obtained, 
by the Medical Management Department Team of Laboratorios Sophia, SA de CV. This report will 
be prepared following the recommendations of the ICH E3 Step 4 Guide. 
13.2 Communication of results  
Regardless of the results of the study, Laboratorios Sophia, SA de CV, is committed to 
communicating the final study report to the principal investigators and COFEPRIS. These results will 
also be shared with the research committee and the IEC. The PI will be responsible for 
communicating the results to the research subjects. 
Laboratorios Sophia, SA de CV will retain at all times the rights to the publication and dissemination 
of the information contained herein. 
13.3 Publication of results  
Laboratorios Sophia, SA de CV, acting as the sponsor of the study, assumes full responsibility for its 
role and retains exclusive ownership rights to the study results, which it may use as it sees fit. 
The PI agrees not to publish or communicate data collected from the study, unless prior written 
agreement is obtained from Laboratorios Sophia, SA de CV. Any manuscript derived from the data 
obtained with this protocol must be reviewed by the sponsor before any attempt to submit it for 
publication in any journal or scientific conference. 
However, if the sponsor is in the process of filing a patent application on the results of the study, 
the sponsor may delay publication or communication of the results of the study until the date of 
registration or when it deems appropriate. 
The assignment of authors for publications derived from this research will be the sole responsibility 
of the sponsor. Researchers invited to participate as authors must express their authorization. All 
researchers who will participate as authors may review the article prior to submission and are free 
to make comments and suggestions within the first 15 calendar days of receipt. 
 
 


14. Financing and insurance 
14.1 Compensation to study participants  
Subjects participating in the study may receive financial compensation for their participation. 
Compensation may vary depending on the activities required during each visit. 
14.2 Insurance for study participants  
Subjects participating in the study will sign the informed consent form, which specifies that 
Laboratorios Sophia, SA de CV agrees to pay for immediate treatment resulting from injuries or 
illnesses caused by the investigational products until resolved, in accordance with medical 
judgment. 
 
 


15. Bibliography 
 












 
 


16. Annexes 
16.1 Efron scale for conjunctival hyperemia 
16.2 SICCA Ocular Staining Grading (modified from Whitcher et al, 2010) 
Lissamine green 
(conjunctiva only)
Fluorescein 
(cornea only) 
Grade      Score 
Grade      Score 
0-9 
1 
10-32 
2 
33-100 
3 
>100 
0 
0 
1 
1-5 
2 
6-30 
3 
>30 
Score by staining pattern 
Additional points for fluorescein 
staining only: 
 
+1 if there are patches of confluent 
staining 
 
+1 if there is staining in the pupillary 
area 
 
+1 if there are filaments 


16.3 Review of articles for sample size calculation 
Reference 
Summary 
Subjects 
Treatment 
Hurwitz P, Thompson JM. Uses of naphazoline ( 
privine ®) in ophthalmology. Arch Ophthal . 1950; 
43(4): 
712-7. 
doi 
:10.1001/archopht.1950.00910010723008. 
Naphazoline hydrochloride has been widely used 
as a nasal decongestant. Considering its potential 
uses as an ocular decongestant, it was decided to 
evaluate its properties in certain clinical 
conditions. A series of experiments were 
conducted to evaluate the effects of naphazoline 
on intraocular pressure, pupillary size, and the 
accommodation and condition of ocular blood 
vessels in 60 patients (both eyes). The 0.1% 
naphazoline solution was well tolerated by the 
eye. A mild to moderate burning sensation was 
experienced 
after 
instillation. 
Occasionally, 
itching occurred, and some patients with lightly 
pigmented irides reported transient blurred 
vision. 
60 
Naphazoline 
0.1% 
Abelson MB, Allansmith MR, Friedlaender MH. 
Effects of topically applied ocular decongestant 
and antihistamine. Am J Ophthalmol . 1980; 90(2): 
254-7. 
In two independent studies involving 25 subjects 
each, 0.05% naphazoline produced significant 
whitening of histamine-induced red eyes and 
ocular itching (but did not prevent itching). 
Antazoline significantly inhibited itching (but not 
red eyes). The combination of naphazoline and 
antazoline produced significant whitening and 
inhibition of itching in all histamine-challenged 
eyes. The combination of both drugs was more 
effective than either component individually in 
preventing 
redness. 
The 
antihistamine/vasoconstrictor combination was 
equally effective for itching. 
25 
Naphazoline 
0.05% 
Abelson MB, Butrus SI, Weston JH, Rosner B. 
Tolerance and absence of rebound vasodilation 
following topical ocular decongestant usage. 
Ophthalmology . 1984; 91(11): 1364-7. doi : 
10.1016/s0161-6420(84)34140-9. 
Two 
commercial 
preparations 
of 
topical 
ophthalmic vasoconstrictors were evaluated for 
their bleaching ability, duration of action, 
tolerability, and rebound vasodilation in 11 
healthy 
volunteers 
. 
Both 
treatments, 
naphazoline 
hydrochloride 
0.02% 
and 
tetrahydrozoline 
hydrochloride 
0.05%, 
significantly decreased initial redness after a 
single application (Part I); however, naphazoline 
produced significantly more bleaching than 
tetrahydrozoline. Only naphazoline maintained 
bleaching after 10 days (Part II). The level of 
11 
Naphazoline 
0.02% 


