Clinical trial NCT03274895
Conditions: Acanthamoeba Keratitis
Keywords: polihexanide propamidine confocal microscopy
Sponsor: SIFI SpA
Start date: 2017-08-13; Completion date: 2022-03-30
Enrollment: 135 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Prospective randomized study to evaluate the efficacy, safety and tolerability of 0.08% polihexanide (PHMB) eye drops in patients affected by acanthamoeba keratitis.

130 subjects were assigned to one of the following 2 treatment groups: Group 1: 0.08% polihexanide (PHMB) + placebo Group 2: 0.02% polihexanide (PHMB) + 0.1% propamidine

Detailed description:
This was a randomized, assessor-masked, active-controlled, multiple center, parallel-group phase 3 study to evaluate the efficacy, safety and tolerability of 0.08% polihexanide (PHMB) ophthalmic solution compared to the conventional 0.02% polihexanide (PHMB) + 0.1% propamidine in patients affected by acanthamoeba keratitis.

The study was designed as a superiority study with the possibility to test for non-inferiority if the superiority hypothesis was not met (CPMP/EWP/482/99).The study consisted of an eligibility screening visit, a treatment period including monthly follow-up visits until a clinical resolution was obtained (within a maximum of 1 year), followed by 2 post-treatment off-therapy visits (30 and 90 days after treatment discontinuation). 130 subjects with confirmed diagnosis of acanthamoeba keratitis (clinical signs plus positive confocal microscopy findings) were assigned to one of the following treatment groups in a ratio of 1:1.

Group 1: 0.08% PHMB + placebo Group 2: 0.02% PHMB + 0.1% propamidine

Arms: arm 1: polihexanide (PHMB) 0.08% and placebo were administered in the affected eye until clinical resolution for a maximum of 12 months arm 2: polihexanide (PHMB) 0.02% and propamidine 0.1% were administered in the affected eye until clinical resolution for a maximum of 12 months
Interventions: intervention 1: PHMB 0.08% intervention 2: Propamidine 0.1% intervention 3: placebo intervention 4: PHMB 0.02%
Intervention descriptions: intervention 1: 16 drops in a day in the affected eye for 5 days followed by 8 drops in a day for 7 days,6 drops in a day for 7 days and 4 drops in a day up to clinical resolution intervention 2: 16 drops in a day in the affected eye for 5 days followed by 8 drops in a day for 7 days,6 drops in a day for 7 days and 4 drops in a day up to clinical resolution intervention 3: 16 drops in a day in the affected eye for 5 days followed by 8 drops in a day for 7 days,6 drops in a day for 7 days and 4 drops in a day up to clinical resolution intervention 4: 16 drops in a day in the affected eye for 5 days followed by 8 drops in a day for 7 days,6 drops in a day for 7 days and 4 drops in a day up to clinical resolution

Protocol documents:
Prot_000.pdf: 


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INVESTIGATOR STATEMENT 
I have read amendment 1 to the protocol entitled “Randomized, assessor-masked, active-controlled, Phase 
3 study to evaluate efficacy, safety and tolerability of 0.08% polyhexamethylene biguanide (PHMB) 
ophthalmic solution in comparison with 0.02% PHMB + 0.1% Propamidine combination therapy in 
subjects affected by Acanthamoeba keratitis.” and the accompanying Investigator’s Brochure. I agree to 
conduct the clinical investigation in compliance with the Final Protocol, the International Conference on 
Harmonisation (ICH) harmonised tripartite guideline E6(R1): Guideline for Good Clinical Practice, 
applicable regulatory/government regulations, and in accordance with the latest revision of the Ethical 
Principles for Medical Research Involving Human Subjects (the Declaration of Helsinki). I will not 
implement any changes to study procedures or conduct without prior approval from the Sponsor and, when 
applicable, the Independent Ethics Committee and Regulatory Authority. 
I agree to maintain the confidentiality of this study protocol, as described on the title page. Further, I will 
not publish results of the study without authorization from the Sponsor. 
 
 
 
 
 
 
 
 
Signature of Principal Investigator 
 
Date 
 
 
 
 
 
 
Printed Name of Principal Investigator 
 
 
 
 
 
 


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Revision History 
Version 
Description 
Rationale for update 
Date 
001 
Final protocol 
 
2017-01-25 
002 
Protocol amendment 1 
Significant changes: treatment of both eyes, corrections 
of number of visits after relapse. Removal of 0.08% 
PHMB treatment after End of Study. Administrative 
updates and textual clarifications. 
2018-10-08 


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TABLE OF CONTENTS 
INVESTIGATOR STATEMENT ...................................................................................... 2 
TABLE OF CONTENTS .................................................................................................. 4 
LIST OF FIGURES.......................................................................................................... 6 
ABBREVIATIONS........................................................................................................... 7 
STUDY SYNOPSIS ......................................................................................................... 8 
SCHEDULE OF ASSESSMENTS................................................................................. 22 
1. 
INTRODUCTION ................................................................................................... 25 
1.1. 
Background ........................................................................................................ 25 
1.1.1. 
Toxicology........................................................................................................ 25 
1.1.2. 
Clinical Studies ................................................................................................ 26 
1.1.3. 
Propamidine ..................................................................................................... 27 
1.2. 
Overall Rationale for the Study ........................................................................... 27 
2. 
OBJECTIVES AND HYPOTHESIS ....................................................................... 28 
2.1. 
Definitions ........................................................................................................... 28 
2.2. 
Objectives ........................................................................................................... 28 
2.3. 
Hypothesis .......................................................................................................... 28 
3. 
STUDY DESIGN AND RATIONALE ..................................................................... 29 
3.1. 
Overview of Study Design .................................................................................. 29 
3.1.1. 
Type of Study ................................................................................................... 29 
3.1.2. 
Screening Visit ................................................................................................. 30 
3.1.3. 
Treatment Period ............................................................................................. 32 
3.2. 
Study Design Rationale ...................................................................................... 32 
4. 
SUBJECT POPULATION...................................................................................... 33 
4.1. 
Inclusion Criteria ................................................................................................. 33 
4.2. 
Exclusion Criteria ................................................................................................ 35 
4.3. 
Concomitant Medication and Other Restrictions during Study ........................... 36 
4.4. 
Subject Completion/Withdrawal .......................................................................... 37 
5. 
TREATMENTS ...................................................................................................... 38 
5.1. 
Treatment Assignment ....................................................................................... 38 
5.2. 
Drug dosage and Administration ........................................................................ 39 
5.3. 
Treatment and Follow-up Protocol ...................................................................... 41 
5.3.1. 
Responding Disease or Treatment Success .................................................... 44 
5.3.2. 
Poor Response at 3 weeks or Treatment Failure ............................................. 46 
5.3.3. 
Relapse ........................................................................................................... 46 
5.3.4. 
End of Study .................................................................................................... 48 
5.4. 
Identity of Investigational Product ....................................................................... 49 
5.5. 
Selection of Doses in the Study .......................................................................... 50 
5.6. 
Masking .............................................................................................................. 50 


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5.7. 
Drug Accountability ............................................................................................. 51 
6. 
STUDY EVALUATIONS ........................................................................................ 51 
6.1. 
Study Procedures ............................................................................................... 51 
6.1.1. 
Overview .......................................................................................................... 51 
6.1.2. 
Screening Measurements ................................................................................ 51 
6.1.3. 
Measurements for Routine Clinical Management ............................................ 52 
6.1.4. 
Baseline Measurements .................................................................................. 52 
6.1.5. 
Efficacy Measurements ................................................................................... 53 
6.1.5.1. 
Ocular Assessments by Investigator ............................................................. 53 
6.1.5.2. 
Subject-reported outcomes ........................................................................... 54 
6.1.6. 
Safety and Tolerability Measurements ............................................................. 54 
6.2. 
Appropriateness of Measurements ..................................................................... 55 
6.2.1. 
Timing of Assessments .................................................................................... 55 
6.3. 
Primary Efficacy Variables .................................................................................. 55 
6.4. 
Secondary Variables .......................................................................................... 56 
7. 
STATISTICAL METHODS..................................................................................... 57 
7.1. 
Study Analysis Sets ............................................................................................ 57 
7.1.1. 
Analysis Data Sets for the Efficacy Analyses .................................................. 57 
7.1.2. 
Analysis Data Set for the Safety Analysis ........................................................ 57 
7.2. 
Statistical Analytical Plan (SAP) for Efficacy, Safety and Tolerability 
Evaluation ........................................................................................................... 57 
7.3. 
Demographic Variables and Baseline Characteristics ........................................ 57 
7.4. 
Evaluation of Efficacy Parameters ...................................................................... 57 
7.4.1. 
Primary Efficacy Variable ................................................................................. 57 
7.4.1.1. 
Justification for choice of the non-inferiority margin  ................................... 58 
7.4.1.1.1. 
Justification on Statistical grounds ............................................................. 58 
7.4.1.1.2. 
Justification on Clinical grounds ................................................................. 59 
7.4.2. 
Secondary Efficacy Variable ............................................................................ 60 
7.5. 
Evaluation of Safety and Tolerability Parameters ............................................... 60 
7.5.1. 
Adverse events ................................................................................................ 60 
7.5.2. 
Other Safety Parameters ................................................................................. 60 
7.6. 
Sample Size Determination ................................................................................ 60 
7.7. 
Interim Analysis .................................................................................................. 61 
8. 
ADVERSE EVENT REPORTING .......................................................................... 61 
8.1. 
Definitions ........................................................................................................... 61 
8.1.1. 
Adverse Event ................................................................................................. 61 
8.1.2. 
Abnormal test findings ..................................................................................... 61 
8.1.3. 
Pre-existing conditions ..................................................................................... 62 
8.1.4. 
Procedures ...................................................................................................... 62 
8.1.5. 
Serious Adverse Events .................................................................................. 62 
8.1.6. 
Hospitalization ................................................................................................. 62 
8.2. 
Adverse Event Reporting Period ........................................................................ 63 
8.2.1. 
Severity Assessment ....................................................................................... 63 
8.2.2. 
Causality Assessment ..................................................................................... 64 
8.3. 
Serious Adverse Event Reporting ....................................................................... 65 
8.4. 
Exposure during Pregnancy ............................................................................... 66 


