Clinical trial NCT03740659
Conditions: Cataract
Sponsor: NTC srl
Start date: 2018-09-04; Completion date: 2018-12-06
Enrollment: 125 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to evaluate the penetration of levofloxacin and dexamethasone 21-phosphate into the aqueous humour after ocular administration in combination or as single active ingredients.

Detailed description:
In most cases, postoperative care after cataract surgery consists of antiinflammatory and antibacterial drug therapy. The use of an ophthalmic solution containing the combination of a steroid and an antibiotic is routinely used in clinical practice; however, treatment duration could favour the emergence of antibiotic resistance. The study treatment associating a broad-spectrum antibiotic with a highly effective corticosteroid that can be used for a short period of time (one week) is therefore of considerable interest.

The aim of this study is to measure the concentrations of levofloxacin and dexamethasone in the aqueous humour after topical application of the combined ophthalmic solution and its single components.

Arms: arm 1: Levofloxacin 5 mg/ml + Dexamethasone 1 mg/ml (30 μl administered twice before Limbal paracentesis).

Limbal paracentesis will be performed prior to cataract surgery. arm 2: Levofloxacin 5 mg/ml (30 μl administered twice before Limbal paracentesis).

Limbal paracentesis will be performed prior to cataract surgery. arm 3: Dexamethasone 1.14 mg/ml (26 μl administered twice before Limbal paracentesis).

Limbal paracentesis will be performed prior to cataract surgery.
Interventions: intervention 1: Levofloxacin + Dexamethasone intervention 2: Levofloxacin intervention 3: Dexamethasone
Intervention descriptions: intervention 1: Levofloxacin + Dexamethasone ophthalmic solution + dexamethasone 21-phosphate administered twice: 90(±15) min. and 60(±15) min. before limbal paracentesis. intervention 2: Levofloxacin ophthalmic solution (Oftaquix®) administered twice: 90 (±15) min. and 60 (±15) min. before limbal paracentesis). intervention 3: Dexamethasone ophthalmic solution (Tamesad®) administered twice: 90 (±15) min. and 60 (±15) min. before Limbal paracentesis.

Protocol documents:
Prot_SAP_000.pdf: 
 
CLINICAL TRIAL PROTOCOL 
Aqueous humour concentrations after topical apPlication 
of combinEd levofloxacin-dexamethasone eye dRops and 
of its single components: a randoMized, assEssor-blinded, 
parallel-group study in patients undergoing cataract 
surgery - iPERME 
 
PROTOCOL NUMBER: LevoDesa_05-2017 
EUDRACT NUMBER: 2018-001149-15 
VERSION: 1.0 
DOCUMENT DATE: 21 March 2018 
 
 
 
CONFIDENTIAL: FURTHER DISSEMINATION MAY ONLY BE 
MADE WITH THE EXPRESS WRITTEN PERMISSION  
OF NTC S.r.l. 
 
 


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Table of Contents 
STATEMENT OF COMPLIANCE .............................................................................................................................. 4 
PROTOCOL APPROVERS .......................................................................................................................................... 5 
INVESTIGATOR APPROVAL SIGNATURES: ......................................................................................................... 6 
1 
PROTOCOL SUMMARY ................................................................................................................................ 7 
1.1 
Synopsis ................................................................................................................................................. 7 
1.2 
Study design ......................................................................................................................................... 11 
1.3 
Schedule of Activities (SoA) ................................................................................................................ 12 
2 
INTRODUCTION .......................................................................................................................................... 13 
2.1 
Study Rationale .................................................................................................................................... 13 
2.2 
Background .......................................................................................................................................... 13 
2.3 
Risk/Benefit Assessment ...................................................................................................................... 15 
2.3.1 
Known Potential Risks ................................................................................................................. 15 
2.3.2 
Known Potential Benefits ............................................................................................................. 16 
2.3.3 
Assessment of Potential Risks and Benefits ................................................................................. 16 
3 
OBJECTIVES AND ENDPOINTS ................................................................................................................ 16 
4 
STUDY DESIGN ............................................................................................................................................ 16 
4.1 
Overall Design ...................................................................................................................................... 16 
4.2 
Scientific Rationale for Study Design .................................................................................................. 17 
4.3 
Justification for Dose............................................................................................................................ 18 
4.4 
End of Study Definition ....................................................................................................................... 18 
5 
STUDY POPULATION ................................................................................................................................. 18 
5.1 
Inclusion Criteria .................................................................................................................................. 19 
5.2 
Exclusion Criteria ................................................................................................................................. 19 
5.3 
Screen Failures ..................................................................................................................................... 20 
5.4 
Strategies for Recruitment and Retention ............................................................................................. 20 
6 
STUDY INTERVENTION ............................................................................................................................. 20 
6.1 
Study Intervention(s) Administration ................................................................................................... 20 
6.1.1 
Study Intervention Description ..................................................................................................... 20 
6.1.2 
Dosing and Administration ........................................................................................................... 20 
6.2 
Preparation/Handling/Storage/Accountability ...................................................................................... 21 
6.2.1 
Acquisition and accountability ..................................................................................................... 21 
6.2.2 
Formulation, Appearance, Packaging, and Labeling .................................................................... 21 
6.2.3 
Product Storage and Stability ....................................................................................................... 21 
6.2.4 
Preparation .................................................................................................................................... 21 
6.3 
Measures to Minimize Bias: Randomization and Blinding .................................................................. 22 
6.4 
Study Intervention Compliance ............................................................................................................ 22 
6.5 
Concomitant medications ..................................................................................................................... 22 
6.5.1 
Rescue therapy .............................................................................................................................. 22 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL ................................................................................................................... 23 
7.1 
Discontinuation of Study Intervention ................................................................................................. 23 
7.2 
Participant Discontinuation/Withdrawal from the Study...................................................................... 23 
7.3 
Lost to Follow-Up ................................................................................................................................ 23 
8 
STUDY ASSESSMENTS AND PROCEDURES .......................................................................................... 23 
8.1 
Assessments ......................................................................................................................................... 23 
8.2 
Laboratory Analyses............................................................................................................................. 24 


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8.3 
Safety .................................................................................................................................................... 25 
8.4 
Adverse Events and Serious Adverse Events ....................................................................................... 25 
8.4.1 
Definition of Adverse Events (AE) .............................................................................................. 25 
8.4.2 
Definition of Serious Adverse Events (SAE) ............................................................................... 25 
8.4.3 
Classification of an Adverse Event ............................................................................................... 25 
8.4.4 
Time Period and Frequency for Event Assessment and Follow-Up ............................................. 26 
8.4.5 
non-serious Adverse Event Reporting .......................................................................................... 26 
8.4.6 
Serious Adverse Event Reporting ................................................................................................. 27 
8.4.7 
Reporting Events to Participants................................................................................................... 27 
8.4.8 
Events of Special Interest ............................................................................................................. 27 
8.4.9 
Reporting of Pregnancy ................................................................................................................ 28 
9 
STATISTICAL CONSIDERATIONS ............................................................................................................ 28 
9.1 
Statistical Hypotheses ........................................................................................................................... 28 
9.2 
Sample Size Determination .................................................................................................................. 28 
9.3 
Populations for Analyses ...................................................................................................................... 29 
9.4 
Statistical Analyses............................................................................................................................... 29 
9.4.1 
General Approach ......................................................................................................................... 29 
9.4.2 
Analysis of the Endpoint .............................................................................................................. 29 
9.4.3 
Safety Analyses ............................................................................................................................ 29 
9.4.4 
Baseline Descriptive Statistics ...................................................................................................... 30 
9.4.5 
Planned Interim Analyses ............................................................................................................. 30 
9.4.6 
Sub-Group Analyses ..................................................................................................................... 30 
9.4.7 
Tabulation of Individual participant Data ..................................................................................... 30 
9.4.8 
Exploratory Analyses.................................................................................................................... 30 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .................................. 30 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ................................................................... 30 
10.1.1 
Informed Consent Process ............................................................................................................ 30 
10.1.2 
Study Discontinuation and Closure .............................................................................................. 31 
10.1.3 
Confidentiality and Privacy .......................................................................................................... 32 
10.1.4 
Future Use of Stored Specimens and Data ................................................................................... 32 
10.1.5 
Safety Oversight ........................................................................................................................... 32 
10.1.6 
Clinical Monitoring ...................................................................................................................... 32 
10.1.7 
Quality Assurance and Quality Control ........................................................................................ 33 
10.1.8 
Data Handling and Record Keeping ............................................................................................. 34 
10.1.9 
Protocol Deviations ...................................................................................................................... 35 
10.1.10 
Insurance ....................................................................................................................................... 36 
10.1.11 
Publication and Data Sharing Policy ............................................................................................ 36 
10.1.12 
Conflict of Interest Policy ............................................................................................................. 37 
10.2 
Abbreviations ....................................................................................................................................... 38 
11 
REFERENCES................................................................................................................................................ 39 
12 
APPENDICES ................................................................................................................................................ 41 
12.1 
Laboratory Protocol 
12.2 
Oxford Grading Scheme 


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STATEMENT OF COMPLIANCE 
The trial will be conducted in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and all applicable laws and regulations. The Principal Investigator 
will assure that no deviation from, or changes to the protocol will take place without prior 
agreement from the sponsor and documented approval from the Independent Ethics Committee 
(IEC) and/or Competent Authority, except where necessary to eliminate an immediate hazard(s) 
to the trial participants. All personnel involved in the conduct of this study have completed ICH 
GCP Training. 
The protocol, informed consent form(s), and all participant materials will be submitted to the IEC 
for review and approval. Approval of both the protocol and the consent form must be obtained 
before any participant is enrolled. Any substantial amendment to the protocol will require review 
and approval by the IEC before the changes are implemented. All changes to the consent form will 
be IEC approved; a determination will be made regarding whether a new consent needs to be 
obtained from participants who provided consent, using a previously approved consent form. 
 