Reference 
Summary 
Subjects 
Treatment 
redness remained significant from baseline for 8 
hours after a single instillation for both 
vasoconstrictors and for 6 hours after multiple 
administrations of naphazoline. 
The decrease in tetrahydrozoline's effectiveness 
after the 10-day study period may encourage its 
overuse. No vasoconstrictor produced rebound 
vasodilation after discontinuation. 
Nayak BK, Kishore K, Gupta SK. Evaluation of 
oxymetazoline and naphazoline in benign red eyes: 
a double blind comparative clinical trial. Indian J 
Ophthalmol . 1987; 35(4): 190-3. 
The efficacy of oxymetazoline and naphazoline 
(0.01%) was evaluated in 20 patients with red 
eye. The decongestive effect of oxymetazoline 
0.01% on days 3 and 7 was 66.66% and 80.55%, 
compared with 22.22% and 50% for naphazoline 
0.01%. Oxymetazoline showed a significant 
improvement in conjunctival symptoms such as 
itching, foreign body sensation, tearing, and 
burning 
with 
naphazoline 
compared 
with 
oxymetazoline. No ocular toxicity or AEs were 
observed during the study. 
20 
Naphazoline 
0.01% 
Hosten LO, Snyder C. Over-the-Counter Ocular 
Decongestants in the United States - Mechanisms 
of Action and Clinical Utility for Management of 
Ocular Redness. Clinical Optometry , 2020; 12: 95 
– 105. 
This review provides a current, clinically relevant 
summary of the mechanism of action, efficacy, 
and safety of available OCT decongestants for the 
reduction of ocular redness caused by minor 
irritations. Currently, the OCT products marketed 
in the US include tetrahydrozoline 0.05%, 
naphazoline 0.012% to 0.03% , and brimonidine 
0.025%. All 3 agents are adrenergic receptor 
agonists but vary in their receptor-binding 
properties: tetrahydrozoline is a selective α1-
adrenergic receptor agonist, naphazoline is a 
combination α1/α2 receptor agonist, and 
brimonidine is a selective α2 receptor agonist. 
These 
OCT 
decongestants 
produce 
vasoconstriction of conjunctival blood vessels, 
resulting in a rapid reduction of ocular redness. In 
general, reported ocular AEs are minimal, mild, 
and transient, with no significant systemic AEs. 
However , ocular decongestants with α1-
adrenergic receptor agonist activity have been 
associated with loss of effectiveness after 
continued use (tachyphylaxis) and rebound 
redness upon discontinuation. In clinical studies, 
146 
Naphazoline 
0.012 
to 
0.03% 


Reference 
Summary 
Subjects 
Treatment 
brimonidine has not been associated with 
tachyphylaxis, and rebound redness has been 
rarely reported. 
 

SAP_001.pdf: 
Statistical  Analysis  Plan  (PAE)
Version  1.0
Phase  I  clinical  study  to  evaluate  the  safety  and  tolerability  
of  PRO-185  ophthalmic  solution
applied  in  clinically  healthy  subjects
Protocol  Code:  SOPH185-0521/I
Version Date: May 1, 2021
Sponsor: Laboratorios Sophia, S.A. de C.V.
This  document  was  prepared  in  accordance  with  FDA,  ICH,  and  GCP  guidelines.


Creation  date:  May  1 ,  2021 
Version:  1.0
Version  date:  May  1 ,  2021
Content
3.3.1  Primary  outcome  variables .........................................................................................................................................5
3.  Study  Design........................................................................................................................................................4
3.1  Objectives .......................................................................................................................................................................4
3.6  Subject  Code  Assignment.......................................................................................................................................8
7.  Change  control ....................................................................................................................................................15
4.  Sample  Size  Calculation.......................................................................................................................................8
3.2  Study  Hypothesis ...................................................................................................................................................5
Clinical  Data  Management................................................................................................................................................10
Introduction.................................................................................................................................................................4
3.4  Subject  duration  in  the  study/treatment..........................................................................................................6
6.3  Exploratory  Analysis.................................................................................................................................................15
Bibliography ...................................................................................................................................................................15
3.5  Randomization ................................................................................................................................................................8
2.
6.4  Other  analyses.......................................................................................................................................................15
Annexes..............................................................................................................................................................................17
1.
3.3.2  Secondary  outcome  variables.......................................................................................................................5
Abbreviations................................................................................................................................................................3
3.3.3  Exploratory  Variables .......................................................................................................................................5
6.1  Population  Analysis................................................................................................................................................13
6.2  Security  Analysis.................................................................................................................................................14
8.
3.3  Study  Variables ....................................................................................................................................................5
5.
9.
6.  Statistical  Methodology................................................................................................................................................11
Machine Translated by Google


PP
Both  eyes
AVMC
ICH
rpm
X
Respiratory  rate
International  Council  for  Harmonization  of
Diastolic  blood  pressure
Intraocular  pressure
Lissamine  green  staining
Investigational  Product
Shapiro-Wilk
TVL
Heart  rate
FCI
bpm
eCRF
KS
PAS
Adverse  event
Source  document  verification
SW
PRO-185  Naphazoline  Hydrochloride  0.03%
SDV
Best  corrected  visual  acuity
OF
Intention-to-treat  population
PAE
Technical  Requirements  of  Pharmaceuticals  for  Human  Use)
PAD
Population  by  protocol
AO
Chi  square
Fluorescein  staining
Informed  Consent  Form
FR
Beats  per  minute
PIO
Kolmogorov-Smirnov
PI
Self-evident  correction
Electronic  Case  Report  Form
FC
TF
SEC
Standard  deviation
EA
ITT
Systolic  blood  pressure
Breaths  per  minute
Statistical  analysis  plan
1.  Abbreviations
2
Machine Translated by Google


To  evaluate  the  safety  and  tolerability  of  PRO-185  ophthalmic  solution  in  ophthalmologically  healthy  
subjects.
•  To  evaluate  the  safety  of  PRO-185  ophthalmic  solution  by  the  incidence  of  subjects  who  present
an  increase  >5  mmHg  in  intraocular  pressure  (IOP)  after  20  minutes  of  its  application  compared  
to  the  initial  value.
3.1.1  Main  objective
•  To  evaluate  the  safety  of  PRO-185  ophthalmic  solution  by  the  incidence  of  subjects  presenting  a
heart  rate  change  >15  beats  per  minute  (bpm)  20  minutes  after  application.
This  is  a  Phase  I,  controlled,  non-comparative,  open-label,  single-center  clinical  study,  see  Figure  1.
3.1.2  Specific  objectives
0521/I  (Phase  I  clinical  study  to  evaluate  the  safety  and  tolerability  of  PRO-185  ophthalmic  solution  
applied  to  clinically  healthy  subjects).  This  PAE  is  intended  to  complement  the  study  protocol.  Any  
deviation  from  this  document  will  be  described  in  the  Final  Clinical  Study  Report  [1].
This  document  describes  the  statistical  analysis  plan  (SAP)  designed  for  the  SOPH185  protocol.
•  To  evaluate  the  safety  of  PRO-185  ophthalmic  solution  by  the  incidence  of  subjects  presenting
an  increase  in  systolic  blood  pressure  (SBP)  >15  mmHg  or  >10  mmHg  in  diastolic  blood  
pressure  (DBP)  after  20  minutes  of  its  application.
•  Evaluate  tolerability  by  the  incidence  of  pharmacological  mydriasis.
•  Evaluate  tolerability  by  the  incidence  of  expected  and  unexpected  adverse  events  (AEs).
expected.
•  Evaluate  tolerability  by  the  incidence  of  grade  3  and  4  conjunctival  hyperemia.
3.  Study  Design
2.  Introduction
3.1  Objectives
Machine Translated by Google