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8.5. 
Follow-up of Adverse Events .............................................................................. 66 
8.6. 
Recording ........................................................................................................... 66 
9. 
QUALITY CONTROL AND QUALITY ASSURANCE ........................................... 67 
10. ETHICAL ASPECTS ............................................................................................. 67 
10.1. Independent Ethics Committee/Institutional Review Board ................................ 67 
10.2. Ethical Conduct of the Study .............................................................................. 67 
10.3. Subject Information and Consent ....................................................................... 67 
10.4. Privacy of Personal Data .................................................................................... 68 
11. STUDY ADMINISTRATIVE STRUCTURE ............................................................ 68 
11.1. Documentation ................................................................................................... 68 
11.1.1. Archiving .......................................................................................................... 68 
11.1.2. Case report form .............................................................................................. 68 
11.1.3. Source data ..................................................................................................... 68 
12. CONFIDENTIALITY AND PUBLICATION POLICY .............................................. 68 
REFERENCES .............................................................................................................. 70 
APPENDIX 1  
TYPICAL CLINICAL FINDINGS OF AN ACANTHAMOEBA 
CORNEAL INFECTION ......................................................................................... 72 
APPENDIX 2  
EUROQOL FIVE-DIMENSION SCALE (EQ-5D) 
QUESTIONNAIRE ................................................................................................. 73 
APPENDIX 3  
VISUAL FUNCTIONING QUESTIONNAIRE 25 (VFQ25) .................. 76 
LAST PAGE .................................................................................................................. 86 
 
LIST OF FIGURES 
Figure 1: Schematic Presentation of the Screening Process ........................................ 31 
Figure 2: Schematic Presentation of the Treatment and Follow-up Protocol ................. 42 
 
 


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ABBREVIATIONS 
 
AE
Adverse event
AK 
ANOVA 
ANCOVA
Acanthamoeba keratitis 
Analysis of variance  
Analysis of covariance
BCVA
Best corrected visual acuity
CHMP
Committee for Medicinal Products for Human Use
CRF
Case report form (an electronic version is used for this study, eCRF)
CRR
Clinical Resolution Rate
CRR_12 
Clinical Resolution Rate at 12 months after randomization, defined as the 
percentage of subjects cured at 30 days after discontinuing all study therapies 
within 12 months of randomization
DLE 
DNA
Dose Limiting Event 
Deoxyribonucleic acid
EMA
European Medicines Agency
EQ-5D
EuroQol five-dimension scale
FAS 
FSH
Full Analysis Set 
Follicle stimulating hormone
GCP
Good Clinical Practice
HSV
Herpes simplex virus
IB
Investigator’s Brochure
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IEC
Independent Ethics Committee
IOP
Intraocular pressure
IRB 
IUD 
IUS
Institutional Review Board 
Intrauterine device 
Intrauterine hormone releasing system
LAM 
LAR
Lactational amenorrhoea method 
Legally authorized representative
NOAEL
No Observed Adverse Effect Level
NSAID
Non-steroidal anti-inflammatory drugs
ODAK
Orphan Drug for Acanthamoeba keratitis
PCR
Polymerase chain reaction
PHMB
Polyhexamethylene biguanide
PPAS
Per-Protocol Analysis Set 
QPPV
Qualified person responsible for pharmacovigilance
SAE
Serious adverse event
SAP
Statistical Analysis Plan
SOC
System organ class
VAS
Visual analog scale
VFQ25
Visual functioning questionnaire 25
 
 
 
 


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STUDY SYNOPSIS 
TABLE OF CONTENT SYNOPSIS 
DEFINITIONS ................................................................................................................................................9 
OBJECTIVES ..................................................................................................................................................9 
HYPOTHESIS .................................................................................................................................................9 
OUTCOME MEASURES ............................................................................................................................. 10 
OVERVIEW OF STUDY DESIGN .............................................................................................................. 10 
SUBJECT POPULATION ............................................................................................................................ 11 
SCREENING PROCEDURE ........................................................................................................................ 11 
INCLUSION CRITERIA .............................................................................................................................. 12 
EXCLUSION CRITERIA ........................................................................................................................... 13 
CONCOMITANT AND PREVIOUS MEDICATIONS ............................................................................... 14 
TREATMENT ASSIGNMENT  ................................................................................................................... 14 
DRUG DOSAGE AND ADMINISTRATION .............................................................................................. 14 
SCHEMATIC OVERVIEW OF THE TREATMENT AND FOLLOW-UP PERIOD  
 ....................................................................................................................................................................... 16 
STUDY EVALUATIONS ............................................................................................................................. 19 
SUBJECT COMPLETION/WITHDRAWAL ............................................................................................... 19 
STATISTICAL METHODS .......................................................................................................................... 20 
QUALITY CONTROL AND QUALITY ASSURANCE ............................................................................. 21 
ETHICAL ASPECTS .................................................................................................................................... 21 
 
 
 


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STUDY TITLE 
Randomized, Assessor-Masked, Active-Controlled, Phase 3 Study to Evaluate Efficacy, Safety and Tolerability of 
0.08% Polyhexamethylene Biguanide (PHMB) Ophthalmic Solution in Comparison with 0.02% PHMB + 0.1% 
Propamidine Combination Therapy in Subjects Affected by Acanthamoeba keratitis. 
DEFINITIONS 
Protocol section 2.1 Definitions, page 28 
Clinical resolution is a clinical definition, resulting from a slit lamp examination, with the following findings: 
 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and oedema) that requires 
treatment, with a healed corneal epithelium and minimal punctate staining (10 dots or less equivalent to Grade 
1 on the Oxford Scale).   
 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal hyperemia): mild 
conjunctival inflammation is acceptable if related to other concurrent conditions such as blepharitis. 
 
No limbitis, scleritis or anterior chamber inflammation.  
 
No relapse within 30 days of discontinuing all topical and systemic therapy given for Acanthamoeba keratitis.  
For regulatory and verification reasons, there is an additional 60 day follow-up to exclude late relapses. 
The CRR_12 is the clinical resolution rate at 12 months from randomization and is defined as the percentage of 
subjects cured 30 days after discontinuing all study therapies, within 12 months of randomization. This is the primary 
outcome measure. Subjects whose disease has not resolved at 12 months are considered treatment failures per protocol.  
OBJECTIVES  
Protocol section 2.2 Objectives, page 28 
The primary objective of the study is to compare the Clinical Resolution Rate (CRR) at 12 months from randomization 
(CRR_12) of 0.08% PHMB + placebo with that of 0.02% PHMB + 0.1% propamidine combination therapy, estimating 
the difference in CRR_12 together with the surrounding degree of uncertainty, and to test for therapeutic superiority 
or non-inferiority of 0.08% PHMB monotherapy.  
A further aim of this study is to obtain additional safety information on 0.08% PHMB ophthalmic solution. 
HYPOTHESIS 
Protocol section 2.3 Hypothesis, page 28 
The primary hypothesis to be tested is that the CRR_12 of subjects treated with 0.08% PHMB monotherapy, is 
superior, or worse by no more than an acceptable pre-defined 0.20 non-inferiority margin (Δ), compared to the 
CRR_12 of 0.02% PHMB + 0.1% propamidine combination therapy, administered according to the Treatment and 
Follow-up Protocol presented below, which is based on a consensus of currently clinical guidelines.  
 
Secondary hypotheses are:  
 
That adverse events, and those relating to toxicity in particular, are less with PHMB 0.08% monotherapy 
compared to the comparator  
 
That time to a cure is shorter in subjects receiving PHMB 0.08% monotherapy compared to the comparator  
 
Note: The estimated CRR_12 from the start of treatment is 67% for the conventional (0.02% PHMB + 0.1% 
propamidine) combination therapy (from the sponsor’s observational, case series retrospective Study 038/SI) (63% 
when assuming a prevalence of late stage diseases in 38% of subjects). 


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OUTCOME MEASURES (Efficacy and Safety Parameters)  
Protocol section 6.3 Primary Efficacy Variables, page 55  
Protocol section 6.4 Secondary Variables, page 56 
The primary efficacy parameter chosen to assess drug efficacy is the CRR_12.  
 