 


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PROTOCOL APPROVERS 
 
CHAIRMAN:   
Prof. Marco Nardi 
  
_________________________          ____________ 
U.O. Oculistica Universitaria  
 
    signature 
 
 
        date   
Azienda Ospedaliera Universitaria Pisana  
Presidio Ospedaliero di Cisanello 
Pisa 
 
 
 
SPONSOR: 
 
 
 
Dr. Federico Bertocchi 
  
_________________________          ____________ 
Head of R&D 
  
    
    signature 
 
 
        date    
NTC – Novelty Technology Care 
 
 
 
CONTRACT RESEARCH 
ORGANIZATION:  
 
 
Dr. Chiara Costantini 
     
 _________________________           ____________ 
Scientific Director 
 
    signature 
 
        
        date 
OPIS s.r.l. 
 
 
Dr. Laura Ambrosoli 
      
 _________________________          ____________ 
Medical Director  
  
 
    signature 
 
        date    
OPIS s.r.l. 
 
 


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INVESTIGATOR APPROVAL SIGNATURES: 
Clinical Study Protocol: LevoDesa_05-2017 
 
Investigator signature 
I have read the protocol and agree to conduct this trial in accordance with all stipulations of the 
protocol, with applicable laws and regulations and in accordance with the ethical principles 
outlined in the Declaration of Helsinki. 
 
 
 
 
 
 
  
Investigator 
 
Signature 
 Date 
 
 
Center name and address: 
 
 
 
 


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1 
 
PROTOCOL SUMMARY 
1.1 SYNOPSIS  
Protocol number 
LevoDesa_05-2017 
EudraCT number 
2018-001149-15 
Title of the Study 
Aqueous humour concentrations after topical apPlication of combinEd 
levofloxacin-dexamethasone eye dRops and of its single components: 
a randoMized, assEssor-blinded, parallel-group study in patients 
undergoing cataract surgery - iPERME 
Phase  
II 
Sponsor 
NTC S.r.l. 
Investigational Sites 
· Prof. Marco Nardi: U.O. Oculistica Universitaria – Azienda 
Ospedaliera Universitaria Pisana – Presidio Ospedaliero di 
Cisanello – Pisa 
· Prof. Luca Rossetti: Clinica Oculistica – Presidio Ospedale San Paolo 
– Milan 
Study Objective 
To evaluate the penetration of levofloxacin and dexamethasone 21-
phosphate into the aqueous humour after ocular administration in 
combination or as single active ingredients. 
Study Endpoint 
Aqueous humour concentration of levofloxacin, dexamethasone 21-
phosphate and dexamethasone. 
Type of Study 
This is a randomized, assessor-blinded, parallel-group study.  
Number of Patients 
The study will enrol 120 patients scheduled for cataract surgery. 
Inclusion Criteria 
1. Written informed consent must be obtained before any 
assessment is performed 
2. Male or female patients, aged ≥ 40 years 
3. Patient undergoing phacoemulsification 
4. Corneal thickness between 450 μm and 600 μm as measured by 
means of pachymetry 


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5. Corneal integrity confirmed by means of fluorescein test 
(Oxford scheme grade 0) 
6. Adequate pupil dilation assessed at screening 
7. Female patients of childbearing potential must have a negative 
pregnancy test 
8. Ability to fully understand all study procedures 
Exclusion Criteria 
1. Corneal epithelium integrity not confirmed by fluorescein test  
2. History of corneal disease or dystrophy (e.g. ocular herpes, 
conjunctivitis, keratitis) 
3. History of ocular trauma with corneal damage 
4. History of acute ocular inflammation (including uveitis) in the 6 
months prior to screening 
5. Previous ocular surgery (including laser treatment)  
6. Glaucoma 
7. Treatment with an ophthalmic investigational drug in the 3 
months prior to screening  
8. Treatment with any topical ocular drug within 12 hours before 
start of cataract surgery other than study drugs and instillation 
of topical anaesthetic (oxybuprocaine hydrochloride 
- 
Novesina®) followed by povidone-iodine (Oftasteril®) within 10 
minutes before start of surgery 
9. Treatment with any topical steroid or antibiotic drug in the 7 
days prior to cataract surgery (artificial tears without BAK are 
allowed) 
10. Treatment with any systemic steroid or antibiotic drug in the 7 
days prior to cataract surgery  
11. Known 
hypersensitivity 
to 
levofloxacin 
or 
other 
fluoroquinolones and/or dexamethasone or other steroids 
12. Pregnant or lactating women  
13. Patients who have received any investigational drug during the 
preceding 30 days or 5 times the plasma half-life, whichever is 
longer, or who have previously participated in this trial 
Study Drugs 
· 
Levofloxacin hemihydrate 5.12 mg/ml, corresponding to 5 
mg/ml levofloxacin + dexamethasone 21-phosphate 1.32 
mg/ml, corresponding to dexamethasone 1 mg/ml: two 30 µl 
doses 30 minutes apart. 
· 
Levofloxacin 
hemihydrate 
5.12 
mg/ml 
(Oftaquix®), 
corresponding to 5 mg/ml levofloxacin: two 30 µl doses 30 
minutes apart. 
· 
Dexamethasone 
21-phosphate 
1,50 
mg/ml 
(Tamesad®), 
corresponding to dexamethasone 1.14 mg/ml: two 26 µl doses 
30 minutes apart.  


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Study Procedures 
After providing informed consent, patients scheduled for cataract 
surgery will undergo screening assessments to verify eligibility for the 
study. Screening must not take place more than 28 days prior to 
surgery. 
Eligible patients will be randomly assigned to levofloxacin 5 mg/ml + 
dexamethasone 1 mg/ml (eye drops, test drug), or to levofloxacin 
(Oftaquix®) or to dexamethasone (Tamesad®) in a 1:1:1 ratio. 
Administration of study drugs will be performed by qualified health 
care personnel using a micropipette and dispenser. Two doses of one 
of the study treatments will be administered, the first 90 ± 15 minutes 
prior to surgery, and the second 60 ± 15 minutes before surgery. The 
two doses must be administered 30 minutes apart. Doses are to be 
administered at the lateral canthus while applying pressure at the 
medial canthus to prevent drainage of the drug. 
Before cataract surgery, a limbal paracentesis will be performed and 
an aqueous humour sample of approximately 0.05 ml will be drawn 
from the anterior chamber with a 30-gauge needle insulin syringe. The 
aqueous humour will be stored immediately in vials at -80 °C and 
subsequently analysed at a central laboratory. Vial labels will contain 
the kit number but no information regarding the drug received. 
Laboratory analysts will thus be blinded to the drug administered and 
samples will be analysed for the concentration of all three molecules 
(levofloxacin, dexamethasone 21-phosphate and dexamethasone) by 
means of LC-MS-MS. 
Following surgery, patients will be treated with antibiotics and/or 
steroids according to local clinical practice. 
Sample size calculation 
The study plans to enrol 120 patients: 40 will be assigned to the 
dexamethasone group, 40 to the levofloxacin group and 40 to the 
dexamethasone-levofloxacin combination. 
The sample size has been estimated based on the expected precision 
(i.e. width of the 95% confidence interval) of the estimates of the study 
drug concentrations.  
Assessment of dexamethasone concentration: When the sample size is 
40, a two-sided 95% confidence interval for a single mean will extend 
4.648 ng/ml from the observed mean, assuming that the standard 
deviation is known to be 15 ng/ml and the confidence interval is based 
on the large sample z statistic. 


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Assessment of levofloxacin concentration: When the sample size is 40, 
a two-sided 95% confidence interval for a single mean will extend 
61.676 ng/ml from the observed mean, assuming that the standard 
deviation is known to be 199.022 ng/ml and the confidence interval is 
based on the large sample z statistic.   
Drug analysis 
All biological samples will be sent to the central laboratory, 
Ticinumlab, Novara (Italy), which will perform in one run the LC-MS-
MS (triple quadrupole) analyses for the simultaneous determination of 
levofloxacin, dexamethasone and dexamethasone 21-phosphate. 
Statistical considerations 
Continuous data will be summarized with standard descriptive 
statistics (i.e. the mean, standard deviation, minimum, median and 
maximum, 95% confidence limits). Categorical data will be 
summarized by frequencies and percentages. 
All analyses will be performed using SAS version 9.4 or later. 
Duration of the study 
Estimated date of the first visit of the first patient (FPFV): Q2, 2018 
Estimated date of the first visit of the last patient (LPFV): Q1, 2019 
Estimated date of the last visit of the last patient (LPLV): Q1, 2019 
Version and date 
V 1.0 – 21 March 2018 


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1.2 STUDY DESIGN 
 
Randomization 
1:1:1 
Levofloxacin 5 mg/ml + Dexamethasone 1 mg/ml 
Levofloxacin 5 mg/ml (Oftaquix®) 
Dexamethasone 1.14 mg/ml (Tamesad®) 
Screening 
Instillation 
-90 (± 15) min 
Instillation 
-60 (± 15) min 
Limbal 
paracentesis 
30 min 
--- 
--- 
Not more than 
28 days prior to 
surgery 


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1.3 SCHEDULE OF ACTIVITIES (SOA) 
 
Procedures 
Screeninga 
Day of surgery 
Pre-dose 
First dose 
-90 (±15) min 
Second dose 
-60 (±15) min 
Limbal 
Paracentesis  
Informed consent 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical history 
X 
 
 
 
 
Concomitant medications 
X 
X 
 
 
 
Pachymetry 
X 
 
 
 
 
Fluorescein test 
X 
 
 
 
 
Inclusion/Exclusion 
criteria 
X 
X 
 
 
 
Pregnancy testb  
 
X 
 
 
 
Randomization 
 
X 
 
 
 
Study drug administrationc 
 
 
X 
X 
 
Limbal paracentesis 
 
 
 
 
X 
Administration of 
Mydrane®d 
 
 
 
 
X 
Adverse event review and 
evaluatione 
X 
X 
X 
X 
X 
a Screening must take place not more than 28 days prior to surgery. 
b Urine pregnancy test (women of childbearing potential). 
c Two doses 30 minutes apart, the first 90 (± 15) minutes prior to surgery, and the second 60 (± 15) minutes prior 
to surgery. 
d Mydrane® is to be administered following withdrawal of aqueous humour. 
e Adverse events are to be reported until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation. 
 