H0=  p  –  p0  ÿÿ
3.3.1  Primary  outcome  variables
•  Intraocular  pressure
•  Pupillary  size
•  Corneal  and  conjunctival  staining  changes  with  fluorescein  (TF)
•  Incidence  of  chemosis
3.2.1  Null  hypothesis  (H0)
•  Heart  rate
H1=  p  –  p0  >ÿ
•  Best  corrected  visual  acuity  (BCVA)
•  Incidence  of  adverse  events
•  Corneal  and  conjunctival  staining  changes  with  lissamine  green  (LG)
3.2.2  Alternative  hypothesis  (H1)
•  Conjunctival  hyperemia
PRO-185  ophthalmic  solution  is  safe  and  tolerable  for  ophthalmic  use,  as  it  did  not  present  any  of  the  following  AEs  in  
more  than  10%  of  subjects:  increased  IOP  (>5  mmHg),  changes  in  HR  (>15  bpm),  increased  systemic  blood  pressure  
(>15  mmHg  in  SBP  or  >10  mmHg  in  DBP),  pharmacological  mydriasis,  or  grade  3  and  4  conjunctival  hyperemia.
3.3.2  Secondary  outcome  variables
•  Respiratory  rate
•  Body  temperature
•  Systemic  blood  pressure
PRO-185  ophthalmic  solution  is  not  safe  and  tolerable  for  ophthalmic  use  because  more  than  10%  of  subjects  experienced  
any  of  the  following  AEs:  increased  IOP  (>5  mmHg),  changes  in  HR  (>15  bpm),  increased  systemic  blood  pressure  (>15  
mmHg  SBP  or  >10  mmHg  DBP),  pharmacological  mydriasis,  or  grade  3  and  4  conjunctival  hyperemia.
•  Incidence  of  unexpected  adverse  events
3.3.3  Exploratory  Variables
3.2  Study  Hypothesis
3.3  Study  Variables
Machine Translated by Google


3.4  Subject's  duration  in  the  study/treatment
Figure  1.  Study  diagram
Up  to  11  days,  1  drop  4  times  a  day  in  both  eyes  (OE)  for  8  days,  see  Table  1.
Machine Translated by Google
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Q 
8 
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~-
o. 
;;; 
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;;; 
o, 
Obtención del consentimiento informado. Cumpl1m1entode todos los criterios de 
inclusión y ninguno de exclusión. 
Evaluac16nclínica pre aplicación del prodi.x:tode investigación: AVMC, diámetro 
pupilar, tmciones oculares, PIO, segmento anterior y posterior, presión arterial 
sistémica, frecuencia cardiaca. 
1 
!;lig~ 
n. ¡ 
:i: 
~-
o 
s·¡ii:~o-03 
i::g:~¡!~ 
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Evaluac1ónclfnica: AVMC, diámetro pupilar, t inc1onesoculares, P!O, segmento 
anterior, presión artenal sistémica, frecuencia cardíaca. 
Evaluación clínica pre aplicación del producto de 1nvest igac1ón: AVMC, diámetro 
pupilar, t1nc1ones oculares, PIO, segmento anterior y posterior, presión artenal 
sistémica, frecuencia cardíaca. 
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Evaluación clínica: AVMC, diámetro pupilar, tmcionesoculares, PIO, segmento 
antenor, presión arterial sistémica, frecuencia cardíaca. 
Evaluación clínica pre aplicadóndel producto de investigación: AVMC, d iámetro 
pupilar, ti ne iones oculares, PIO, segmento anterior y posterior, presión arterial 
sistémica, frecuencia cardíaca. 
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~ ~ ll 
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~ • 
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:! ! 
o 
:) 
~ 
e g, i:- i ;. 
Evaluaciónclfnica: AVMC, diámetro pupilar, t1nciones oculares, PIO, segmento 
anterior, presión arterial s1stém1ca, frecuencia cardíaca. 
Evaluación clínica final: AVMC, diámetro pupilar, t1nciones oculares, PIO, segmento 
anterior y posterior, presión arterial sistémica, frecuencia cardíaca. 
"D 
::o 
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o.: 
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Statistical Analysis Plan


Procedures
Table  1.  Study  schedule
X
Pre-Tx
respiratory
X
Evaluation  of
X
X
X
X
Medical  record
X
X
X
Post-Tx
X
posterior  segment
study
X
study
X
X
X
X
X
X
X
X
Pre-Tx
Evaluation  of  the
Evaluation  of  the
medicine  of  the
Day  1
Frequency
X
X
X
X
X
X
FCI  Signature
X
Day  3  (+1)
AVMC
Intraocular  pressure
Return  of  the
Baseline  visit
Heart  rate
X
X
X
Application  of  the  
medicine  in
X
X
X
X
X
in  urine
Eye  stains
adherence
Without  treatment
X
X
X
X
X
X
X
Pre-Tx:  pretreatment,  indicates  the  assessments  to  be  performed  before  administration  of  the  investigational  drug.  Post-Tx:  post-treatment,  indicates  
the  evaluations  to  be  performed  within  20–40  minutes  after  administration  of  the  investigational  drug.  ICF:  informed  consent  form.  BCVA:  best-corrected  
visual  acuity.
Post-Tx
X
X
X
Visit  1
Evaluation  of  the
Day  10  (+1)
X
Pupillary  diameter  
measurement
Delivery  of  
the  medication
X
X
X
X
X
Pre-Tx
X
X
X
X
Pregnancy  test
X
X
anterior  segment
Delivery  of  material  for  
the  subject
X
X
X
Adverse  events
X
Day  8
X
X
subject
X
Temperature  (°C)
X
concomitants
Final  visit
visit
X
X
X
Subject  code  assignment
X
X
X
X
Withdrawal  from  the  diary  of
Post-Tx
Blood  pressure
X
medications
X
X
X
X
X
Eligibility  
criteria
X
X
X
Visit  2
Machine Translated by Google