Secondary efficacy parameters are:  
 
Best corrected visual acuity (BCVA) 
 
Corneal scarring as identified by slit lamp examination 
 
Ulceration severity as identified by slit lamp examination using a 2 grade scoring procedure 
 
Anterior chamber inflammation as identified by ophthalmoscopy using a 3 grade scoring procedure  
 
EQ-5D questionnaire 
 
VFQ25 questionnaire 
 
Secondary safety parameters are: 
 
Adverse events  
 
Clinical laboratory tests 
 
Intraocular pressure (IOP) 
 
Ophthalmoscopy 
 
Worsening of the corneal epithelial defect and definable inflammatory signs (development of ring abscess 
and hypopyon) despite >30 days of treatment with the study drug) 
 
Rate of subjects with a relapse 
 
Rate of subjects requiring surgery, including amniotic membrane transplants, superficial keratectomy, 
application of cyanoacrylate glue, therapeutic penetrating, lamellar keratoplasty, cataract surgery, 
evisceration, or enucleation 
 
Rate of subjects requiring non-study therapies, e.g., topical steroids and NSAIDs 
 
Rate of subject discontinuation from study: to permit alteration of anti-amoebic therapy or for other unrelated 
specified reasons.   
 
Incidence of secondary complications, such as significant corneal neovascularization, corneal scarring, 
corneal perforation, scleritis, secondary glaucoma, cataract, retinopathy. 
 
Note: The estimated CRR_12 is 67% for the conventional (0.02% PHMB + 0.1% propamidine) combination therapy 
(from the sponsor’s observational, case series retrospective Study 038/SI) (63% when assuming a prevalence of late 
stage diseases in 38% of subjects). 
OVERVIEW OF STUDY DESIGN  
Protocol section 3.1 Overview of Study Design, page 29 
This is a randomized, assessor-masked, active-controlled, multiple center, parallel-group Phase 3 study to evaluate 
the efficacy, safety and tolerability of 0.08% PHMB ophthalmic solution compared to the conventional 0.02% PHMB 
+ 0.1% propamidine combination therapy in male and female subjects affected by Acanthamoeba keratitis.  
The study is designed as a superiority study with the possibility to test for non-inferiority if the superiority hypothesis 
is not met, according to the requirements of the guidance from the European Agency for the Evaluation of Medicinal 
Products (EMA) (CPMP/EWP/482/99).21 The specific conditions for this outcome are described in Section 7.4.  
The study consists of an eligibility screening visit, a treatment period including short ambulant visits, and follow-up 
visits. 
A total of 130 subjects affected by Acanthamoeba keratitis will be assigned to one of the following 2 treatment groups 
in a ratio of 1:1.  
Group 1: 0.08% PHMB + placebo 
Group 2: 0.02% PHMB + 0.1% propamidine combination therapy 


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SUBJECT POPULATION 
Protocol section 4, SUBJECT POPULATION, page 33 
The study will be performed in male and female subjects affected by Acanthamoeba keratitis, ≥12 years of age, 
inclusive. 
SCREENING PROCEDURE 
Protocol section 3.1.2 Screening Visit, page  30 
A schematic overview of the screening procedure is presented below. 
 
All subjects with clinical signs consistent with Acanthamoeba keratitis and positive confocal findings consistent with 
Acanthamoeba keratitis will be eligible for randomization providing they have not been using a topical biguanide or 
diamidine. 
All subjects at screening, meeting inclusion criteria and including those with negative confocal microscopy findings 
will have corneal scrapes for culture and histology of Acanthamoeba, and to perform Acanthamoeba deoxyribonucleic 
acid (DNA) identification by polymerase chain reaction (PCR).  
Note: this is being done for study quality, for correlation with confocal microscopy findings, and to identify patients 
with fungal and viral infection. Because culturing and PCR will take 2 to 8 days, during which time subjects thought 
clinically to have Acanthamoeba keratitis would be starting treatment before randomization, this assessment is not 
part of the inclusion criteria. Subjects who have been proven to have fungal and viral infections, if randomized on the 
basis of clinical and confocal microscope findings, will be removed from the study, even though they may have 
concomitant Acanthamoeba keratitis. 
Subjects with a negative confocal microscopy evaluation but a positive PCR/Culture (confocal false negative) will 
not be enrolled in the study but treated with the best clinically available treatment outside the study.  
Subjects meeting the inclusion criteria, including findings on slit lamp examination and confocal microscopy 
consistent with Acanthamoeba keratitis are entered into the study and randomized to one of the two treatment arms. 
They will then enter the Treatment Plan and Follow-up protocol as presented on page 16.  


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INCLUSION CRITERIA  
Protocol section 4.1 Inclusion Criteria, page 33 
Subjects must meet all of the following criteria to be considered eligible to participate in the study: 
1. 
Subject must be able and willing to give informed consent. 
2. 
Subject must be a man or woman of any race and ≥12 years of age, inclusive. Subjects <18 years will only be 
enrolled in selected study sites 
3. 
Subject must be able to understand and willing to comply with study procedures, restrictions and requirements, 
as judged by the investigator. 
4. 
Clinical findings consistent with Acanthamoeba keratitis 
Clinical findings include the following: 
 
Epithelial lesions: epithelial punctate keratopathy, epithelial infiltrates, epithelial defects, dendritiform 
epithelial ulcers.  
 
Extracorneal lesions: limbal inflammation (limbitis), anterior scleral inflammation, diffuse or nodular.  
 
Stromal lesions: perineural infiltrates, anterior stromal infiltrates, disciform corneal swelling, stromal 
ulceration, ring abscess.  
 
Anterior chamber lesions: keratic precipitates, hypopyon.  
 
Late findings: fixed dilated pupil, mature cataract 
5. 
Confocal microscopy findings consistent with Acanthamoeba keratitis (performed within 7 days prior to study 
entry or as part of screening procedures) 
 
Confocal microscopy findings include: cysts are round or ovoid, may show a double wall and are 15-30 
µm in size. 
6. 
Subjects using the following previous treatments for Acanthamoeba keratitis are eligible for the study: 
 
Antibiotics: subjects who have an ocular bacterial infection at baseline are eligible for the study. 
However, only topical moxifloxacin is permitted, unless resistant  or contraindicated. 
Note: subjects who develop intercurrent bacterial infections will be retained in the study and treated with 
topical moxifloxacin. 
 
Antiviral drugs and antifungal drugs: subjects are often misdiagnosed as having these infections when 
they have Acanthamoeba keratitis. Subjects taking antivirals and antifungal agents (except for any using 
PHMB or Chlorhexidine) for a misdiagnosis can be included, but must discontinue these drugs after 
entry into the study. 
Note: subjects who are thought to have combined Acanthamoeba keratitis with herpes or fungal keratitis 
are excluded from the study. 
 
Anti-inflammatory drugs: Subjects using topical steroids and/or oral NSAIDs before the diagnosis of 
Acanthamoeba keratitis are eligible for the study. However, these subjects must agree to change therapy 
to the topical steroids and oral NSAIDs that are specified for use in the study (see Concomitant and 
Previous Medications). 
7. 
Females of childbearing potential will be included if they are either sexually inactive (sexually abstinent for 
14 days prior to the first study drug dose continuing through 28 days after the last study drug dose, or using one 
of the following highly effective contraceptive (i.e. results in <1% failure rate when used consistently and 
correctly) methods in this study: 
a. 
intrauterine device (IUD); 
b. surgical sterilization of the partner (vasectomy for 6 months minimum); 
c. 
combined (estrogen or progestogen containing) hormonal contraception associated with the inhibition 
of ovulation (either oral, intravaginal, or transdermal); 


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d. progestogen only hormonal contraception associated with the inhibition of ovulation (either oral, 
injectable, or implantable); 
e. 
intrauterine hormone releasing system (IUS);  
f. 
bilateral tubal occlusion. 
Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual 
intercourse during the entire period of risk associated with the study treatments. In this study, abstinence is only 
acceptable if in line with the subjects preferred and usual lifestyle. 
Periodic abstinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), 
spermicides only, and lactational amenorrhoea method (LAM) are not acceptable methods of contraception. As 
well, a female condom and a male condom should not be used together. 
8. 
Females of childbearing potential agree to remain sexually inactive or to keep the same birth control method for 
at least 28 days following the last study drug dose. 
9. 
A female of non-childbearing potential must have undergone one of the following sterilization procedures at 
least 6 months prior to the first study drug dose: 
a. 
hysteroscopic sterilization; 
b. 
bilateral tubal ligation or bilateral salpingectomy; 
c. 
hysterectomy; 
d. 
bilateral oophorectomy; 
or be postmenopausal with amenorrhea for at least 1 year prior to the first study drug dose and follicle 
stimulating hormone (FSH) serum levels consistent with postmenopausal status 
10. A non-vasectomized male subject agrees to use a condom with spermicide or abstain from sexual intercourse 
during the study until 90 days beyond the last dose of study drug and the female partner agrees to comply with 
inclusion 7 or 8. For a vasectomized male who has had his vasectomy 6 months or more prior to study start, it is 
required that they use a condom during sexual intercourse. A male who has been vasectomized less than 
6 months prior to study start must follow the same restrictions as a non-vasectomized male. 
Periodic abstinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), 
spermicides only, and LAM are not acceptable methods of contraception. As well, female condom and male 
condom should not be used together. 
11. If male, they must agree not to donate sperm from the first study drug dose until 90 days after dosing. 
 