 


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2 
INTRODUCTION 
 
2.1 STUDY RATIONALE  
In most cases, postoperative care after cataract surgery consists of anti-inflammatory and 
antibacterial drug therapy. The use of an ophthalmic solution containing the combination of a 
steroid and an antibiotic is routinely used in clinical practice also because it favours the proper 
administration of the two agents, reduces the possibility of inaccurate dosage and improves patient 
compliance with medication. To reduce the emergence of antibiotic resistance, treatment with an 
antibiotic should however not be longer than the time necessary for the healing of surgically 
induced epithelial lesions, and therefore should not last more than a week.  
Based on these considerations, there is clear interest in having a combination of a broad-spectrum 
antibiotic with a highly effective corticosteroid that can be used for a short period of time (one 
week). The combination of dexamethasone and levofloxacin tested in this study is the first 
combination of a steroid and a quinolone under development and represents a major step forward 
in the prevention and treatment of post-operative inflammation and prevention of post-operative 
infections. 
Levofloxacin 0.5% – dexamethasone 0.1% ophthalmic solution is an anti-inflammatory/antibiotic 
combination indicated for the prevention and treatment of inflammation and prevention of 
infection associated with cataract surgery in adults. The active components of this combination are 
currently available in marketed ophthalmic products. The purpose of this study is to measure the 
concentrations of levofloxacin and dexamethasone in the aqueous humour (AH) after topical 
application of the combined ophthalmic solution and its single components. 
2.2 BACKGROUND  
Cataract is an ocular condition that causes vision impairment due to changes in the opacity of the 
ocular lens. Cataract is an age-related condition, but smoking, ocular trauma, exposure to 
ultraviolet light, diabetes and drug induced metabolic changes in the lens are risk factors for lens 
opacification causing cataract.1 
The only cure for cataract is surgical, with phacoemulsification with lens implantation as the 
treatment of choice.2 Recovery after surgery is easier than it was in the past and patients usually 
no longer require in-patient hospital care, with recovery usually uneventful and pain-free. 
However, adverse events may occur due to corneal trauma, ocular inflammation and dryness, 
which can result in ocular irritation during the recovery period.3-5 These conditions may cause 
symptoms such as pain, burning, stinging, foreign body sensation, itchiness and glare in the 
operated eye. The incidence of ocular irritation symptoms experienced during the first 
postoperative hours may be as high as 98%.6 Conjunctival hyperaemia, anterior chamber cells and 
flare and cystoid macular oedema are some possible signs of inflammation after cataract surgery. 


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In many cases, inflammation is self-limiting, but drug therapy is often used to shorten resolution 
time and improve ocular comfort.7 
Steroidal and non-steroidal anti-inflammatory drugs are used to prevent and treat postoperative 
inflammation. Topical corticosteroids are usually administered for 1 to 6 weeks after cataract 
surgery. According to the practice pattern studies published by the American Society of Cataract 
Refractive Surgery (2012), Canadian Ophthalmological Society members study 20118 and 
European Society of Cataract and Refractive Surgeons (ESCRS), there are variations in 
postoperative drug therapy provided to individual patients and among different countries. In 
Europe, dexamethasone was the most commonly used steroidal anti-inflammatory drug and was 
chosen as the preferred drug by 71% of ophthalmological surgeons.9 
Endophthalmitis, is a rare yet severe condition that occurs in 0.03–0.3% of patients undergoing 
cataract surgery.10,11 This condition usually appears during the first days after surgery, and in 80% 
of cases, endophthalmitis will have developed during the first 6 weeks after surgery.11,12 Topically 
applied antibiotics are routinely used for the prophylaxis of postoperative bacterial ocular 
infections such as endophthalmitis. Intraocular, usually intracameral or subconjunctival antibiotics 
are used during surgery, and both pre- and postoperative topical applications are also provided.10 
To increase the efficacy of this approach, a broad-spectrum antibiotic should be chosen.  
A prospective observational study was conducted to determine the antibiotic susceptibility patterns 
of conjunctival bacterial flora isolated preoperatively from patients undergoing anterior segment 
surgery. Of the 120 eyes studied, 21 (18%) showed no bacterial growth. Of the 143 bacterial strains 
isolated from the remaining 99 eyes, 112 (78%) were coagulase-negative staphylococci (CNS). 
Among the CNS, greater than 90% were susceptible to cefotaxime, levofloxacin, imipenem, 
meropenem, vancomycin, and each of the aminoglycosides except neomycin. Between 70% and 
90% of the CNS were susceptible to cefazolin, neomycin, ciprofloxacin, ofloxacin, norfloxacin, 
and chloramphenicol.13 
Based on these results, quinolones clearly represent a rational approach to the prevention of ocular 
infections after cataract surgery. Indeed, among available antibiotics for ophthalmic use, 
quinolones are characterized by a broad spectrum that includes both the Gram-positive and Gram-
negative bacteria that are most frequently responsible for ocular bacterial infections. 
In most cases, postoperative care after cataract surgery consists of anti-inflammatory and 
antibacterial drug therapy. Topical anti-inflammatory agents and antibiotics are both routinely 
provided after cataract surgery. The combined administration of corticosteroids and antibiotics 
guarantees a powerful anti-inflammatory effect, and contemporarily a preventive/therapeutic 
action against infections related either to the intensity of the inflammatory process or to the 
immunosuppressive effect of the steroid.  
However, use of such a combination should also be in line with the modern approach to 
antimicrobial therapy, which advocates preventing increasing bacterial resistance caused by the 


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inappropriate use of antibiotics. The ESCRS guidelines do not indicate a specific antibiotic to be 
used for topical postoperative treatment, nor a precise duration of such treatment, although they 
emphasize the importance of rational use to limit the development of bacterial resistance, 
especially when a broad-spectrum antibiotic is used.14 For this reason, antibiotic treatment should 
not be prolonged beyond the time necessary for the healing of surgically induced epithelial lesions, 
and therefore should not last more than a week.15 
In light of these considerations, having the combination of a broad-spectrum antibiotic and a highly 
effective corticosteroid to be used for a short period of time would be of great clinical interest. The 
association of dexamethasone 1 mg/ml and levofloxacin 5 mg/ml tested in this study is the first 
combination of a steroid and a quinolone under development and represents a major step forward 
in this direction.  
The concentration of dexamethasone (1 mg/ml) is well-established in ophthalmic solutions both 
as sole active ingredient or in combination with an antibiotic to prevent ocular inflammation 
following cataract surgery. After topical administration of 0.1% solutions of this agent, the 
concentration in the aqueous humour is adequate to exert its potent anti-inflammatory activity at 
the site of action.16 The dexamethasone 21-phosphate salt is freely soluble in water and thus the 
solution does not require shaking before application and guarantees consistency of the content of 
the active ingredient at each application.  
The pharmacokinetics of both levofloxacin and dexamethasone administered individually confirm 
that after application as an eye drop solution, both active drugs reach significant concentrations in 
the aqueous humour, with peaks of aqueous levels between 90 and 150 minutes, and detectable 
concentrations after 12 hours from dosing.16-21 Conversely, the concentrations in plasma are 
negligible, which guarantees lack of systemic effects during a short-course treatment.  
The purpose of this study is to analyse the aqueous humour drug concentrations of levofloxacin 
and dexamethasone administered topically in combination and as single components to assess the 
penetration of these agents and provide data for clinical use. 
2.3 RISK/BENEFIT ASSESSMENT 
 
2.3.1 KNOWN POTENTIAL RISKS  
In toxicity studies, the only toxicity data reported for dexamethasone were those related to an 
exaggerated pharmacological activity including a negative feedback on the pituitary-adrenal axis. 
Systemic effects are however not expected or are negligible when the drug is topically 
administered as eye drops and for a limited period of time. Nevertheless, its potential effect on 
foetal development and on newborns suggests it be used very carefully in pregnant women and to 
discard milk during lactation.22,23 


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Possible adverse effects of topical ocular corticosteroid therapy consist of an increase of 
intraocular pressure (IOP), inhibition of corneal wound healing and increased likelihood of 
infection and serious complications, but systemic adverse effects are rare.7,24 
Levofloxacin is not toxic on reproduction and does not have mutagenic or carcinogenic potential. 
It is not irritating after eye instillation, but it is phototoxic at very high doses and photosensitization 
may not be excluded. Thus, it is suggested not to expose patients to direct sunlight during treatment 
with eye drops containing levofloxacin.  
2.3.2 KNOWN POTENTIAL BENEFITS  
The efficacy of dexamethasone as anti-inflammatory agent and of levofloxacin as antibiotic in 
ophthalmology is well established. Combining the two agents would favour their proper 
administration, reduce the possibility of inaccurate dosage and improve patient compliance. The 
combination would also allow the implementation of a shorter antibiotic treatment period as 
compared to other marketed products and thus prevent the emergence of antibiotic resistance. 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
All things considered, the potential benefits of this trial surely outweigh their potential risks. 
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVE 
ENDPOINT 
JUSTIFICATION FOR 
ENDPOINT 
To evaluate the penetration of 
levofloxacin/dexamethasone 21-
phosphate into the aqueous 
humour after ocular 
administration in combination or 
as single active ingredients. 
Aqueous humour concentration 
of levofloxacin and 
dexamethasone. 
The endpoint is 
standard for penetration 
studies of ophthalmic 
preparations into the 
aqueous humour. 
 