3.6  Subject  code  assignment
3.5  Randomization
4.1  Calculation  methodology
4.  Sample  Size  Calculation
B.  Juan  De  la  Torre  Orozco
Example:
TO.
•  two  digits  corresponding  to  the  research  center  number.
•  three  digits  of  the  consecutive  number  assigned  to  its  inclusion  in  the  research  center.
Arieh  Daniel  Mercado  Carrizalez
Example:
After  verifying  the  inclusion/exclusion  criteria,  eligible  subjects  will  be  identified  by  a  number  and  their  
initials.
Once  the  subject  has  been  selected,  they  will  be  assigned  a  number  that  will  identify  them  throughout  the  
study.  This  code  will  consist  of  eight  numbers  in  the  following  order  from  left  to  right:
A  search  was  carried  out  in  the  open  access  search  engines  PubMed  (pubmed.ncbi.nlm.nih.gov)  and  
Web  of  Science  (webofscience.com),  finding  until  May  2021,  102  results  in  PubMed  and  24  in  Web  of  
Science  that  matched  the  keywords  of
•  three  digits  of  the  molecule  under  study  according  to  the  sponsor's  name.
The  initials  of  the  subject  of  study  will  be  obtained  starting  with  the  first  letter  of  the  name,  followed  by  the  
first  letter  of  the  first  surname  and  the  first  letter  of  the  second  surname,  obtaining  a  maximum  of  three  
letters.  In  case  the  person  has  two  names  or  a  compound  surname,  the  first  letter  will  always  be  used.
As  this  is  an  open,  non-comparative  study,  randomization  of  subjects  entering  the  study  is  not  necessary.
b.  Initials:  JDO
to.
The  sample  size  calculation  was  based  on  the  study's  primary  objective:  to  evaluate  the  safety  and  
tolerability  of  PRO-185  ophthalmic  solution  (naphazoline  hydrochloride  0.03%)  in  ophthalmologically  
healthy  subjects.  To  meet  the  study's  primary  objective,  22  subjects  are  estimated  to  contribute  OA  to  the  
study.
Initials:  AMC
185-01-001
Protocol  SOPH185-0521/I
Sophia  Laboratories,  SA  de  CV
Version  0.1
8
Machine Translated by Google


4.2  Proposal
Search:  “Naphazoline  Ophthalmic”.  The  extract  was  generated  from  this  search,  and  the  integrated  
database  was  reviewed  to  eliminate  duplicate  matches  and  filter  out  those  articles  that  were  
consistent  with  the  study  objective.  After  reviewing  the  abstracts  (available  only  in  89  articles),  7  
articles  were  selected  for  full-text  review.  Of  these,  5  articles  were  selected  to  make  the  sample  
size  proposal.
PRO-185  ophthalmic  solution  is  expected  to  present  in  less  than  10%  of  subjects  any  of  the  following  AEs:  increased  IOP  
(>5  mmHg),  heart  rate  changes  (>15  bpm),  increased  systemic  blood  pressure  (>15  mmHg  in  SBP  or  >10  mmHg  in  DBP),  
pharmacological  mydriasis  or  grade  3  and  4  conjunctival  hyperemia,  20  minutes  after  its  application  with  respect  to  its  initial  
value.
The  sample  size  was  calculated  using  the  equation  for  a  non-superiority  proportion  [8].
Equations
This  equation  is  useful  for  non-inferiority/superiority  testing,  if  we  want  to  test  whether  a  proportion  
(p)  is  not  superior  to  a  reference  value,  p0.  The  idea  is  that  statistically  significant  differences
between  the  proportion  and  the  reference  value  cannot  be  of  interest  unless  the  difference  is
greater  than  a  threshold  ÿ  (10%  in  this  case).  This  type  of  calculation  is  useful  in  clinical  studies
where  the  value  of  ÿ  is  chosen  based  on  clinical  judgment  and  subject  matter  expertise  (see
Appendix  9.1).
The  calculation  to  estimate  the  sample  size  and  power  was  performed  using  an  online  tool  and  
following  the  equations  [8]:
Finally,  the  calculated  value  was  increased  by  20%  to  account  for  possible  losses.
[2,  3,  4,  5],  one  of  them  is  a  review  article  [6],  this  was  used  only  as  a  reference  (see  Annex  9.1).
The  mean  ±  standard  deviation  (SD)  sample  size  used  in  the  selected  studies  was  24.4  ±  21.24  
subjects  (range  6–60).  Considering  a  10%  proportion  in  the  incidence  of  AEs,  these  values  were  
entered  into  an  online  tool  [7]  to  estimate  the  sample  size  and  power,  following  the  specific  
equations  for  the  calculation  [8].
The  hypothesis  test  is:
Protocol  SOPH185-0521/I
Sophia  Laboratories,  SA  de  CV
Version  0.1
9
Machine Translated by Google


-1
5.  Clinical  Data  Management
Where:
is  the  function  of  the  standard  normal  distribution,
ÿ  is  the  Type  I  error,
According  to  the  previous  calculation,  18  subjects  are  estimated  per  treatment  arm.  Considering  potential  
losses,  the  estimate  increases  by  20%.  This  estimate  is  consistent  with  the  average  number  of  subjects  
used  in  the  clinical  studies  reviewed  in  the  PubMed  and  Web  of  Science  searches  (24  subjects).
The  CDM  team  will  include  the  following  roles:
•  Data  Manager,
ÿ  is  the  Type  II  error,  which  means  that,  1-ÿ  is  the  power,  and
•  Database  designer/programmer,
is  the  function  of  the  standard  normal  distribution,
In  accordance  with  roles  and  responsibilities,  multiple  users  can  be  created,  whose  access  types  to  the  
eCRF  can  be  limited  to  data  entry  (principal  investigator,  PI),  medical  coding,  database  design,  or  quality  
control  (QC)  [9,  10].  Discrepancy  handling
will  be  performed  based  on  the  flow  in  Figure  2.
n  is  the  sample  size,
•  Quality  control,  and
p0  is  the  reference  value,
Clinical  data  management  (CDM)  enables  the  generation  of  high-quality,  reliable,  and  statistically  
valuable  data.  CDM  is  the  process  of  collecting,  cleaning,  and  managing  subject  information  in  a  study  in  
compliance  with  regulatory  standards  (21  CFR  Part  11,  ICH,  and  GCP  guidelines).  It  encompasses  eCRF  
design,  eCRF  commenting,  database  design,  data  entry ,  source  document  verification  ( SDV),  
discrepancy  management  ( queries),  medical  coding,  soft  lock,  and  hard  lock  [ 9].
•  Capturist  (data  entry  associate).
ÿ  is  the  test  margin.
•  Clinical  coder  (Medical  coder),
•  Clinical  data  coordinator,
Protocol  SOPH185-0521/I
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Version  0.1
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Machine Translated by Google