 
 
EXCLUSION CRITERIA  
Protocol section 4.2 Exclusion Criteria, page 35 
Subjects who meet any of the following criteria will be excluded from participation in the study:  
1. Subject with documented history and/or clinical signs of concomitant presence of an ocular infection caused by 
viruses (herpes simplex virus [HSV]) or fungi.  
2. Subject treated with drugs having effects on Acanthamoeba cysts prior to study entry, including biguanides 
(PHMB, chlorhexidine) and diamidines (propamidine, hexamidine). 
3. Subjects requiring systemic immunosuppression for Acanthamoeba associated scleritis.  
4. Subjects requiring urgent surgical intervention for advanced Acanthamoeba keratitis in either eye (e.g., for 
advanced corneal thinning/melting etc.). 
5. Subject with known or suspected allergy to biguanides, diamidines or intolerance to any other ingredient of the 
investigational treatments. 
6. Subject affected by immunodeficiency diseases or taking systemic immunosuppressive therapy. 


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7. Subject with a major systemic disease or other illness that would, in the opinion of the investigator, compromise 
subject’s safety or interfere with the collection or interpretation of study results. 
8. If female, pregnancy, planned pregnancy, or breast-feeding  
9. Subject is participating in another interventional clinical study with an experimental or unapproved/unlicensed 
therapy or has participated in another interventional clinical study within 4 weeks prior to this study. 
The investigator must ensure that all study enrolment criteria have been met at randomization. If a subject's status 
changes after randomization, but before the first dose of study drug is given such that he or she no longer meets all 
eligibility criteria, then the subject should be excluded from participation in the study. 
CONCOMITANT AND PREVIOUS MEDICATIONS 
Protocol section 4.3 Concomitant Medication and Other Restrictions during Study, page 36 
Subjects are allowed to use the following concomitant medications during the study: 
Antibiotics: Topical moxifloxacin is permitted for the treatment of intercurrent bacterial infections (unless culture 
and sensitivity, or clinical progress demands a change). Topical moxifloxacin is not permitted for use as a prophylactic 
antibiotic in patients with corneal ulcers; PHMB is a good broad spectrum anti-bacterial and an additional antibacterial 
is not needed for this. The value of prophylaxis is unproven. 
Antiviral drugs and antifungal drugs: The use of these drugs is not allowed during the study. In case subjects use 
antiviral or antifungal drugs at study entry, they must be discontinued.  
Anti-inflammatory drugs: For subjects on steroids at study entry (already using topical steroids for e.g. mistaken 
diagnosis of HSV keratitis or as adjunctive treatment for bacterial keratitis), there are the following options: 
a. 
Either stop steroids, OR maintain, OR reduce the doses (at the investigator’s discretion). Unpreserved 
Dexamethasone (0.1% or 0.15%) is the only topical steroid permitted for use in this trial.  Patients using any 
other topical steroids on trial entry should be changed to this at the appropriate frequency.  Diclofenac is the 
only oral NSAID permitted in the trial and will be ADDED at the appropriate dose (75 mg to 150 mg daily, 
divided in two or three doses) and continued at any level while topical steroids are in use during the study. 
b. Subjects using topical NSAIDs and ciclosporin at study entry should have these discontinued after 
randomization. 
c. 
Subjects using no topical steroids at study entry can have these started together with oral NSAIDs 
(recommended diclofenac; 75 mg to 150 mg daily, divided in two or three  doses) during the study as specified 
in the schematic overview of the Treatment and Follow-up Protocol. 
Other permitted topical mediations: Unpreserved lubricants, mydriatics (cyclopentolate, homatropine or 
atropine) and glaucoma medications are permitted. 
TREATMENT ASSIGNMENT  
Protocol section 5.1 Treatment Assignment , page 38 
Randomization to one of the treatment groups will be in a 1:1 ratio. The randomization schedule will be generated 
using a computer program and verified for accuracy using strict quality control procedures. Eligible patients will 
receive a masked treatment assignment with a unique randomization code based on the randomization list. The 
assigned randomization code will be captured in the electronic Case Report Form (eCRF).  
Protocol for bilateral disease: if both eyes are affected, only one eye will be treated with the study treatment 
and will be considered for the study. This will be the right eye, unless the severity of the infection differs in both 
eyes, in which case the worst affected eye should be selected. The fellow eye should be treated with the best 
treatment according to clinical practice. 
DRUG DOSAGE AND ADMINISTRATION 
Protocol section 5.2 Drug dosage and Administration, page 39 
On Day 0, subjects will receive instructions on how to apply the eye drops. Subjects will receive the assigned treatment 


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to be administered in the affected eye until resolution. Subjects will be treated for a maximum of 1 year after 
randomization and subjects meeting the criteria for clinical resolution at or before 12 months will be followed up for 
90 days before completing the study. 
Subjects will receive an initial intensive course of 0.08% PHMB + placebo or 0.02% PHMB + 0.1% propamidine 
combination therapy according to the following schedule. Subjects will be treated for a maximum of 1 year after 
randomization. 
Treatment throughout the study will be given using 1 drop of each ophthalmic solution in the study eye at different 
frequencies during the daytime only. At study entry, an intensive 19-day antiamoebic treatment protocol will be 
initiated as follows: 
Day 0 to 5 
1-hourly drops (16 drops in a day) for 5 days 
Day 6 to 12 
2-hourly drops (8 drops in a day) for 7 days 
Day 13 to 19 
3-hourly drops (6 drops in a day) for 7 days 
Day 20  
Then 4x daily thereafter  
Notes on treatment 
i. 
The frequency will be maintained at 4x daily unless there is a relapse (see Box XI, relapse, Figure 2 on page 
16). In the event of a relapse, culture negative subjects will receive the same intensive 19-day treatment 
course as given at study entry.  
ii. 
Antiamoebic drops will be discontinued when clinical resolution is reached (see Figure 2 and Legend of  
BOX IV DISCONTINUE TREATMENT on page 16).  
iii. 
Clinical resolution is a clinical definition, resulting from a slit lamp examination, with the following findings:  
 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and oedema) that requires 
treatment, with a healed corneal epithelium and minimal punctate staining (10 dots or less equivalent to Grade 
1 on the Oxford Scale).   
 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal hyperemia): mild 
conjunctival inflammation is acceptable if related to other concurrent conditions such as blepharitis. 
 
No limbitis, scleritis or anterior chamber inflammation.  
For subjects using steroids at study entry (already using topical steroids for e.g. mistaken diagnosis of HSV keratitis 
or as adjunctive treatment for bacterial keratitis), the same intensive 19-day antiamoebic treatment protocol will be 
used. For their steroid use there are the following options: 
 
Either stop steroids, OR maintain, OR reduce the doses (at the investigator’s discretion). Unpreserved 
Dexamethasone (0.1% or 0.15%) is the only topical steroid permitted for use in this trial. The frequency of 
use is at the discretion of the investigator.  
 
Patients using any other topical steroids on trial entry should be changed to this at the appropriate 
frequency.  Diclofenac is the only oral NSAID permitted in the trial and will be ADDED at the dose 
determined by the investigator (75 mg to 150 mg daily, divided in two or three doses) and continued at any 
level while topical steroids are in use during the study. 
Subjects using antifungals or antivirals on study entry must discontinue these after randomization.  


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SCHEMATIC OVERVIEW OF THE TREATMENT AND FOLLOW-UP PERIOD  
Protocol section 5.3 Treatment and Follow-up Protocol, page 41 
 
 
. Legend for Figure 2: Schematic Overview of the Treatment and Follow-up Period 


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The pathways are colour coded:  
 
Pale blue text boxes and lines for responding disease or treatment success and STUDY END POINT 
 
Red text boxes and lines for poorly responsive and recurrent / relapsing disease presumed to be due to 
replication of Acanthamoeba  
 
Pink text box for exacerbations of inflammation presumed to be due to flare ups of inflammation 
secondary to the immune response to non-viable Acanthamoeba 
 
Black text boxes for treatment failure endpoints ALL leading to TRIAL EXIT 
 
Each box is labelled with a Roman numeral I to XVIII. The legend below describes the rationale for each step: 
I. 
FOLLOW UP PROTOCOL from day zero at TRIAL ENTRY.  
Treatment throughout the study is given using 1 drop of each ophthalmic solution in the study eye at different 
frequencies during the daytime only: 
At trial entry initiate an intensive 19 day anti-amoebic treatment protocol:  
Day 0-5:     every hour daytime only hourly drops (16 drops a day) for five days. 
Day 6-12:   2 hourly (8 drops a day) for 7 days 
Day 12-19: 3 hourly (6 drops a day) for 7 days  
Day 20:       reduce to 4x daily and continue thereafter.  
a. 
This frequency is maintained at 4x daily unless there is a relapse of infection (XI RELAPSE of 
infection) when culture negative patients receive the same intensive 19 day treatment course as given 
at trial entry.  
b. Anti-amoebic drops are discontinued as when the clinical findings are as described in IV 
DISCONTINUE TREATMENT. 
In patients on steroids at TRIAL ENTRY, (already using topical steroids for e.g. mistaken diagnosis of 
herpes simplex virus keratitis or as adjunctive treatment for bacterial keratitis) the same intensive 19 day 
anti-amoebic treatment protocol is used, there are the options: 
Either stop steroids, OR maintain, OR reduce the doses (at the Principal Investigator’s discretion). ADD an 
oral non-steroidal anti-inflammatory drug (NSAID) WHEN steroid is to be continued at any level.  
In patients using antifungals or antivirals on trial entry these must be discontinued. 
 