4 
STUDY DESIGN  
 
4.1 OVERALL DESIGN 
This is a randomized, assessor-blinded, parallel-group clinical study.  
One hundred and twenty (120) patients scheduled for cataract surgery at 2 centres who have 
provided written informed consent will undergo screening procedures during a preoperative visit. 
Prior to surgery, and once eligibility criteria have been confirmed, eligible patients will be assigned 
to one of the three following treatment groups in a 1:1:1 ratio: 
· Levofloxacin 5 mg/ml + dexamethasone 1 mg/ml (test drug) 
· Levofloxacin (Oftaquix®) 


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· Dexamethasone (Tamesad®) 
Administration of study drugs will be performed by qualified health care personnel using a 
micropipette and dispenser. Two doses of one of the study treatments will be administered, the 
first 90 ± 15 minutes prior to surgery, and the second 60 ± 15 minutes before surgery. The two 
doses must be administered 30 minutes apart. Eye drops are to be administered at the lateral 
canthus while applying pressure at the medial canthus to prevent drainage of the drug. 
Phacoemulsification and limbal paracentesis will be performed by an experienced surgeon. Before 
the main incisions of phacoemulsification, approximately 0.05 ml of aqueous humour will be 
drawn from the anterior chamber with a 30-gauge needle syringe. Mydrane®, a combined solution 
(phenylephrine [0.31%], tropicamide [0.02%] and lidocaine [1%]) for intracameral injection 
indicated for cataract surgery to obtain mydriasis and intraocular anaesthesia during the surgical 
procedure is to be administered following paracentesis. The patient’s participation in the study will 
end with the administration of Mydrane®. Following surgery, patients will be treated with 
antibiotics and/or steroids according to local clinical practice. 
The aqueous humour will be stored immediately in vials at -80°C and subsequently analysed at a 
central laboratory. Vial labels will contain the study kit number but no information regarding the 
drug received. The laboratory analyst will be blinded to the drug administered and samples will be 
analysed for the concentration of all three molecules (levofloxacin, dexamethasone 21-phosphate 
and dexamethasone) by means of an LC tandem mass spectrometry method. Quality control 
samples blinded to the analyst will be included following advice from BfArM. To that end, the 
randomization list will include kit numbers corresponding to quality control samples. A different 
analyst will prepare twelve samples (4 of each active principle) with synthetic aqueous humour 
and a known concentration of the active ingredients in pre-labelled vials at the central laboratory 
in the presence of personnel belonging to a Contract Research Organization (CRO) (OPIS s.r.l.) 
working on behalf of the Sponsor. The twelve quality control samples correspond to 10% of the 
true biological samples obtained from the patients. 
4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
The randomized design is standard for studies investigating the penetration of ophthalmic 
preparations into the aqueous humour. The laboratory analyst will be blinded to the drug 
administered to ensure an unbiased analysis (see Section 4.1). 
 
 


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4.3 JUSTIFICATION FOR DOSE 
The concentration of dexamethasone (1 mg/ml) in the combination treatment is well established 
in ophthalmic solutions, both as sole active ingredient or in combination with an antibiotic. 
Levofloxacin is also well established as sole active ingredient of ophthalmic solutions at a 
concentration of 5 mg/ml. 
Two 30 µl doses administered by micropipette and dispenser of combined levofloxacin 
hemihydrate 5.12 mg/ml, corresponding to 5 mg /ml levofloxacin + dexamethasone 21-phosphate 
1.32 mg/ml, corresponding to dexamethasone 1 mg/ml administered 90 minutes and 60 minutes 
prior to surgery were deemed suitable for investigating the penetration of the active principles into 
the aqueous humour. AH concentration of levofloxacin and dexamethasone will be analysed also 
using marketed products containing the single active principles; levofloxacin hemihydrate 5.12 
mg/ml, corresponding to 5mg/ml levofloxacin (Oftaquix®) will be administered at the same dose 
(two 30 µl instillations) given the same concentration of the active principle, whereas 
dexamethasone 21-phosphate 1.50 mg/ml, corresponding to dexamethasone 1.14 mg / ml 
(Tamesad®) will be administered at a dose of two 26 µl instillations due to the higher concentration 
of the active principle in this product respect to that of the combination product. The dose of 26 µl 
was calculated as follows: 
Tamesad® 0.15%:1 ml contains 1.5 mg of dexamethasone sodium phosphate 
1.5 mg / molecular weight dexamethasone sodium phosphate x molecular weight dexamethasone 
= 1.14 mg dexamethasone. 
Tamesad® therefore contains 1.14 mg dexamethasone / ml. 
30 µl x 0.1% / 0.114% = 26.3 µl, i.e. 26.3 µl of the 1.5 mg/ml solution will have the same content 
in dexamethasone as 30 µl of the 1 mg/ml solution. 
4.4 END OF STUDY DEFINITION 
A participant is considered as having completed the study if he or she has completed all phases of 
the study including the last procedure as indicated in the Schedule of Activities (SoA) (paracentesis 
and administration of Mydrane®), Section 1.3. The patient will subsequently be treated according 
to local clinical practice. 
The end of the study is defined as completion of the last procedure in the trial globally. 
5 
STUDY POPULATION 
The study population will be made up of 120 patients scheduled for cataract surgery. 
 
 


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5.1 INCLUSION CRITERIA 
To be eligible to participate in this study, a subject must meet all the following criteria: 
1. Written informed consent must be obtained before any assessment is performed 
2. Male or female patients, aged ≥40 years 
3. Patient undergoing phacoemulsification 
4. Corneal thickness between 450 μm and 600 μm as measured by means of pachymetry 
5. Corneal integrity confirmed by means of fluorescein test (Oxford scheme grade 0) 
6. Adequate pupil dilation assessed at screening 
7. Female patients of childbearing potential must have a negative pregnancy test 
8. Ability to fully understand all study procedures 
 
5.2 EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this 
study: 
1. Corneal epithelium integrity not confirmed by fluorescein test  
2. History of corneal disease or dystrophy (e.g. ocular herpes, conjunctivitis, keratitis) 
3. History of ocular trauma with corneal damage 
4. History of acute ocular inflammation (including uveitis) in the 6 months prior to screening 
5. Previous ocular surgery (including laser treatment)  
6. Glaucoma 
7. Treatment with an ophthalmic investigational drug in the 3 months prior to screening  
8. Treatment with any topical ocular drug within 12 hours before start of cataract surgery other 
than study drugs and instillation of topical anaesthetic (oxybuprocaine hydrochloride - 
Novesina®) followed by povidone-iodine (Oftasteril®) within 10 minutes before start of 
surgery 
9. Treatment with any topical steroid or antibiotic drug in the 7 days prior to cataract surgery 
(artificial tears without BAK are allowed) 
10. Treatment with any systemic steroid or antibiotic drug in the 7 days prior to cataract surgery  
11. Known hypersensitivity to levofloxacin, other fluoroquinolones or dexamethasone  
12. Pregnant or lactating women  
13. Patients who have received any investigational drug during the preceding 30 days or 5 times 
the plasma half-life, whichever is longer, or who have previously participated in this trial 
 
 