The  PAE  was  developed  considering  the  evaluation  criteria  described  in  the  study  protocol.
Figure  2.  Discrepancy  management  (DCF,  medical  note;  CRA,  clinical  monitor;  SEC,  self-evident  
correction)  [9]
Statistical  analysis  will  be  performed  by  personnel  from  Laboratorios  Sophia,  SA  de  CV.  The  SPSS  
statistical  package  version  19.0  for  Windows  (IBM  Corporation,  Armonk,  NY,  USA)  will  be  used.  
Coding  will  be  performed  using  consecutive  numbers.  Data  will  be  collected  and  organized  in  an  Excel  
spreadsheet  (Microsoft®  Office).  The  data  will  then  be  exported  to  the  SPSS  package  platform.  
Variables  will  be  categorized  according  to  their  nature  (see  Table  2).  The  statistical  tests  used  will  
follow  the  corresponding  assumptions  for  nonparametric  statistics.
6.  Statistical  Methodology
Table  2.  Operational  Definition  of  the  Variables
statistics
Variable
Proof
Operational  Definition
Type  of  
measurement
reference
Value  of
Discrepancy?
11
Conceptual  Definition
Data
AVE
IP
manager
Resolution
Database
YEAH
DCF
NO
Data  validation
SDV
SEC
Smirnov  or  U
Mann's
Whitney*
Kolmogorov-
Version  0.1
Kolmogorov-
Protocol  SOPH185-0521/I
Sophia  Laboratories,  SA  de  CV
Smirnov  or  U
discreet
The  pulse  is  a  measurement  of  the
10  –  21
FC,  that  is,  the  amount  of
Intraocular
Quantitative
For  each  heartbeat,  the  left  
ventricle  contracts  and  expels  
blood  into  the  heart.
Heart  rate
60  –  100  bpm
By  means  of  tonometry
Goldmann  based  on  the  Imberk-
Fick  principle.
Pressure
Discrete  
quantitative
mmHg
Tonometry  is  the  objective  
measurement  of  IOP,  based  on  the  
force  required  to  flatten  the  cornea  or  
the  degree  of  corneal  
indentation  produced  by  a  fixed  force.
Machine Translated by Google


discreet
Direct  observation  with
slit  lamp,  it  will  be  graded  
according  to  the
PAS:  120  –
Discrete  
quantitative
AVMC
It  is  defined  as  the  simplest  reaction  of  
the  conjunctiva  to  a  stimulus,  a  red  
appearance  secondary  to  the  vasodilation  
of  the  vessels  is  observed.
Conjunctival  
hyperemia
0  to  6
times  the  heart  beats  per  minute.
0  to  6
pupillary
PAD:  80  –  89
blood  pressure  monitor  
and  a  stethoscope.
Corneal  and  
conjunctival  TVL
resting  and  filling  with
Quantitative
Changes  in
SICCA  (see  Annex  9.3)  [12].
2  –  6  mm
The  natural  pupil  of  the  human  
eye  is  usually
Snellen  chart
Classification  by  Efron  scale  (see  
Annex  9.2).
Detection  of  epithelial  defects  
in  the  cornea  and  conjunctiva.
Quantitative
It  is  the  force  exerted  by  blood  against  
the  walls  of  the  arteries.  SBP  is  
the  pressure  inside  the  artery  when  
the
Pressure
heart  contracts  and  pumps  blood  
through  the  body;  while  DBP  is  the  
pressure  inside  the  artery  when  
the  heart  is  in
Measure  with  a
approximately  circular,  for  a  given  subject,  
the  pattern  of  aberrations,  diffraction,  
depth  of  field  and  retinal  illumination  
depend  on  the  pupil  diameter,  
which  in  turn  varies  depending  on  the  
ambient  lighting.
autonomous  [11].
Changes  in  the
Discrete  
quantitative
systemic  
arterial
139  mmHg
Diameter
Qualitative  
ordinal
Continuous  
quantitative
Changes  in
of  the  aorta.  This  forceful  expulsion  
of  blood  creates  a  wave  
that  is  transmitted  to  the  
periphery  of  the  body  through  the  
arteries.  Measured  with  a
mmHg
SICCA  (see  Annex  9.3)  [12].
blood.
the  conjunctiva  of  variable  intensity.
Direct  observation.
continues
Corneal  and  
conjunctival  TF
Direct  observation  with  slit  
lamp,  will  be  graded  according  to  
the
The  pupillary  diameter  results  from  
the  balance  between  the  
sphincter  muscle  of  the  pupil  
and  the  radial  fibers  of  the  iris  
that  have  only  innervation
electronic  or  manual  blood  
pressure  monitor.
Detection  of  epithelial  defects  
in  the  cornea  and  conjunctiva.
Spatial  visual  acuity  is  the  ability  to  
distinguish  separate  elements  
of  an  object  and  identify  them  as  a  
whole.  It  is  quantified  as  the  minimum  
angle  of  separation  (located  at  the  
nodal  point  of  the  eye)  between  two  
objects  that  allows  them  to  be  
perceived  as  separate  objects.
0.6  to  2.0
0=  Normal
2=  Mild
1=  Very  mild
Degrees:
4=  Severe
3=  Moderate
Reference  value
Operational  Definition
Statistical  test
Type  of  
measurement
Conceptual  Definition
12
Variable
Whitney*
Sophia  Laboratories,  SA  de  CV
Mann's
Protocol  SOPH185-0521/I
Smirnov  or  U
Whitney*
Kolmogorov-
Fisher*
Kolmogorov-
Smirnov  or  U
Kolmogorov-
Smirnov  or  U
Smirnov  or  U
Mann's
Whitney*
Pearson's  X  2
Whitney*
or  Exact  of
Kolmogorov-
Mann's
Mann's
Kolmogorov-
Version  0.1
Whitney*
Smirnov  or  
Mann  U
Machine Translated by Google