II. 
STABLE (defined as no deterioration but not improving, as opposed to a poor response as defined in VIII). 
These subjects may improve (meeting the criteria described in Box III IMPROVEMENT: see below) or not 
over the next few weeks. If there is no improvement but they remain stable, then these patients may be started 
on anti-inflammatory treatment as described in IX. Alternatively, the inflammation may deteriorate (this 
may occur at any stage of treatment) and the rationale for managing this problem is described here: 
 
 
DETERIORATION of inflammation (after excluding co-infection with other organisms). 
 
The two possibilities which the PI needs to differentiate between are:  
 
a. 
Relapse of infection (Box XI) presumed or proven to be due to continued replication of 
Acanthamoeba) OR  
b. Exacerbation of inflammation (Box XII) presumed to be due to inflammation secondary to the 
immune response to non-viable Acanthamoeba 
 


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XI RELAPSE of infection is identified by a positive culture, unfortunately very insensitive due to the 
persistence of deep organisms, supported by an increase in cysts on confocal (also insensitive in severe 
disease) or using clinical criteria: development of more severe corneal inflammation, melting, ulceration, 
hypopyon, development of ring abscess necessitating another intensive course of therapy. This is as 
opposed to:  
 
XII EXACERBATION of inflammation: which is accompanied by mild conjunctival or corneal 
inflammation (sometimes coarse anterior stromal infiltrates like those following adenovirus keratitis) 
and/or mild scleritis. XV SCLERITIS unresponsive to topical steroids and oral NSAIDS is another cause 
of this: these patients are complex to manage and as a result we decided that these should exit the trial.  
 
Once a decision has been made between these two the appropriate protocol is followed for each scenario as 
outlined in XI and XII.  
 
Note: if this occurs after 11 month (335 Days) it is a treatment failure XVII (as patients relapsing at this 
point will not have time to respond to treatment) and the patient exits the trial. 
 
III. 
IMPROVEMENT reducing inflammation and some healing of any ulcers and/or improving symptoms. 
These subjects may continue to improve to the point they go to IV Discontinue treatment.  
 
 
 
Deterioration of inflammation: Patients in this subset may deteriorate due to: XI Relapse of Infection or 
XII Exacerbation of inflammation in which case they are managed as described in paragraph II a, b for 
these issues.   
 
IV. 
DISCONTINUE TREATMENT (includes topical steroids, oral NSAIDs and topical anti-
amoebics/microbials): when any ulcers have healed, and all signs of AK related inflammation have 
resolved (see detailed description in protocol, mild conjunctival inflammation related to drugs or other 
condition such as blepharitis are acceptable) at or before 12 months (365 days) from Trial entry.  Clinical 
resolution (CR) after 365 days is a failure (see Box XVI). We recommend discontinuing topical steroids 2-
4 weeks before discontinuing topical anti-amoebics.  
 
V. 
Follow up visit at Day 30 after last treatment.  
 
Deterioration of inflammation: Patients in this subset may deteriorate due to: XI Relapse of Infection or 
XII Exacerbation of inflammation in which case they are managed as described in paragraph II a, b for 
these issues.   
 
VI. 
Follow up visit at Day 90 after last treatment. If clinical resolution has been maintained by 15 months (day 
455 at the latest) these are treatment successes and have reached the study endpoint (Box VII). 
 
VII. 
TREATMENT SUCCESS.  
 
VIII. 
POOR RESPONSE at 3 weeks from I Trial entry or XIII RESTART: patients are managed using the 
guidelines in IX (this is the earliest that anti-inflammatory treatment can be started for those not on this 
when included in the Trial). Criteria for a poor response are described in detail in the protocol and include 
enlarging ulcer, stromal thinning, deteriorating corneal inflammation (infiltrate including ring abscess), 
intraocular inflammation (uveitis and hypopyon) and/or scleritis. 
 
IX. 
ANTI-INFLAMMATORY TREATMENT 
a. 
For patients NOT using topical steroids start these WITH oral NSAIDS (this is requested to simplify 
anti-inflammatory treatment - rather than ad hoc switching). 
b. For patients already on topical steroids and oral NSAIDS, increase the potency or frequency of the 
steroids and NSAIDS. 
If this results in III. IMPROVEMENT the management continues down the blue pathway. 


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X. 
Alternatively, if CONTINUED DETERIORATION occurs cases move to XVII TREATMENT FAILURE 
and EXIT Trial (see below). 
XI. 
RELAPSE of Infection within 11 months, after an initial response is common. Cultures for Acanthamoeba 
and other organisms (fungi bacteria and herpes) must be repeated. Carry out a corneal biopsy, if PI thinks 
this is appropriate.  
a. 
If the CULTURES are NEGATIVE patients this may mean that the anti-amoebic therapy is having 
some effect although we have no way of being certain about this (confocal and PCR don’t help as 
these can be positive when the organisms are non-viable). These patients are managed as described 
in XIII below with another 19 day course of intensive therapy, with or without anti-inflammatory 
therapy depending on status.  
b. If CULTURES are POSITIVE patients have TREATMENT FAILURE and managed as described in 
XIV and XVII below. 
XII. 
EXACERBATION of inflammation is presumed to be due to inflammation secondary to the immune 
response to non-viable Acanthamoeba and anti-inflammatory treatment can be re-started or increased at the 
discretion of the PI without the need for re-culture and a further course of intensive anti-amoebic therapy. 
IMPORTANT: if anti-amoebic treatment had been stopped restart this when anti-inflammatory treatment 
is re-started.  
Distinguishing between XI and XII is often difficult and guidelines for this are given in II a, b above.  
XIII. 
Restart with another 19 day intensive course of anti-amoebic therapy with or without topical steroids and 
oral NSAIDS:  
a. 
If patients were not on these at the time of the relapse of infection we recommend not using these 
until after the 19 day course of intensive treatment and depending on response move to as above. 
b. If they were on them at the time of the relapse continue at a dose determined by the PI and follow 
pathways II, III, IX or X. 
XIV. 
CULTURE POSITIVE these patients are Exit Trial and are managed as described in XVII below.  
XV. 
SCLERITIS UNRESPONSIVE to treatment in IX. This is a failure (see XVII below). 
XVI. 
RELAPSE after day 335 or ingoing inflammation and treatment at day 365: this is a treatment failure and is 
managed as described in XVII below 
XVII. 
TREATMENT FAILURE and EXIT TRIAL.  
XVIII. 
Summary of Study end points. 
 
STUDY EVALUATIONS 
Protocol section 6 STUDY EVALUATIONS, page  51 
Efficacy evaluations will consist of assessment of best corrected visual acuity (BCVA), pupil test (swinging light test), 
slit lamp examination, EuroQol five-dimension scale (EQ-5D) questionnaire, and visual functioning questionnaire 25 
(VFQ25). 
Safety and tolerability evaluations will consist of adverse event (AE) reporting, clinical laboratory (hematology, 
biochemistry and urinalysis), intraocular pressure (IOP), ophthalmoscopy, and photography of affected cornea.  
Detailed information about adverse event reported is provided in Section 0.  
See the Schedule of Assessments on page 22, Section 6 and Appendices 1 to 3 in the full protocol, and the Study 
Operations Manual for details of the evaluations. 
SUBJECT COMPLETION/WITHDRAWAL 
 
Detailed information is provided in Section 4.4 of the full protocol 


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STATISTICAL METHODS 
Protocol section 7 STATISTICAL METHODS, page 57 
A statistical analysis plan (SAP) with more technical and detailed elaboration of the principal features of the proposed 
statistical analysis, presentations and the way in which anticipated analysis problems will be handled, will be written 
before the code is broken. See Section 7 for detailed information. 
Sample Size Determination 
From the results of the sponsor’s observational, case series retrospective study 038/SI, a CRR_12 of 67% for the 
conventional combination therapy is expected. This figure is in the range of cure rates described in the literature. If 
the true difference in CRR_12 is 0.20 (Δ) (or more) in favor of PHMB monotherapy (0.08% PHMB), a total sample 
size of 116 subjects (allowing for 10% loss to follow-up) should give the study at least 80% power to detect superiority, 
with 2-sided alpha=0.10 (or equivalently 1-sided alpha=0.05). 
Assuming a prevalence of late stage diseases in 38% of subjects, the expected CRR_12 in the control group will be 
reduced from 67% to 63%. To account for the inclusion of this group, the sample size has been adjusted to 130 subjects 
(allowing for 10% loss to follow-up). 
Efficacy 
All efficacy evaluations will be based on the Full Analysis Set. Moreover, the primary efficacy variable, CRR_12, 
will be evaluated also for the Per-Protocol Analysis Set. 
Since it is also possible to test for non-inferiority if the superiority hypothesis is not met, additional conditions are 
specified (see list below) to meet the strict requirements of a non-inferiority study, making it feasible to test for non-
inferiority using the results from the superiority analysis, without a further separate analysis. The 90% confidence 
interval for the difference between the treatments obtained from the superiority analysis gives the necessary 
information. Position of the lower end of the confidence interval relative to a pre-defined agreed 0.20 non-inferiority 
margin (Δ) provides the key information for making decisions (conclusions) about non-inferiority. The additional 
conditions specified to make testing for non-inferiority feasible are the following: 
 
Pre-defined non-inferiority margin (Δ=0.20) based on both clinical and statistical considerations, i.e., chosen 
on the basis of external information and not chosen to fit the data. 
 