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5.3 SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently randomly assigned to the study intervention. A minimum set of screen failure 
information is required to ensure transparent reporting of screen failure participants, to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond 
to queries from regulatory authorities. Minimal information includes demography, screen failure 
details, eligibility criteria, and any serious adverse event (SAE). 
5.4 STRATEGIES FOR RECRUITMENT AND RETENTION 
This study will enrol subjects already scheduled for cataract surgery and no other recruitment 
strategies are required. No strategy for retaining subjects is necessary given the duration of the 
study. 
6 
STUDY INTERVENTION 
 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
The study drugs are as follows: 
· Levofloxacin hemihydrate 5.12 mg/ml + dexamethasone 21-phosphate 1.32 mg/ml (eye 
drops solution) 
· Levofloxacin hemihydrate 5.12 mg/ml (Oftaquix® eye drops) 
· Dexamethasone 21-phosphate 1,50 mg/ml (Tamesad® eye drops) 
6.1.2 DOSING AND ADMINISTRATION 
Eligible patients will be randomly assigned to one of the three following treatment groups in a 
1:1:1 ratio: 
· Levofloxacin 5 mg/ml + dexamethasone 1 mg/ml (test drug): two 30 µl doses, 30 minutes 
apart, one 90 ± 15 minutes prior to surgery and the other 60 ± 15 minutes prior to surgery. 
· Levofloxacin (Oftaquix®): two 30 µl doses, 30 minutes apart, one 90 ± 15 minutes prior to 
surgery and the other 60 ± 15 minutes prior to surgery. 
· Dexamethasone (Tamesad®): two 26 µl doses, 30 minutes apart, one 90 ± 15 minutes prior 
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Administration of study drugs will be performed by qualified health care personnel using a 
micropipette and dispenser. Doses are to be administered at the lateral canthus while applying 
pressure at the medial canthus to prevent drainage of the study drug. 
Should the treatment schedule not be respected, subjects will not undergo aqueous humour 
extraction and will be followed for safety purposes only.  
6.2 PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
Study treatments will be provided by the Sponsor. The investigator, or a pharmacist or other 
appropriate individual who is designated by the investigator, will maintain accurate records of the 
product’s delivery to the study site, the inventory at the site, the use for each subject, and the return 
to the sponsor or alternative disposition of unused products. Drug-related documentation will be 
reviewed by the field monitor during the study conduct. 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING   
· Test drug: levofloxacin 5 mg/ml – dexamethasone 1 mg/ml eye drops 
Composition in active ingredients: levofloxacin hemihydrate 5.12 mg/ml, corresponding to 
5mg/ml, and dexamethasone 21-phosphate 1.32 mg/ml, corresponding to dexamethasone 1 mg/ml. 
The Investigational Medicinal Product (IMP) is a sterile, clear, greenish-yellow isotonic 
ophthalmic solution with a pH of 7.2. The finished product is packaged into a light-resistant LDPE 
bottle with ophthalmic dropper and PP screw cap. 
· Levofloxacin 5.0 mg/ml (Oftaquix®) 0.5% eye drops  
Transparent, light yellow or light yellow-green solution in a multi-dose 5 ml vial. 1 ml contains: 
5,12 mg di levofloxacin hemihydrate equal to 5 mg of levofloxacin. 
· Dexamethasone 1.5 mg/ml (Tamesad®) 0,15% eye drops 
Solution in multi-dose vial. One ml of solution contains: dexamethasone sodium phosphate 1.5 
mg. 
6.2.3 PRODUCT STORAGE AND STABILITY 
All drugs to be administered to patients enrolled in this trial as per study protocol are to be stored 
at room temperature not higher than 25o C, not frozen and not refrigerated. 
6.2.4 PREPARATION 
Study drugs are ready for use and do not require preparation. 
 
 


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6.3 MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
Patients will be randomized to one of the three treatment groups described in the sections above 
in a 1:1:1 ratio. Randomization is stratified by centre. 
The randomization numbers will be generated using procedures that ensure that treatment 
assignment is unbiased. A patient randomization list will be produced using a validated system 
that automates the random assignment of patient numbers to randomization numbers. 
Prior to surgery, patients who fulfil all inclusion criteria and none of the exclusion criteria will be 
randomized via an Interactive Web Response System (IWRS) that will assign the patient a 
randomization number linked to a treatment arm.  
Vials containing frozen humour aqueous samples will be labelled and shipped to a centralized 
laboratory for analysis. Vial labels will contain the kit numbers but no information regarding the 
drug received. Laboratory analysts will thus be blinded to the drug administered and samples will 
be analysed for the concentration of all three molecules (levofloxacin, dexamethasone 21-
phosphate and dexamethasone. Quality control samples blinded to the analyst will be included. To 
that end, the randomization list will include kit numbers corresponding to quality control samples. 
Twelve samples (4 of each active principle) will be prepared with synthetic aqueous humour and 
a known concentration of the active ingredients in pre-labelled vials at the central laboratory by a 
different analyst in presence of OPIS personnel.  
6.4 STUDY INTERVENTION COMPLIANCE 
Study personnel will be responsible for respecting administration times and recording them in a 
Dose and Sampling Log. Compliance will not be evaluated as patients will receive two doses of 
treatment within the first two hours prior to cataract surgery. 
6.5 CONCOMITANT MEDICATIONS 
Medications to be reported in the electronic Case Report Form (eCRF) are concomitant 
prescription medications, over-the-counter medications and supplements. 
Treatment with any topical ocular drug other than study drugs before start of cataract surgery the 
day of surgery is prohibited [a topical anaesthetic (oxybuprocaine hydrochloride - Novesina®) 
followed by povidone-iodine (Oftasteril®) instillation is allowed within 10 minutes before start of 
surgery). Treatment with any topical or systemic steroid or antibiotic drug in the 7 days prior to 
cataract surgery is also not permitted (artificial tears without BAK are allowed). 
6.5.1 RESCUE THERAPY 
Not applicable. 
 


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7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 DISCONTINUATION OF STUDY INTERVENTION 
If a patient receives only one dose of the study drug (but not the second), this does not mean 
discontinuation from the study, however aqueous humour will not be extracted from patients if the 
treatment schedule is not respected. These patients will be followed for safety purposes only. If a 
clinically significant finding is identified after enrolment, the investigator or qualified designee 
will determine if any change in participant management is needed. Any new clinically relevant 
finding leading to discontinuation from study treatment will be reported as an adverse event (AE). 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. An 
investigator may discontinue or withdraw a participant from the study for the following reasons: 
· Pregnancy. 
· If any clinical adverse event (AE) or other medical condition or situation occurs such that 
continued participation in the study would not be in the best interest of the participant. 
· If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation. 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
eCRF. Subjects who sign the informed consent form and are randomized but do not complete the 
study will not be replaced.  
7.3 LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if he or she does not complete study procedures.  
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 ASSESSMENTS  
At screening, which must take place not more than 28 days before surgery, information will be 
collected concerning demographics, medical history (a diagnosis should be provided and not 
symptoms) and the use of concomitant medications. Pachymetry will be performed to measure 
corneal thickness and a fluorescein test will be performed to confirm corneal epithelium integrity, 
defined as Oxford scheme grade 0. 
Prior to surgery, a pre-dose visit will be performed to confirm eligibility criteria are met. Women 
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administered prior to surgery by qualified health care personnel using a micropipette and 
dispenser. Two doses of one of the study treatments will be administered, the first 90 ± 15 minutes 
prior to surgery, and the second 60 ± 15 minutes before surgery. The two doses must be 
administered 30 minutes apart. Eye drops are to be administered at the lateral canthus while 
applying pressure at the medial canthus to prevent drainage of the drug. 
Immediately before cataract surgery, a limbal paracentesis will be performed and an aqueous 
humour sample of approximately 0.05 ml will be drawn from the anterior chamber with a 30-gauge 
needle insulin syringe. The aqueous humour will be stored immediately in vials at -80 °C. 
Treatment with any topical drug (including mydriatic eye drops) other than study drugs within 12 
hours before the start of cataract surgery is prohibited to prevent variations of the penetration of 
the study drugs. Oxybuprocaine hydrochloride (Novesina®) followed by povidone-iodine 
(Oftasteril®) instillation are allowed within 10 minutes before the start of surgery. Mydrane® is a 
combined solution (phenylephrine [0.31%], tropicamide [0.02%] and lidocaine [1%]) for 
intracameral injection indicated for cataract surgery to obtain mydriasis and intraocular anaesthesia 
during the surgical procedure. Mydrane® is to be administered following paracentesis to avoid 
potential interference with the penetration of study drugs. Mydrane® will be provided free of 
charge by the Sponsor. 
8.2 LABORATORY ANALYSES 
The concentration of levofloxacin, dexamethasone 21-phosphate and dexamethasone will be 
measured by LC tandem mass spectrometry at the centralised laboratory Ticinumlab (Novara, 
Italy). Ticinumlab is a laboratory working according to Good Laboratory Practice (GLP) and 
qualified by the Health Authority for the analysis of biological samples during human 
pharmacokinetic studies. The laboratory will develop and validate a specific analytical procedure 
via LC tandem mass spectrometry by triple quadrupole. The method is able to detect the three 
analytes at once, namely levofloxacin, dexamethasone and dexamethasone 21-phosphate. This 
method will allow a minimum quantity of aqueous humour (i.e. 50 µl) to be withdrawn from each 
patient. The vials containing frozen aqueous humour will contain no information regarding the 
drug received. Quality control samples blinded to the analyst will be included following advice 
from BfArM. To that end, the randomization list will include kit numbers corresponding to quality 
control samples. Twelve samples will be prepared with synthetic aqueous humour and a known 
concentration of the active ingredients in pre-labelled vials at the central laboratory by a different 
analyst in presence of OPIS personnel. The twelve quality control samples correspond to 10% of 
the true biological samples obtained from the patients. 
The laboratory analyst will thus be blinded to the drug administered and samples will be analysed 
for the concentration of all three molecules (levofloxacin, dexamethasone 21-phosphate and 
dexamethasone). All the analyses will be done in a single run and will not be duplicated. 