Chemosis
EA
continues
rpm
treatments
Present /
The  FR  is  the  amount  of
37.4°C
Frequency
treatment  [13].
at  rest.
discreet
Any  adverse  medical  event  that  
occurs  in  a  patient  or  clinical  research  
subject  who  has  been  
administered  a  pharmaceutical  
product  and  that  does  not  
necessarily  have  a  causal  
relationship  with  it.
clinical  parameter  that  reflects  the  
physiological  state  of  the  
organism.  It  is  the  balance  
between  heat  produced  and  heat  
lost.
breaths  a  person  takes  per  
minute
present  or  absent.
It  is  conjunctival  edema,  the  
result  of  an  inflammatory  reaction.  
It  is  classified  as
discreet
•  Causality
Incidence  of
•  Incidence
12  –  16
Incidence  of
Body  temperature
Quantitative
•  Nominal
qualitative
Nominal  
qualitative
36.7  –
Absent
The  evaluator  will  use  a  narrow  
beam  of  light  at  60°  and  measure  
whether  the  conjunctiva  
separates  by  ÿ1/3  of  the  entire  
eyelid  opening  or  if  it  extends  
beyond  the  gray  line.
•  Quantitative
respiratory
The  process  involves  
inspiration  and  expiration,  
diffusion  of  oxygen  from  the  pulmonary  
alveoli  to  the  blood  and  carbon  dioxide  
from  the  blood  to  the  alveoli,  and  the  
transport  of  oxygen  to  body  tissues  
and  organs.
•  Seriousness
(gravity)
All  AEs  will  be  reported  (according  
to  the  protocol)  and  not  
only  those  where  the  PI  
suspects  a  causal  relationship  
with  the
Quantitative
Degree  of  heat  or  cold,  
expressed  in  terms  of  a  specific  
scale,  using  a  thermometer.
6.1  Population  analysis
Statistical  test
Operational  Definition
Reference  value
Type  of  
measurement
Conceptual  Definition
13
Variable
Pearson  or
Smirnov  or  U
Mann's
Sophia  Laboratories,  SA  de  CV
Mann's
Exact  of
Whitney*
Protocol  SOPH185-0521/I
Whitney*
*When  applicable.
•  X  2  of
Kolmogorov-
,
Smirnov  or  U
Abbreviations:  BCVA,  best-corrected  visual  acuity;  AE,  adverse  event;  HR,  heart  rate;  RR,  respiratory  rate;  PI,  principal  investigator;  bpm,  beats  per  minute;  
DBP,  diastolic  blood  pressure;  SBP,  systolic  blood  pressure;  IOP,  intraocular  pressure;  rpm,  breaths  per  minute;  TF,  fluorescein  staining;  TVL,  lissamine  
green  staining;  X  2
•  Mann  University
Chi-square.
Whitney
Mann's
Fisher*
McNemar
Version  0.1
Kolmogorov-
Whitney*
The  results  of  the  quantitative  variables  will  be  presented  in  measures  of  central  tendency:  mean,  
standard  deviation,  maximum  and  minimum.  The  changes  in  pupil  diameter,  BCVA,  and  in  the
The  results  of  nominal  and  ordinal  qualitative  variables  will  be  presented  as  frequencies,  
proportions,  and/or  percentages.  For  these,  2  x  2  frequency  tables  will  be  constructed.  All  
percentages  will  be  presented  with  one  decimal  place.
study  and  an  overview  of  the  safety  and  tolerability  of  its  results.  The  data  provided  by  the  research  
site  will  be  summarized  for  this  purpose,  according  to  their  nature.  The  Shapiro-Wilk  test  will  be  
performed  to  determine  whether  the  quantitative  data  are  normally  distributed.
Statistical  analysis  will  be  presented  to  give  an  overall  summary  of  the  subjects  entered  into  the
Body  temperature  will  be  expressed  as  continuous  variables.  Changes  in  IOP,  HR,  systemic  blood  
pressure,  TVL,  and  TF  will  be  expressed  as  discrete  variables.
Machine Translated by Google