Analysis of the Full Analysis Set (FAS) and the Per-Protocol Analysis Set (PPAS), giving similar results 
with respect to p-values and confidence intervals for the efficacy measure at issue. 
 
Study design and implementation directed at minimizing deviation from protocol (a particularly important 
source of bias in non-inferiority studies), e.g., special care to minimize violations of: entry criteria; non-
compliance; withdrawals; loss; missing data; etc. This should help ensure the second bullet point.  
 
Evidence that the standard treatment is showing its usual (expected) level of efficacy. 
These specifications are in accord with the recommendations by the Committee for Proprietary Medicinal Products 
(CPMP) 482/99. 
Null hypothesis superiority: 
 
No difference between test treatment and reference treatment 
Non-inferiority clinical studies aim to demonstrate that the test treatment is no worse than the reference by more than 
a pre-specified small amount. 
Null hypothesis non-inferiority: 
 
Test treatment is inferior to the reference by  or more 
CRR_12 will be analyzed with a logistic regression model and model-based estimates of the difference between 
treatments, odds ratio with corresponding confidence intervals and p-values will be provided. Time-to-event (clinical 
resolution) analysis will also be performed for the comparison of CRR_12s between treatments, including the Cox 


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Proportional Hazards regression (subject to validity of the 'proportional hazards' assumption) and Kaplan-Meier 
survival plots. If the proportional hazard assumption is not fulfilled a logrank test will be performed. 
Which test to be used in the analyses of secondary efficacy variables depends on the type of variable, e.g., logistic 
regression for binary variables, proportional odds model for an ordinary scaled variable, analysis of variance 
(ANOVA) or covariance (ANCOVA) for continuous variables as appropriate. If the underlying assumptions are not 
fulfilled, data transformation or non-parametric test will be performed. For comparison of proportions, Chi square 
tests, Fisher's exact test, or Mantel-Haenszel procedures, as appropriate, will be considered. For continuous variables, 
the Wilcoxon rank-sum test or rank ANOVA/ANCOVA will be used. The STATA or SAS statistical software will be 
used for the analysis. 
EQ-5D and VFQ25 will be evaluated according to their respective user protocols. 
Safety 
AEs data will be captured and encoded by system organ class (SOC) and lowest level term (Medical Dictionary for 
Regulatory Activities, current version). A listing of AEs will be created. This listing, at minimum, will contain a 
description of AEs as to seriousness, severity, onset date and end date, duration, action taken (if any), outcome and 
likelihood of drug causation (“relation"). A frequency table will be compiled for AEs by treatment group, SOC, and 
lowest level term. Frequency tables will be compiled showing the number of subjects per treatment group affected by 
one or more (related) AEs, including percentages, the total number of AEs per treatment group and the average number 
of AEs per subject exposed to the respective treatment.  
Clinical laboratory tests will be presented with summary statistics. In addition, the tests will be analyzed with 
ANCOVA (baseline value of respective laboratory test will be included in the model). All results will be regarded as 
descriptive only. The p-values will be used to flag safety and tolerability variables worthy of further attention.  
Null hypotheses:  
 
No difference between test treatment and reference treatment.  
QUALITY CONTROL AND QUALITY ASSURANCE 
Detailed information is provided in Section 9 of the full protocol. 
ETHICAL ASPECTS 
Detailed information is provided in Section 10 of the full protocol. 
 


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SCHEDULE OF ASSESSMENTS 
Visits 1 
Screening
 
Treatment Period 
Follow-up Period 
Relapse 3 End-of-study 4
Study Procedure  
Study Day 
-2 to 0 5 
0 7 ± 2 14 ± 2 21 ± 2 30 ± 3 60 ± 3 and 
every 30 ± 
5 until 
resolution
30 ± 3 
after end 
of 
treatment
90 ± 5 after 
end of 
treatment2
 
 
 
Screening/Administrative/Baseline 
Informed consent 
x 
 
 
 
 
 
 
 
 
 
 
Parent(s), legal guardian(s), or legally authorized 
representative (LAR) Informed consent 6 
x 
 
 
 
 
 
 
 
 
 
 
Demographics 
x 
 
 
 
 
 
 
 
 
 
 
Review medical history 7 
x 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
x 
 
 
 
 
 
 
 
 
 
 
Clinical suspicion of an Acanthamoeba infection by 
typical clinical findings  
x 
 
 
 
 
 
 
 
 
 
 
Confirmation of Acanthamoeba infection by 
confocal microscopy 8 
x 
 
 
 
 
 
 
 
 
 
 
Corneal scraping 9 
x 
 
 
 
 
 
 
 
 
x 
 
Vital signs 10 
x 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
Randomization 11 
 
x 
 
 
 
 
 
 
 
 
 
Dispense study drug 12 
 
x 
 
 
 
x 
x 
 
 
x 
 
Study drug administration 13 
 
x 
x 
x 
x 
x 
x 
 
 
x 
 
 
Efficacy Assessments  
Best corrected visual acuity 
 
x11
 
 
 
 
 
 
 
 
x 
Slit lamp examination  
 
x11
x 
x 
x 
x 
x 
x 
x 
x 
x 
Pupil test (swinging light test) 14 
 
x11
x 
x 
x 
x 
x 
x 
x 
x 
x 


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Visits 1 
Screening
 
Treatment Period 
Follow-up Period 
Relapse 3 End-of-study 4
Study Procedure  
Study Day 
-2 to 0 5 
0 7 ± 2 14 ± 2 21 ± 2 30 ± 3 60 ± 3 and 
every 30 ± 
5 until 
resolution
30 ± 3 
after end 
of 
treatment
90 ± 5 after 
end of 
treatment2
 
 
EQ-5D questionnaire 15,16 
 
x11
x 
x 
x 
x 
x 
x 
x 
x 
x 
Visual functioning questionnaire (VFQ25) 15,17 
 
x11
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Safety Assessments 
Hematology, biochemistry  
x 
 
 
 
 
 
 
 
 
 
x 
Urinalysis (dipstick)  
x 
 
 
 
 
 
 
 
 
 
x 
Urine pregnancy dipstick (women of childbearing 
potential only 
x 
 
 
 
 
x 
x 
x 
x 
x 
x 
Use of topical steroids and oral NSAIDs 18 
x 
 
 
 
x 
 
 
 
 
 
 
IOP  
 
x11
x 
x 
x 
x 
x 
x 
x 
x 
x 
Ophthalmoscopy 19 
 
x11
 
 
 
 
 
 
 
 
x 
Photography of affected cornea  
 
x11
 
 
 
x 
 
 
x 
 
x 
 
Ongoing Subject Review 
Concomitant therapy 7 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Adverse events 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
1. During each visit, subjects will enter the clinical research center and will leave after completion of all assessments.  
2. If subjects are cured 90 days after end-of-treatment visit, the study-end-point is reached and the End-of-Study assessments will be performed.  
3. If within 90 days after end-of-treatment and within 11 months (Day 335) after randomization the subject relapses, and cultures are negative for Acanthamoeba, treatment with the study 
drug will be re-started according to the treatment schedule, starting with the Day 0 treatment regime. Assessments and visits will also be restarted  at Day 0, Day 30 and all consecutive 
visits of the treatment phase. A detailed treatment plan and follow-up protocol is provided in Section 5.3. 
4. End-of-study is defined as: (a) the last follow-up visit at 90 days after end of treatment, (b) the day of study drug unmasking in the event of the subject  becoming a Treatment failure, 
(c) if withdrawn from the trial of other reasons, (d) if a subject has been treated for 12 months after randomization (also a Treatment failure).  
5. Screening and baseline assessments will take place on Day -2 to 0, before randomization and study drug administration.  
6. For subjects who have not reached the age of majority (i.e. 18 years of age in most jurisdictions), the subject must provide assent (written or verbal) and the parent/guardian/legally 
authorized representative must provide written informed consent before the subject’s participation in the study. 
7. All medical and medication history related to the current eye infection will be reported. For the remaining medical and medication history, only the last 30 days before signing the ICF 
will be reported in the case report form (CRF). 
8. Confocal microscopy is optional if already performed within 7 days prior to study entry as part of standard care providing the images are available to the Investigator to assess.  