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Further details are provided in the laboratory protocol (Appendix 1). 
8.3 SAFETY 
The assessment of safety consists in evaluating all adverse events. 
8.4 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.4.1 DEFINITION OF ADVERSE EVENTS (AE) 
An adverse event can be defined as any untoward medical occurrence associated with the use of 
an intervention in humans after providing written informed consent for participation in the study 
until the end of study visit, whether considered intervention-related or not.  
8.4.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes: death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions, or a congenital anomaly/birth defect. Important medical events that may not result in 
death, be life-threatening, or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the participant and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition.  
8.4.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.4.3.1 SEVERITY OF EVENT 
The following guidelines will be used to describe severity.  
· Mild – Events require minimal or no treatment and do not interfere with the participant’s 
daily activities.  
· Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
· Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating. Of note, the term “severe” does not necessarily equate to “serious”. 
8.4.3.2 RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) must have their relationship to study intervention assessed by the 
clinician who examines and evaluates the participant based on temporal relationship and his/her 


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clinical judgment. Investigators are to judge the causal relationship of the event with the study 
intervention as “suspected”, “unsuspected” or “unknown”. 
8.4.3.3 EXPECTEDNESS  
The Sponsor will be responsible for determining whether an adverse event (AE) is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency of the event 
is not consistent with the risk information previously described for the study intervention. 
8.4.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND 
FOLLOW-UP 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention 
of study personnel during study visits and interviews of a study participant presenting for medical 
care, or upon review by a study monitor. 
All AEs including local and systemic reactions will be captured on the appropriate case report 
form (eCRF). Information to be collected includes event description (a diagnosis and not 
symptoms should be provided, if possible), time of onset, clinician’s assessment of severity, 
relationship to study product (assessed only by those with the training and authority to make a 
diagnosis), and time of resolution/stabilization of the event. All AEs occurring while on study must 
be documented appropriately regardless of relationship. All AEs will be followed to adequate 
resolution. 
Any medical condition that is present at the time that the participant is screened will be considered 
as baseline and not reported as an AE. However, if the study participant’s condition deteriorates 
at any time during the study, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the duration of the 
event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
The investigator will record all reportable events with onset dates occurring any time after 
informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day 
of study participation. Events will be followed for outcome information until resolution or 
stabilization. 
8.4.5 NON-SERIOUS ADVERSE EVENT REPORTING  
All identified non-serious AEs (related and unrelated) must be recorded and described on the 
eCRF. 
 
 


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8.4.6 SERIOUS ADVERSE EVENT REPORTING  
Every SAE, regardless of suspected causality, occurring after the subject has provided informed 
consent and until 30 days after the last day of study participation must be reported to the sponsor 
within 24 hours of site awareness. 
Any SAE experienced after this 30-day period should only be reported to the sponsor if the 
investigator suspects a causal relationship to the study treatment. Recurrent episodes, 
complications, or progression of the initial SAE must be reported as follow-up to the original 
episode within 24 hours of the investigator receiving the follow-up information. 
Information about all SAEs will be recorded on the eCRF. In case of technical difficulties, SAE 
notification can be carried out by contacting the CRO (OPIS) in charge of Pharmacovigilance, via 
email at all_phv@opis.it or by fax using the following number: +39 0362 633622. 
All SAEs will be followed until satisfactory resolution or until the site investigator deems the event 
to be chronic or the adherence to be stable. Other supporting documentation of the event may be 
requested by the study sponsor and should be provided as soon as possible. The study sponsor will 
be responsible for notifying Health Authorities of any unexpected fatal or life-threatening 
suspected adverse reaction as soon as possible, but in no case later than 7 calendar days after the 
sponsor's initial receipt of the information. 
Suspected Unexpected Serious Adverse Reactions (SUSARs) will be collected and reported to the 
competent authorities and relevant Ethics Committees in accordance with Directive 2001/20/EC 
or as per national regulatory requirements. 
8.4.7 REPORTING EVENTS TO PARTICIPANTS  
Should an event occur that changes the overall benefit/risk ratio of the study, the Sponsor shall 
evaluate if a risk minimization measure is needed. Should this measure require a substantial 
amendment to the protocol, the informed consent and patient information will be revised and 
submitted to the patient for written consent. 
8.4.8 EVENTS OF SPECIAL INTEREST  
Not applicable. 
 
 


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8.4.9 REPORTING OF PREGNANCY  
Pregnant women will not be permitted to participate in this study. A negative pregnancy test will 
be required to enter the study and, given the very short duration of the study, the occurrence of a 
pregnancy is highly unlikely. Nevertheless, the investigator shall report all pregnancy exposure 
occurring in a female patient within 24 hours to the sponsor using the Pregnancy Reporting Form 
of the eCRF. In case of technical difficulties, pregnancy notification can be carried out by 
contacting the Pharmacovigilance Officer via email at all_phv@opis.it or by fax using the 
following number: +39 0362 633622. The investigator should counsel the subject; discuss the risks 
of continuing with the pregnancy and the possible effects on the foetus potentially induced by 
participation in the study. Monitoring of the subject should continue until conclusion of the 
pregnancy. 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 STATISTICAL HYPOTHESES 
No formal statistical hypothesis has been formulated. All analyses will be descriptive in nature and 
no inferential statistical analyses are planned. 
9.2 SAMPLE SIZE DETERMINATION 
The study plans to enrol 120 patients: 40 will be assigned to the dexamethasone group, 40 to the 
levofloxacin group and 40 to the dexamethasone-levofloxacin combination. 
The sample size has been estimated based on the expected precision (i.e. width of the 95% 
confidence interval) of the estimates of the study drug concentrations.  
For assessment of dexamethasone concentration: 
When the sample size is 40, a two-sided 95% confidence interval for a single mean will extend 
4.648 ng/ml from the observed mean, assuming that the standard deviation is known to be 15 ng/ml 
25 and the confidence interval is based on the large sample z statistic. 
For assessment of levofloxacin concentration: 
When the sample size is 40, a two-sided 95% confidence interval for a single mean will extend 
61.676 ng/ml from the observed mean, assuming that the standard deviation is known to be 
199.022 ng/ml26 and the confidence interval is based on the large sample z statistic.  
 
 


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9.3 POPULATIONS FOR ANALYSES 
The descriptive analysis will be carried out on the following populations: 
- Per Protocol (PP): all patients who have completed the study without any major protocol 
deviations.  
- Full analysis Set (FAS): all randomized patients. According to Intention To Treat (ITT) 
principles all patients will be analyzed considering the randomized study treatment assigned by 
IWRS. 
The safety analysis will be performed on the Safety Population, made up of all patients who have 
received at least one dose of study treatment. 
9.4 STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
Continuous data will be summarized with standard descriptive statistics (i.e. the mean, standard 
deviation, minimum, median and maximum, 95% confidence limits). Categorical data will be 
summarized by frequencies and percentages. 
Missing values will not be replaced. 
All statistical tables, listings and analyses will be produced using SAS® release 9.4 or later (SAS 
Institute, Inc., Cary, NC, USA). 
9.4.2 ANALYSIS OF THE ENDPOINT 
Aqueous humour concentration of levofloxacin and dexamethasone, measured by LC-MS-MS, 
will be descriptively summarized by treatment group and 95% confidence limits will be provided.  
9.4.3 SAFETY ANALYSES 
The incidence of Adverse Events (AEs) and Serious Adverse Events (SAEs) recorded throughout 
the study will be presented overall and by treatment group respectively. 
Depending on the onset date of the event, AEs will be defined as follows: 
· Treatment-emergent AEs, those events with an onset date after any treatment initiation. 
· Non-treatment-emergent AEs, those events with an onset date between informed consent 
and any treatment initiation. 
Non-treatment-emergent AEs will be listed only. 


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Treatment-emergent AEs will be summarized using the Medical Dictionary for Regulatory 
Activities (MedDRA) System Organ Class (SOC) and Preferred Term (PT). A summary of 
treatment-emergent AEs by SOC, PT and severity will be also provided. All related treatment-
emergent AEs, treatment-emergent SAEs, treatment-emergent AEs with an outcome of death, 
treatment-emergent AEs leading to discontinuation of treatment will be summarized by MedDRA 
SOC and PT and will also be listed. 
9.4.4 BASELINE DESCRIPTIVE STATISTICS 
Medical history will be described according to SOC and PT using MedDRA and will be presented 
by treatment group. 
Previous/concomitant medications will be described according to ATC 2 level and PT using the 
World Health Organization Drug Dictionary (WHO-DD) and will be presented by treatment group. 
9.4.5 PLANNED INTERIM ANALYSES  
Not applicable 
9.4.6 SUB-GROUP ANALYSES 
No sub-group analysis is planned. 
9.4.7 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
Patient listings will be provided. 
9.4.8 EXPLORATORY ANALYSES 
No exploratory analysis is planned. 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS 
PROVIDED TO PARTICIPANTS 
Consent forms describing in detail the study intervention, study procedures, benefits and risks are 
given to the participant and written documentation of informed consent is required prior to 
undertaking any study-related procedures.  
 