The  analysis  of  safety  and  tolerability  outcomes  will  be  conducted  in  the  safety  population,  defined  
as  all  subjects  who  received  at  least  one  dose  of  the  investigational  product  (PRO-185),  regardless  
of  their  adherence  to  the  protocol  (intention-to-treat,  ITT,  population).
The  level  of  difference  considered  significant  will  be  an  alpha  (ÿ)  of  0.05  or  less.  The  triangulation  
between  the  type  of  variable  and  the  measurements  is  shown  in  Table  3.
Statistical  analysis  for  quantitative  primary  variables  will  be  estimated  using  the  Kolmogorov-
Smirnov  statistic  for  the  difference  in  measurements,  once  the  values  have  been  adjusted  for  
baseline  within  each  individual.  Where  applicable,  the  nonparametric  Mann-Whitney  U  test  will  be  
used.
TM  TM  TM  TM  TM  T  TM  TM  T  TM  TM  TM
Secondary  Safety  Outcome
B
B2
Selection
Abbreviations:  BCVA,  best-corrected  visual  acuity;  B,  bivariate  analysis;  D,  descriptive  statistics;  AE,  adverse  event;  HR,  heart  rate;  RR,  respiratory  rate;  M,  multivariate  
analysis;  DBP,  diastolic  blood  pressure;  SBP,  systolic  blood  pressure;  IOP,  intraocular  pressure;  T,  2x2  contingency  table.
D3
B
TM
comprehensive  ophthalmological
Variable  
type
B
B
T
T
Medical  
history/Selection  criteria
C3
C2  C3  C4  D1  D2  D3  D4
B
B
Pupillary  diameter
B
B1
Exploratory
D1
PIO
Security
A1
B
D4
TM
TM
Variable
B
TM  B
T
T
DTB
A2
E1
TM
Conjunctival  
hyperemia
T
Vital  signs
B
B
E1
AVMC
B
C1
Demographics
Chemosis
Corneal  and  conjunctival  
stains
TM
B
A1  A2  B1
Body  temperature
T
TB
DT
E2
TM
T
B
TM
B
B
Assessment
FR
B
C4
C2
DT
Incidence  of  AE
E2
D2
B
B2  C1
Basal
HR,  SBP,  DBP
6.2  Safety  &  Tolerability  Analysis
Table  3.  Triangulation  of  concepts  [1]
6.2.1  Analysis  for  primary  variables
Protocol  SOPH185-0521/I
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The  analysis  of  exploratory  variables,  FR  and  body  temperature,  will  be  performed  on  those  subjects  who  
complete  their  participation  without  deviations  from  the  study  protocol  (per-protocol  population,  PP).
The  final  results  report  will  be  displayed  in  tables  or  graphs,  as  appropriate.
For  the  analysis  of  these  variables,  the  same  primary  analysis  will  be  performed  as  long  as  the  necessary  
measurements  are  available.
expected  under  5.
An  imputation  procedure  for  missing  data  is  not  contemplated.
Newly  created  document,  no  changes  apply.
Author  of  the  document
6.3  Exploratory  analysis
6.4  Other  analyses
6.2.2  Analysis  for  secondary  variables
6.4.2  Procedure  for  handling  missing  data
2  Pearson  or  Fisher  exact  values  For  
conjunctival  hyperemia,  the  X  test  will  be  used
Protocol  SOPH185-0521/I
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Version  0.1
8.  Bibliography
7.  Change  control
[1]  ICH  Harmonized  Tripartite  Guideline.  Clinical  safety  data  management:  definitions  and  standards  for  expedited
[2]  Hurwitz  P,  Thompson  JM.  Uses  of  naphazoline  (Privine®)  in  ophthalmology.  Arch  Ophthal,1950;  43(4):  712-7.
[3]  Abelson  MB,  Allansmith  MR,  Friedlaender  MH.  Effects  of  topically  applied  ocular  decongestant  and  antihistamine.  Am  J
Ophthalmol,  1980;90(2):  254-7.
[4]  Abelson  MB,  Butrus  SI,  Weston  JH,  Rosner  B.  Tolerance  and  absence  of  rebound  vasodilation  following  topical
Biostatistics  Manager
reporting,  E2A.  1994.
Medical  Management,  Sophia  Laboratories,  SA  de  CV
Dr.  Patricia  del  Carmen  Muñoz  Villegas
[5]  Nayak  BK,  Kishore  K,  Gupta  SK.  Evaluation  of  oxymetazoline  and  naphazoline  in  benign  red  eyes:  a  double  blind
comparative  clinical  trial.  Indian  J  Ophthalmol,  1987;  35(4):  190-3.
ocular  decongestant  usage.  Ophthalmology,  1984;  91(11):  1364-7.
15
Machine Translated by Google