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9. All subjects at screening, meeting the inclusion criteria and including those with negative confocal microscopy findings will have corneal scrapes for culture and histology of 
Acanthamoeba, and to perform Acanthamoeba deoxyribonucleic acid (DNA) identification by polymerase chain reaction (PCR). Note: this is being done for study quality, for correlation 
with confocal microscopy findings, and to identify subjects with fungal and viral infections. Because culturing and PCR will take 2 to 8 days, during which time subjects thought clinically 
to have Acanthamoeba keratitis and would be starting treatment before randomization, this assessment is not part of the inclusion criteria. This assessment is optional if already performed 
within 7 days prior to study entry as part of standard care. Corneal scraping for culture and histology of Acanthamoeba, and to perform Acanthamoeba DNA identification by PCR will 
be repeated in case of a relapse within 90 days after end-of-treatment and within 11 months (Day 355) after randomization. 
10. Blood pressure, pulse rate and body temperature. 
11. Before first study drug application. 
12. Study drug will be provided for 30 days  at the Day 0 visit. At other dispensing visits, study drug will be provided for 90 days of treatment. 
13. On Day 0, the first study drug application will be at the clinical research center. Thereafter, study drug will be self-administered at home. When the subject is at the clinical research 
center for assessments, subjects will apply the study drug themselves at the clinical research center. On Days 0 to 5, subjects will apply study drug every hour daytime only (1 drop of 
each ophthalmic solution in the affected eye). On Days 6 to 12, (1 week), subjects will apply study drug every 2 hours daytime only. On Days 13 to 19, (1 week), subjects will apply 
study drug every 3 hours daytime only. On Day 20 until resolution, subjects will apply study drug 4 times a day at daytime only. Subjects will be treated for the maximum of 1 year after 
randomization.  
14. The Pupil test (swinging light test), to detect an afferent pupillary defect, is used to detect retinal or optic nerve involvement when the retina cannot be examined.  
15. If the subject is unable to complete the questionnaires without assistance, the questionnaires may be verbally answered and the subject’s responses recorded by site personnel or a 
chaperone. When multiple assessments are scheduled at the same time, completion of questionnaires by the subject will be done before the clinical examinations. 
16. Subjects being ≤15 years of age will complete the youth version of this questionnaire.  
17. Only to be performed by subjects of ≥18 years. 
18. The use of topical steroids and oral NSAIDs is complex and described in the Synopsis and Protocol. For subjects not already using topical steroids or NSAIDs on trial entry these may 
be introduced at day 21 in subjects whose inflammatory signs are not improving. Please see the Synopsis and Protocol for detailed guidelines.  
19. Ophthalmoscopy will be performed at baseline on all patients and at any time thereafter if the pupil tests or intraocular pressure are abnormal.  
 
 


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1. 
INTRODUCTION 
The Orphan Drug for Acanthamoeba keratitis (ODAK) is a project aiming to investigate the 
potential of polyhexamethylene biguanide (PHMB) as a safe and effective drug for the treatment 
of the rare eye disease Acanthamoeba keratitis. This debilitating infectious disease is caused by a 
commonly occurring protozoan and in the absence of treatment can result in blindness. There are 
currently no approved drugs to treat this disease. 
PHMB has received the orphan drug designation (EU/3/07/498) according to EC regulations 
141/2000. 
1.1. 
Background 
PHMB is a poly-cationic polymer composed by hexamethylene biguanide units (n varies 2 to 40 
with a mean of 5.5).1 Biguanides are an important class of cationic surface-active antimicrobial 
agents, which have been used for the preservation of many aqueous formulations in addition to the 
use as disinfectants and antiseptics. PHMB is currently used as an environmental biocide and 
antiseptic in a variety of products including wound care dressings, contact lens cleaning solutions, 
perioperative cleansing products and swimming pool cleaners. It has a broad spectrum of activity, 
being effective against gram-positive and gram-negative bacteria. At a cellular level in Escherichia 
coli, PHMB interacts with the cytoplasmic membrane, causing leakage of cellular components and 
inhibition of respiratory enzymes considered essential for survival.1,2,3 
PHMB has been shown to have excellent in vitro activity against a broad range of fungal 
pathogens. Antimicrobial effectiveness has been demonstrated on Acanthamoeba polyphaga, 
Acanthamoeba castellanii and Acanthamoeba hatchetti. Against these protozoa, PHMB acts by 
binding of its highly charged positive molecules to the mucopolysaccharide plug of the ostiole. 
This results in penetration through the ostiole to the internalized amoeba, where the drug binds to 
the phospholipid bilayer of the amoeba cell membrane causing membrane damage, cell lysis and 
death.4 PHMB is effective and well tolerated at concentrations of 200 to 600 mg/L (0.02%-0.06%) 
when used as treatment of patients with Acanthamoeba keratitis.5,6 
1.1.1. 
Toxicology 
PHMB is considered safe when used as preservative in cosmetics up to a concentration of 0.3%.7 
In addition, PHMB is considered of moderate acute oral toxicity with a no observed adverse effect 
level (NOAEL) of 36 mg/kg/bw.7 Despite the weak evidence of the carcinogenetic potential, 
PHMB is classified in the category “Suggestive Evidence of Carcinogenicity, but Not Sufficient 
to Assess Human Carcinogenic Potential” for inciting vascular tumors in laboratory animals by 
the oral and dermal routes.7 No quantitative or qualitative susceptibility was evident in the 
available prenatal developmental toxicity studies. Moreover, a 2-generation reproductive study did 
not show quantitative or qualitative evidence of increased susceptibility to abnormalities in the 
offspring when adults were exposed to PHMB. No mutagenic or genotoxic responses were 
observed.7 At ocular level, pure or powder PHMB induced irreversible ocular damage and was 
considered as corrosive to the rabbit eye, whereas a 20% solutions induced conjunctivitis, corneal 
opacity, iritis and was considered as moderately irritating to the eye.7 Concentrations of 0.05% and 
a 0.1% PHMB solution have been tested in an irritation test on enucleated pig eyes without causing 


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desquamation of epithelial cells or damage to the microvilli. In addition, the administration of a 
0.2% PHMB 8 times a day for 4 weeks into the conjunctival sac of the rabbit eye caused mild 
transient conjunctival redness and discharge. However, the above toxicity signs were not observed 
if administered twice daily. Accordingly, PHMB (0.1% and 0.2%) was categorized as non-irritant 
(Draize test) after a twice daily application over a period of 28 days in the rabbit.8 A repeated 
intensive ocular administration in rabbits of 0.08%, 0.25% and 0.8% PHMB for 2 weeks showed 
no toxicity of PHMB up to 0.25%. Only PHMB at 0.8% induced moderate/severe treatment-related 
and irreversible effects.9 The safety of 0.08% PHMB following repeated ocular administration to 
the rabbit over a period of 26 weeks was also investigated. The treatment regimen was 16 times/day 
at approximately 1 hour intervals from Day 1 to 5 of the study, 8 times/day at approximately 2 
hours intervals from Day 6 to Week 3 and 4 times/day at approximately 4 hours intervals from 
Week 4 to 26. The results did not report any significant findings with the exception of a light 
conjunctival redness, a slight conjunctival chemosis and slight discharge during the first 4 days. 
No fluorescein staining has been observed. No treatment-related changes were reported at the 
histopathological examination of the eyes, adnexa or in the remaining organs/tissues.9 For 
additional non-clinical information, refer to the Investigator's Brochure for PHMB.10 
1.1.2. 
Clinical Studies 
To date, the use of PHMB has been shown to be well tolerated and safe in ophthalmology. 
Moreover, 0.02% PHMB topical toxicity has been reported rarely and it is limited to mild punctate 
keratitis.11  
A prospective, randomized comparative study was conducted to compare the therapeutic efficacy 
of two agents, PHMB and chlorhexidine, in the treatment of Acanthamoeba keratitis. According 
to the Cochrane Database Review, this is to date the only randomized, controlled study conducted 
in patients with Acanthamoeba keratitis.12 Fifty-six eyes of 55 patients with Acanthamoeba 
keratitis were randomized to receive 0.02% PHMB or 0.02% chlorhexidine. Treatments were 
started immediately after a clinical diagnosis of Acanthamoeba keratitis was established. PHMB 
0.02% or chlorhexidine 0.02% was commenced at a frequency of hourly day and night for the first 
two days, then frequency was reduced to hourly by day only for the next five days. The frequency 
of instillation was further reduced to four times daily according to the clinical response to treatment 
over succeeding weeks. The results showed that 78% PHMB patients were treatment successes 
compared with 85.7% chlorhexidine patients (p=0.71). No serious toxic side effects occurred in 
any of the eyes in the study.13 Results yielded no difference with respect to outcomes reported 
between PHMB and chlorhexidine. However, the sample size was small to detect clinically 
meaningful differences, as indicated by the wide confidence intervals of effect estimates. 
 