 


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10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be 
approved by the IEC and the participant will be asked to read and review the document. The 
investigator will explain the research study to the participant and answer any questions that may 
arise. A verbal explanation will be provided in terms suited to the participant’s comprehension of 
the purposes, procedures, and potential risks of the study and of their rights as research participants. 
Participants will have the opportunity to carefully review the written consent form and ask 
questions prior to signing. The participants should have the opportunity to discuss the study with 
their family or surrogates and think about it prior to agreeing to participate. The participant will 
sign the informed consent document prior to any procedures being done specifically for the study. 
Participants must be informed that participation is voluntary and that they may withdraw from the 
study at any time, without prejudice. A copy of the informed consent document will be given to 
the participants for their records. The informed consent process will be conducted and documented 
in the source document (including the date), and the form signed, before the participant undergoes 
any study-specific procedures. The rights and welfare of the participants will be protected by 
emphasizing to them that the quality of their medical care will not be adversely affected if they 
decline to participate in this study. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, 
investigator, IEC, sponsor and regulatory authorities. If the study is prematurely terminated or 
suspended, the Principal Investigator will promptly inform study participants, the IEC, and sponsor 
and will provide the reason(s) for the termination or suspension. Study participants will be 
contacted, as applicable, and be informed of changes to the study visit schedule. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
· Determination of unexpected, significant, or unacceptable risk to participants 
· Insufficient compliance to protocol requirements 
· Data that are not sufficiently complete and/or evaluable 
The study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IEC and/or regulatory authorities. 
 
 


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10.1.3 CONFIDENTIALITY AND PRIVACY  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover 
testing of biological samples in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the IEC, 
regulatory agencies or pharmaceutical company supplying study product may inspect all 
documents and records required to be maintained by the investigator, including but not limited to, 
medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. 
The clinical study site will permit access to such records. 
The study participant’s contact information will be securely stored at each clinical site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by the reviewing IEC, Institutional policies, or sponsor 
requirements. 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, 
will be transmitted to and stored at the CRO (OPIS) working on behalf of the Sponsor. This will 
not include the participant’s contact or identifying information. Rather, individual participants and 
their research data will be identified by a unique study identification number. Only the study centre 
will be able to link the study ID number to the patient’s identity. The study data entry and study 
management systems used by clinical sites and by OPIS research staff will be secured and 
password protected.  
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
Not applicable, samples of aqueous humour will be destroyed after the concentration analyses. 
10.1.5 SAFETY OVERSIGHT 
Given the nature of the study, oversight of safety by a Data and Safety Monitoring Board was not 
deemed necessary. 
10.1.6 CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants 
are protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
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Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s).  
The field monitor will visit the site to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrolment, and to 
ensure that study treatment is being stored, dispensed, and accounted for according to 
specifications. Key study personnel must be available to assist the field monitor during these visits.  
The investigator must maintain source documents for each patient in the study, consisting of case 
and visit notes (hospital or clinic medical records) containing demographic and medical 
information, and Dose and Sampling Logs. All information on eCRFs must be traceable to these 
source documents in the patient's file. The investigator must also keep the original informed 
consent form signed by the patient (a signed copy is given to the patient). 
Monitoring standards require full verification for the presence of informed consent, adherence to 
the inclusion/exclusion criteria, documentation of SAEs, and of data that will be used for all 
primary variables. Additional checks of the consistency of the source data with the eCRFs are 
performed according to the study-specific Monitoring Plan (MP). No information in source 
documents about the identity of the patients/subjects will be disclosed. 
10.1.7 QUALITY ASSURANCE AND QUALITY CONTROL 
Following written Standard Operating Procedures (SOPs), monitors will verify that the clinical 
trial is conducted and data are generated, documented (recorded), and reported in compliance with 
the protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), GLP 
and applicable regulatory requirements. 
The investigational site and central laboratory will provide direct access to all trial-related 
facilities, source data/documents, and reports for the purpose of monitoring and auditing by the 
sponsor, and inspection by local and regulatory authorities. 
Independent audits may be conducted to ensure monitoring practices are performed consistently 
across all participating sites and that monitors are following the MP. Independent audits may be 
conducted by the Sponsor to ensure monitoring practices are performed consistently across all 
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10.1.8 DATA HANDLING AND RECORD KEEPING  
 
10.1.8.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data collection 
Designated investigator staff will enter the data required by the protocol into the eCRF using fully 
validated software that conforms to 21 CFR Part 11 requirements. Designated investigator site 
staff will not be given access to the electronic data capture system until they are trained.  
Web-based software will be used and no installation procedure is needed. Each site will be 
authorized by the administrator to access the eCRF. Each site-qualified personnel will be allowed 
to access the eCRF by means of a ‘login mask’ requiring user ID and password and may read, 
modify, and update only the information previously reported at his or her site and according to 
their profile. Each page reports site code and subject code. 
On-line validation programs will check for data discrepancies and, by generating appropriate error 
messages, allow the data to be confirmed or corrected before transfer to the CRO OPIS working 
on behalf of the sponsor. The investigator will certify that the data entered into the eCRF are 
complete and accurate.  
Data generated by the central laboratory will automatically be transmitted to the study database 
according to a predefined transmission protocol. 
After database lock, the investigator will receive a CD-ROM of subject data for archiving at the 
investigational site. 
Database management and quality control 
The CRO (OPIS) working on behalf of the Sponsor will review the data entered in the eCRF by 
investigational staff for completeness and accuracy and instruct site personnel to make any 
necessary corrections or additions. The Data Manager will perform the cleaning session by 
reviewing the warning messages raised by on-line checks and by running post-entry checks by 
means of validation programs and data listings specific for the study. If clarifications are needed, 
the Data Manager will raise queries by means of data query forms through the web application. 
Designated investigator site staff will be required to respond to queries and the Data Manager will 
make the correction to the database according to the responses. 
Data collection and query flows, as well as the on-line and off-line checks, are detailed in the Data 
Management Plan and Data Validation documents. 
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classification system. Medical history/current medical conditions and AEs will be coded using the 
MedDRA. 
Randomization codes are tracked using the eCRF. The system is supplied by a CRO, OPIS s.r.l., 
who also manages the database. 
The occurrence of any protocol deviations will be checked and the database will be locked and 
made available for data analysis after these actions have been completed and the database has been 
declared complete and accurate. 
10.1.8.2 STUDY RECORDS RETENTION  
The investigator/institution/central laboratory should maintain the trial documents as specified in 
Essential Documents for the Conduct of a Clinical Trial (ICH E6 Section 8) and as required by 
applicable regulations and/or guidelines. The investigator/institution/central laboratory should 
take measures to prevent accidental or premature destruction of these documents.  
Essential documents (written and electronic) should be retained for a period of not less than 
twenty-five (25) years from the completion of the study unless the sponsor provides written 
permission to dispose of them or, requires their retention for an additional period because of 
applicable laws, regulations and/or guidelines. The subjects’ medical files will be archived in 
accordance with the national laws. 
10.1.9 PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, International 
Conference on Harmonisation Good Clinical Practice (ICH GCP). The noncompliance may be 
either on the part of the participant, the investigator, or the study site staff. As a result of deviations, 
corrective actions are to be developed by the site and implemented promptly.  
These practices are consistent with ICH GCP:  
· 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
· 5.1 Quality Assurance and Quality Control, section 5.1.1  
· 5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
It is the responsibility of the site to avoid any protocol deviations and of the site staff and monitors 
to use continuous vigilance to identify and report deviations. All deviations must be addressed in 
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10.1.10 
INSURANCE 
The Sponsor certifies that it has taken out a liability insurance policy covering this clinical trial. 
This insurance policy is in accordance with local laws and requirements. The insurance of the 
Sponsor does not relieve the Investigator and the collaborators from any obligation to maintain 
their own liability insurance policy. An insurance certificate will be provided to the IEC and/or 
regulatory authorities. 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
All data and results and all intellectual property rights in the data and results derived from the 
study will be the property of the Sponsor, who may utilize them in various ways, such as for 
submission to government regulatory authorities or disclosure to other investigators. 
This study will ensure that the public has access to the published results of the research.  
As the study involves more than one center, the first publication must be related to data 
collected from all patients enrolled and analyzed under the Sponsor’s responsibility. The 
investigator shall not publish or communicate data collected in only one center or part of the 
centers before the publication of the complete results of the study, unless prior written 
authorization from the Sponsor has been provided. 
Any publication and/or communication project regarding the study and/or its results, whether 
obtained during the study or after the study end, shall be submitted to the Sponsor at least 30 
days for a publication and 15 days for an abstract before the planned date of communication 
and/or submission for a publication. The Sponsor shall make comments on the project within 
15 days of receipt of the project for a publication and within 7 days for an abstract. The 
investigator who submitted the project shall take the Sponsor's comments into due consideration. 
Nevertheless, should the investigator who submitted the project decide not to modify the project 
according to the Sponsor's comments, he/she shall provide the Sponsor with the grounds for 
his/her decision in writing. 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a clinical trials registration policy as a condition for publication. The ICMJE defines a clinical trial 
as any research project that prospectively assigns human subjects to intervention or concurrent 
comparison or control groups to study the cause-and-effect relationship between a medical 
intervention and a health outcome. Medical interventions include drugs, surgical procedures, 
devices, behavioural treatments, process-of-care changes, and the like. Health outcomes include 
any biomedical or health-related measures obtained in patients or subjects, including 
pharmacokinetic measures and adverse events. The ICMJE policy requires that all clinical trials 
be registered in a public trials registry such as ClinicalTrials.gov, which is sponsored by the 
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10.1.12 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the design and conduct 
of this trial. The study leadership has established policies and procedures for all study group 
members to disclose all conflicts of interest and will establish a mechanism for the management 
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10.2 ABBREVIATIONS 
 