[6]  Hosten  LO,  Snyder  C.  Over-the-Counter  Ocular  Decongestants  in  the  United  States  -  Mechanisms  of  Action  and
[7]  HyLow  Consulting  http://
powerandsamplesize.com.  [Last  accessed:  May  10,  2021].
[8]  Chow  S,  Shao  J,  Wang  H.  Sample  size  calculations  in  clinical  research,  2nd  ed.,  New  York,  Chapman  and  Hall/CRC
biostatistics  series,  2008.
perspectives.Open  Access  Journal  of  Clinical  Trials,  2010;  2:  93-105.
[13]  Mexican  Official  Standard,  NOM-220-SSA1-2016,  Pharmacovigilance  Facilities  and  Operations,  CDMX:
Official  Gazette  of  the  Federation,  2017.
[9]  Krishnankutty  B,  Bellary  S,  Kumar  NB,  Moodahadu  LS.  Data  management  in  clinical  research:  An  overview.  Indian  J
Pharmacol,  2012;  44(2):  168-72.
16
line].
[12]  Whitcher  JP,  Shiboski  CH,  Shiboski  SC,  Heidenreich  AM,  Kitagawa  K,  Zhang  S,  Hamann  S,  Larkin  G,  McNamara  NA,
Greenspan  JS,  Daniels  TE;  Sjögren's  International  Collaborative  Clinical  Alliance  Research  Groups.  A  simplified
quantitative  method  for  assessing  keratoconjunctivitis  sicca  from  the  Sjögren's  Syndrome  International  Registry.
Am  J  Ophthalmol,  2010;  149(3):  405-15.
Available:
Clinical  Utility  for  Management  of  Ocular  Redness.  Clin  Optom  (Auckl),  2020;  12:  95-105.
[11]  Leon-Sarmiento  F,  Prada  DG,  Gutiérrez  C.  Pupil,  pupillometry  and  pupilography.  Beurol  Colomb  Act,  2008;  24:
LLC,  Power  and  Sample  Size  2013-2021.  [In
188-97.
[10]  Lu  Z,  Su.  Clinical  data  management:  Current  status,  challenges,  and  future  directions  from  industry
Protocol  SOPH185-0521/I
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Version  0.1
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9.1  Review  of  Articles  for  sample  size  calculation,  PRO-185  (Naphazoline  Hydrochloride  0.03%)
17
9.  Annexes
60
Naphazoline  hydrochloride  has  been  widely  used  as  a  nasal  decongestant.  Considering  its  potential  
uses  as  an  ocular  decongestant,  it  was  decided  to  evaluate  its  properties  in  certain  clinical  conditions.  
A  series  of  experiments  were  conducted  to  evaluate  the  effects  of  naphazoline  on  intraocular  pressure,  
pupillary  size,  and  the  accommodation  and  condition  of  ocular  blood  vessels  in  60  patients  (both  eyes).  
The  0.1%  naphazoline  solution  was  well  tolerated  by  the  eye.  A  mild  to  moderate  burning  sensation  
was  experienced  after  instillation.  Occasionally,  itching  occurred,  and  some  patients  with  lightly  
pigmented  irides  reported  blurred  vision.
Sophia  Laboratories,  SA  de  CV
Hurwitz  P,  Thompson  JM.  Uses  of  naphazoline  (privine®)  in  ophthalmology.  
Arch  Ophthal.  1950;  43(4):  712-7.  doi:  10.1001/archopht.1950.00910010723008.
Naphazoline
0.05%
Protocol  SOPH185-0521/I
Naphazoline
Summary
Two  commercial  preparations  of  topical  ophthalmic  vasoconstrictors  were  evaluated  for  their  bleaching  
ability,  duration  of  action,  tolerability,  and  rebound  vasodilation  in  11  healthy  volunteers.  Both  treatments,  
naphazoline  hydrochloride  0.02%  and  tetrahydrozoline  hydrochloride  0.05%,  significantly  decreased  
initial  redness  after  a  single  application  (Part  I);  however,  naphazoline  produced  significantly  more  
bleaching  than  tetrahydrozoline.  Only  naphazoline  maintained  bleaching  after  10  days  (Part  II).  The  
level  of  redness  remained  significant  after  baseline  for  8  hours  after  a  single  instillation  for  both  
vasoconstrictors  and  for  6  hours  after  multiple  administrations  of  naphazoline.
25
The  decrease  in  tetrahydrozoline's  effectiveness  after  the  10-day  study  period  may  encourage  its  
overuse.  No  vasoconstrictor  produced  rebound  vasodilation  after  discontinuation.
Abelson  MB,  Allansmith  MR,  Friedlaender  MH.  Effects  of  topically  applied  ocular  
decongestant  and  antihistamine.  Am  J  Ophthalmol.  1980;  90(2):  254-7.
Subjects  Treatment
The  efficacy  of  oxymetazoline  and  naphazoline  (0.01%)  was  evaluated  in  20  patients  with  red  eye.  The  
decongestive  action  of  oxymetazoline  0.01%  on  days  3  and  7  was  66.66%  and  80.55%  compared  to  
22.22%  and  50%  for  naphazoline  0.01%.
Nayak  BK,  Kishore  K,  Gupta  SK.  Evaluation  of  oxymetazoline  and  naphazoline  
in  benign  red  eyes:  a  double  blind  comparative  clinical  trial.  Indian  J  Ophthalmol.  
1987;  35(4):  190-3.
Naphazoline
transitory.
Reference
In  two  independent  studies  involving  25  subjects  each,  0.05%  naphazoline  produced  significant  
whitening  of  histamine-induced  red  eyes  and  ocular  itching  (but  did  not  prevent  itching).  Antazoline  
significantly  inhibited  itching  (but  not  red  eyes).  The  combination  of  naphazoline  and  antazoline  
produced  significant  whitening  and  inhibition  of  itching  in  all  histamine-challenged  eyes.  The  combination  
of  both  drugs  was  more  effective  than  either  component  individually  in  preventing  redness.  The  
antihistamine/vasoconstrictor  combination  was  equally  effective  for  itching.
0.02%
11
0.01%
0.1%
Version  0.1
Abelson  MB,  Butrus  SI,  Weston  JH,  Rosner  B.  Tolerance  and  absence  of  
rebound  vasodilation  following  topical  ocular  decongestant  usage.  Ophthalmology.  
1984;  91(11):  1364-7.  doi:  10.1016/s0161-6420(84)34140-9.
20
Naphazoline
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18
Hosten  LO,  Snyder  C.  Over-the-Counter  Ocular  Decongestants  in  the  United  States  -  
Mechanisms  of  Action  and  Clinical  Utility  for  Management  of  Ocular  Redness.  Clinical  
Optometry,  2020;  12:  95  –  105.
Naphazoline
This  review  provides  a  current,  clinically  relevant  summary  of  the  mechanism  of  action,  efficacy,  and  
safety  of  available  OCT  decongestants  for  the  reduction  of  ocular  redness  caused  by  minor  irritations.  
Currently,  the  OCT  products  marketed  in  the  US  include  tetrahydrozoline  0.05%,  naphazoline  0.012%  
to  0.03%,  and  brimonidine  0.025%.  All  3  agents  are  adrenergic  receptor  agonists  but  vary  in  their  
receptor-binding  properties:  tetrahydrozoline  is  a  selective  ÿ1-adrenergic  receptor  agonist,  naphazoline  
is  a  combination  ÿ1/ÿ2  receptor  agonist,  and  brimonidine  is  a  selective  ÿ2  receptor  agonist.  These  
OCT  decongestants  produce  vasoconstriction  of  conjunctival  blood  vessels,  resulting  in  a  rapid  
reduction  of  ocular  redness.  In  general,  reported  ocular  AEs  are  minimal,  mild,  and  transient,  with  no  
significant  systemic  AEs.  However,  ocular  decongestants  with  ÿ1-adrenergic  receptor  agonist  activity  
have  been  associated  with  loss  of  effectiveness  after  continued  use  (tachyphylaxis)  and  rebound  
redness  upon  discontinuation.  In  clinical  studies,  brimonidine  has  not  been  associated  with  
tachyphylaxis,  and  rebound  redness  has  been  rarely  reported.
0.012  to  0.03%
Version  0.1
0.01%.  A  significant  improvement  in  conjunctival  symptoms  such  as  itching,  foreign  body  sensation,  
tearing,  and  burning  was  observed  with  oxymetazoline  compared  with  naphazoline.  No  ocular  toxicity  
or  AEs  were  observed  during  the  study.
Protocol  SOPH185-0521/I
Sophia  Laboratories,  SA  de  CV
146
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(conjunctiva  only)
Grade  Points
0
1-5
3
1
0
>30
0-9
3
+1  if  there  is  staining  in  the  pupillary  area
10-32
(cornea  only)
>100
+1  if  there  are  filaments
Grade  Points
Score  by  staining  pattern:
0
6-30
2
19
Additional  points  for  fluorescein  staining  
only:
Lissamine  green
33-100
+1  if  there  are  patches  of  confluent  staining
2
Fluorescein
9.2  Efron  scale  for  conjunctival  hyperemia
9.3  SICCA  Ocular  Staining  Score  (modified  from  Whitcher  et  al,  2010)  [12].
Version  0.1
Sophia  Laboratories,  SA  de  CV
Protocol  SOPH185-0521/I
1
Machine Translated by Google