AE 
Adverse Event 
AH 
Aqueous Humour 
ATC 
Anatomic Therapeutic Chemical 
CNS 
Coagulase-Negative Staphylococci 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
ESCRS 
European Society of Cataract and Refractive Surgeons 
eCRF 
electronic Case Report Forms 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
HPLC -MS 
High Performance Liquid Chromatography – Mass Spectrometry 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IEC 
Independent Ethics Committee 
IOP 
Intraocular Pressure 
IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
MP 
Monitoring Plan 
PT 
Preferred Term 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SoA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
WHO-DD 
World Health Organization Drug Dictionary 
 
 
 


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11 REFERENCES  
1. West et at., Epidemiology of risk factors for age-related cataracts. Surv Ophthalmol 
1995;39:323–334. 
2. Kelman CD et al., Phaco-emulsification and aspiration. A new technique of cataract removal. 
A preliminary report. Am J Ophthalmol 1967;64:23–35. 
3. Li XM et., Investigation of dry eye disease and analysis of the pathogenic factors in patients 
after cataract surgery. Cornea 2007;26:S16–S20. 
4. Rosado-Adames et al., The changing fate of the corneal endothelium in cataract surgery. Curr 
Opin Ophthalmol 2012;23:3–6. 
5. Kasetsuwan N et al., Incidence and pattern of dry eye after cataract surgery. PLoS ONE 
2013;8:e78657. 
6. Dell SJ et al., Randomized comparison of postoperative use of hydrogel ocular bandage and 
collagen corneal shield for wound protection and patient tolerability after cataract surgery. J 
Cataract Refract Surg 2011;37:113–121. 
7. Gonzales JA, Gritz DC, Channa R, et al. (2013): Nonsteroidal anti-inflammatory drugs versus 
corticosteroids for controlling inflammation after uncomplicated cataract surgery. Cochrane 
Database of Syst Rev (5): CD010516. doi: 10.1002/14651858.CD010 516. 
8. Ong-Tone L, Bell A & Tan YY. Practice patterns of Canadian Ophthalmological Society 
members in cataract surgery: 2011 survey. Can J Ophthalmol 2012;47:124– 130. 
9. European Society of Cataract Refractive Surgery (2012): ESCRS Survey 2012 Available at: 
http://www.analeyz.com. 
10. Behndig A, Cochener B, Guell JL et al. Endophthalmitis prophylaxis in cataract surgery: 
overview of current practice patterns in 9 European countries. J Cataract Refract Surg. 
2013;39:1421–1431.  
11. Gower EW, Lindsley K, Nanji AA et al. (2013): Perioperative antibiotics for prevention of 
acute endophthalmitis after cataract surgery. Cochrane Database of Syst Rev (7): CD006364. 
doi:10.1002/ 14651858.CD006364.pub2.  
12. Barry P, Gardner S, Seal D, Gettinby G, Lees F, Peterson M & Revie C; ESCRS Endoph-
thalmitis Study Group (2009): Clinical observations associated with proven and unproven 
cases in the ESCRS study of prophylaxis of post-operative endophthalmitis after cataract 
surgery. 
13. Ta CN, Chang RT, Singh K et al. Antibiotic resistance patterns of ocular bacterial flora: a 
prospective study of patients undergoing anterior segment surgery; Ophthalmology. 2003;110: 
1946-1951. 
14. Barry P et Al. ESCRS Guidelines for prevention and treatment of endophthalmitis following 
cataract surgery: data, dilemmas and conclusions 2013. Available at: www. escrs.org. 
15. Porela-Tiihonen S. Recovery after cataract surgery Acta Ophthalmol. 2016: 94, thesis 2: 1–34. 
16. Cagini C, Cometa F, Torroni G et al. Dexamethasone Disodium Phosphate Penetration Into 
the Human Aqueous Humor After Topical Application. Curr Eye Res. 2016;41(7):897-9. 


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17. Bucci FA. An in vivo study comparing the ocular absorption of levofloxacin and ciprofloxacin 
prior to phacoemulsification; Am J Ophthalmol. 2004;137:308-312. 
18. Healy DP, Holland EJ, Nordlund ML et al. Concentrations of levofloxacin, ofloxacin, and 
ciprofloxacin in human corneal stromal tissue and aqueous humor alter topical administration; 
Cornea. 2004;23:255-263. 
19. Puustjarvi T, Terasvirta M, Nurmenniemi P et al. Penetration of topically applied levofloxacin 
0.5% and ofloxacin 0.3% into the vitreous of the non-inflamed human eye; Graefes Arch Clin 
Exp Ophthalmol 2006;244:1633-1637. 
20. Yamada M, Mochizuki H, Yamada K et al. Aqueous humor levels of topically applied 
levofloxacin in human eyes; Curr Eye Res. 2002;24;403-406. 
21. Rosenblum C, Dengler RE Jr, Geoffroy RF. Ocular absorption of dexamethasone phosphate 
disodium by the rabbit. Arch Ophthalmol 1967;77(2):234-7. 
22. McDonald TO, Kasten K, Hervey R, Gregg S, Smith D, Robb CA. Comparative toxicity of 
dexamethasone and its tertiary butyl acetate ester after topical ocular instillation in rabbits. Am 
J Ophthalmol. 1973 Jul;76(1):117-25. 
23. Weijtens O, Schoemaker RC, Romijn FP, Cohen AF, Lentjes EG, van Meurs JC. Intraocular 
penetration and systemic absorption after topical application of dexamethasone disodium 
phosphate. Ophthalmology. 2002;109(10):1887-91. 
24. DeCroos FC & Afshari NA: Perioperative antibiotics and anti-inflammatory agents in cataract 
surgery. Curr Opin Ophthalmol. 2008;19:22–26. 
25. O. Weijtens, Schoemaker RC, Fred P et al. Intraocular penetration and systemic absorption 
after 
topical 
application 
of 
dexamethasone 
disodium 
phosphate 
Ophthalmology 
2002;109:1887-1891. 
26. K. Sundelin Seal D, Gardner S et al. Increased anterior chamber penetration of topical 
levofloxacin 0.5% after pulse dosing in cataract patients. Acta Ophthalmol. 2009;87:160-165. 
 
 
 


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12 APPENDICES 
12.1 
Laboratory Protocol 
12.2 
Oxford Grading Scheme 
 
 


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Appendix 12.2 
 
1 
12.2 OXFORD GRADING SCHEME 
Staining is represented by punctate dots on a series of panels (A-E). Staining ranges from 0-5 for 
each panel and 0-15 for the total exposed inter-palpebral conjunctiva and cornea. The dots are 
ordered on a log scale. 
 
Conduct of Test: 
· Dye is instilled. 
· Slit-lamp is set (e.g.16 magnification with x10 oculars with Haag-Streit). 
· Cornea: The upper eyelid is lifted slightly to grade the whole corneal surface. 
· Conjunctiva: To grade the temporal zone, the subject looks nasally; to grade the nasal zone the 
subject looks temporally. 
· (The upper and lower conjunctiva can also be graded). 
 
 


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2 
Fluorescein sodium 
With fluorescein, staining must be graded as quickly as possible after instillation, since the dye 
then diffuses rapidly into the tissue and its high luminosity blurring the stain margin. 
1. Quantified drop instillation 
E.g. 2 ml of 2 % sterile fluorescein instilled into each conjunctival sac with a micro-pipette (using 
a sterile tip). In very dry eye, larger volumes risk the possibility of inadequate dilution into the 
fluorescent range. 
2. Unquantified instillation – impregnated paper strips 
This is a convenient approach in the clinic using the following method of application: 
· A single drop of unit dose saline is instilled onto a fluorescein-impregnated strip. 
· When the drop has saturated the impregnated tip, the excess is shaken into a waste bin with 
a sharp flick. 
· The right lower lid is then pulled down and the strip is tapped onto the lower tarsal 
conjunctiva. A similar procedure is carried out on the left. 
If too large a volume is delivered then the concentration in the tear film will be too high, and the 
tear film and staining pattern will be non-fluorescent. 
3. Timing 
The fluorescein break-up time (FBUT) is usually performed prior to grading. Since fluorescein 
diffuses rapidly into tissues, punctate staining blurs after a short period. It is therefore essential to 
assess staining rapidly, in sequence, in the right and then the left eye, so that the staining patterns 
observed are equally crisp. 
If it is intended to photograph the staining pattern for grading, then photography should follow 
immediately after each instillation. 
Exciter and Barrier Filters 
The absorption peak of fluorescein sodium occurs between 465 - 490 nm and the emission peak 
between 520 - 530 nm. A suggested filter pair for detection of fluorescein staining is a yellow, 
Kodak Wratten 12 barrier filter (transmitting above 495 nm) or an orange Wratten 15 filter 
(transmitting above 510 nm) in combination with a blue Wratten 47 or 47A exciter filter. The 47A 
shows greater transmittance than the Wratten 47 over the absorption range. The ‘cobalt’ filter of 
many slit-lamps is suitable to use with a Wratten 12 or 15 barrier. 
Where more light is required for photographic purposes, narrow band-pass, interference filters can 
be used. 


Sponsor: NTC S.r.l. 
Version 1.0 
Protocol No.: LevoDesa_05-2017 
21 March 2018 
Appendix 12.2 
 
3 
The use of both exciter and barrier filters allows both the cornea and conjunctiva to be assessed 
using a single stain. This is a major advantage in clinical trials where it is otherwise customary to 
employ fluorescein to grade corneal staining and rose bengal or lissamine green to grade 
conjunctival staining. 
References: Bron A, Evans VE, Smith JA. (2003). Grading of corneal and conjunctival staining 
in the context of other dry eye tests. Cornea 22(7): 640-50.