A randomized, double-masked, placebo-controlled Phase 1 study was conducted to establish the 
ocular safety and tolerability, and systemic safety of 3 different concentrations of PHMB in healthy 
subjects. Safety and tolerability were compared to those of a placebo. In total 90 subjects were 
assigned to one of 4 treatment groups: 0.04% PHMB (n=27), 0.06% PHMB (n=27), 0.08% PHMB 
(n=27), or placebo (n=9). In each group, subjects received the study drug/placebo 12 times daily 
(1 drop every hour, daytime only) for 7 days and, if well tolerated, followed by 6 times daily (1 


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drop every 2 hours, daytime only) for an additional 7 days. The primary safety variable of the 
study was the rate of dosing limiting events (DLEs) in each treatment group. DLEs were defined 
as all serious adverse events (AEs) and all AEs that prompts interruption of the study drug. The 
purpose of the analysis was to state whether each treatment group is at significantly larger risk 
than placebo. During the study no SAEs occurred. Five out of the 90 subjects enrolled had a DLE 
(DLE rate=5.6%). The total rate of AEs was 61.1%. No DLEs occurred in the group treated with 
placebo and 0.04% PHMB; two DLEs occurred in the group treated with 0.06% PHMB and three 
DLEs in the group treated with 0.08% PHMB. For the latter group, the DLE rate was =11.1%. The 
events causing the withdrawn from the study were all of ocular origin and of mild to moderate 
intensity. The most frequent AEs reported in the study were conjunctival staining, corneal staining, 
pain after instillation and conjunctival hyperemia. In conclusion, only mild to moderate ocular AEs 
occurred after an intensive treatment with 0.08% PHMB. In addition, these AEs in patients with 
Acanthamoeba keratitis are acceptable in patients with this severe infection in whom a more 
concentrated solution is expected to provide a higher cure rate. For comprehensive clinical 
information, refer to the Investigator's Brochure for PHMB.10  
1.1.3. 
Propamidine  
Propamidine isethionate is an aromatic diamidine licensed in several EU countries as an 
antimicrobial. The antimicrobial effects of the diamidines result from the cationic surface-active 
properties inducing structural membrane changes affecting cell permeability. Propamidine is 
active against Gram-positive non-spore forming organisms, but less active against Gram-negative 
bacteria and spore forming organisms. It also has antifungal properties and is effective against both 
the trophozoite and cyst forms of Acanthamoeba but it is used only in combination with PHMB or 
chlorhexidine. Propamidine is well tolerated by ocular tissues although prolonged treatment with 
propamidine may lead to toxic keratopathy in vitro and in vivo.5,14,15 
1.2. 
Overall Rationale for the Study 
Acanthamoeba keratitis is difficult to treat. If left untreated, the condition results in blindness or 
corneal transplant surgery in most patients (only 1 of the first 20 patients reported before the 
introduction of biguanide therapy had a good outcome, 3 had medical cures of whom 1 required a 
corneal transplant, 3 had their eyes removed and another 14 had 1 or more therapeutic corneal 
transplants.16 There are currently no drugs that are licensed for use in Acanthamoeba keratitis. The 
diamidines and biguanides are currently the most effective cysticidal anti-amoebic agents. 
However, the biguanides have the best amoebicidal and cysticidal activity. As a result, the current 
approach for the treatment for Acanthamoeba keratitis includes biguanides (PHMB or 
chlorhexidine) with or without the addition of diamidines (propamidine or hexamidine).5 The 
combination of 0.02% PHMB and 0.1% propamidine is the most commonly used combination.17 
However, cases with a very poor clinical response have been reported.5,6,13,18 Such failures are 
possibly related to poor drug penetration into the deep cornea.19 The bioavailability of PHMB in 
the corneal stroma is likely to be increased by increasing the concentration of PHMB above 0.02%. 
Therefore, the use of PHMB 0.08% can be expected to reduce the treatment failures seen with 
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2. 
OBJECTIVES AND HYPOTHESIS 
2.1. 
Definitions 
Clinical resolution is a clinical definition, resulting from a slit lamp examination, with the 
following findings: 
 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and 
oedema) that requires treatment, with a healed corneal epithelium and minimal punctate 
staining (10 dots or less equivalent to Grade 1 on the Oxford Scale).   
 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal 
hyperemia): mild conjunctival inflammation is acceptable if related to other concurrent 
conditions such as blepharitis. 
 
No limbitis, scleritis or anterior chamber inflammation.  
 
No relapse within 30 days of discontinuing all topical and systemic therapy given for 
Acanthamoeba keratitis.  
For regulatory and verification reasons there is an additional 60 day follow-up to exclude late 
relapses. 
The CRR_12 is the clinical resolution rate at 12 months from randomization and is defined as the 
percentage of subjects cured 30 days after discontinuing all study therapies, within 12 months of 
randomization. This is the primary outcome measure. Subjects whose disease has not resolved at 
12 months are considered treatment failures per protocol. 
2.2. 
Objectives 
The primary objective of the study is to compare the Clinical Resolution Rate (CRR) at 12 months 
from randomization (CRR_12) of 0.08% PHMB + placebo with that of 0.02% PHMB + 0.1% 
propamidine combination therapy, estimating the difference in CRR_12 together with the 
surrounding degree of uncertainty, and to test for therapeutic superiority or non-inferiority of 
0.08% PHMB monotherapy. 
A further aim of this study is to obtain additional safety information on 0.08% PHMB ophthalmic 
solution. 
2.3. 
Hypothesis 
The primary hypothesis to be tested is that the CRR_12 of subjects treated with 0.08% PHMB 
monotherapy, is superior, or worse by no more than an acceptable pre-defined 0.20 non-inferiority 
margin (Δ), compared to the CRR_12 of a 0.20 PHMB + 0.1% propamidine combination therapy, 
administered according to the Treatment and Follow-up Protocol described in Section 5.3, which 
is based on a consensus of currently clinical guidelines. 
Secondary hypotheses are:  


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 That adverse events, and those relating to toxicity in particular, are less with PHMB 0.08% 
monotherapy compared to the comparator.  
 That time to a cure is shorter in subjects receiving PHMB 0.08% monotherapy compared to 
the comparator. 
Note: The estimated CRR_12 from the start of treatment is 67% for the conventional (0.02% 
PHMB + 0.1% propamidine) combination therapy (from the sponsor’s observational, case series 
retrospective study 038/SI20) (63% when assuming a prevalence of late stage diseases in 38% of 
subjects).  
3. 
STUDY DESIGN AND RATIONALE 
3.1. 
Overview of Study Design 
3.1.1. 
Type of Study 
This is a randomized, double-masked, double-dummy, active-controlled, multiple center, parallel-
group Phase 3 study to evaluate the efficacy, safety and tolerability of 0.08% PHMB ophthalmic 
solution compared to the conventional 0.02% PHMB + 0.1% propamidine combination therapy in 
male and female subjects affected by Acanthamoeba keratitis.  
The study is designed as a superiority study with the possibility to test for non-inferiority if the 
superiority hypothesis is not met, according to the requirements of the guidance from the European 
Agency for the Evaluation of Medicinal Products (EMA) (CPMP/EWP/482/99 ).21 The specific 
conditions for this outcome are described in Section 7.4.  
The study consists of an eligibility screening visit, a treatment period including short ambulant 
visits, and a follow-up visit. 
A total of 130 subjects affected by Acanthamoeba keratitis will be assigned to one of the following 
2 treatment groups in a ratio of 1:1.  
Group 1: 0.08% PHMB + placebo  
Group 2: 0.02% PHMB + 0.1% propamidine combination therapy 
Efficacy evaluations will consist of assessment of best corrected visual acuity (BCVA), pupil test 
(swinging light test), slit lamp examination, EuroQol five-dimension scale (EQ-5D) questionnaire 
and visual functioning questionnaire 25 (VFQ25).  
Safety and tolerability evaluations will consist of adverse event (AE) reporting, clinical laboratory 
results (hematology, biochemistry and urinalysis), intraocular pressure (IOP), ophthalmoscopy, 
and photography of affected cornea. 


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3.1.2. 
Screening Visit 
Subjects will report to the research center for the eligibility screening according to the inclusion 
and exclusion criteria defined in Section 4.1 and 4.2, respectively, on the day of the first drug 
administration (Day 0).  
Subjects will receive an explanation of the purpose and nature of the study, and will be asked to 
review and voluntarily sign the study specific Informed Consent Form (ICF) prior to any study 
specific screening procedures being performed. The written informed consent will be obtained for 
all subjects, regardless of their eligibility for the study; the signed ICFs will be retained and 
archived and another copy will be provided to the subject. 
For subjects who have not reached the age of majority (i.e., 18 years of age in most jurisdictions), 
written informed consent will also be obtained from the subject’s parent(s), legal guardian(s), or 
legally authorized representative (LAR) (hereafter referred to as parent/guardian/LAR) prior to 
any study specific screening procedures being performed. The parent/guardian/LAR must also be 
willing to provide safety and efficacy information about the subject and oversee the administration 
of the investigational product. The parent/guardian/LAR must commit to accompanying the patient 
to each study visit. 
Eligibility screening will consist of assessments as presented in the schedule of assessments (page 
22).  
 
 


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A schematic overview of the screening procedure is presented in Figure 1. 
 
Figure 1: Schematic Presentation of the Screening Process  
 
All subjects with clinical signs consistent with Acanthamoeba keratitis and positive confocal 
findings consistent with Acanthamoeba keratitis will be eligible for randomization providing they 
have not been using a topical biguanide or diamidine (see Sections 4.1 and Synopsis).  
Protocol for bilateral disease: if both eyes are affected, only one eye will be treated with the study 
treatment and will be considered for the study. This will be the right eye, unless the severity of the 
infection differs in both eyes, in which case the worst affected eye should be selected. The fellow 
eye should be treated with the best treatment according to the clinical practice. 
All subjects at screening meeting the inclusion criteria and including those with negative confocal 
microscopy findings will have corneal scrapes for culture and histology of Acanthamoeba, and to 
perform Acanthamoeba deoxyribonucleic acid (DNA) identification by polymerase chain reaction 
(PCR).  
 
 
A. One or more clinical features of Acanthamoeba keratitis 
(Acanthamoeba keratitis suspects) 
Confocal microscopy 
Positive 
RANDOMIZATION 
0.08% PHMB monotherapy + placebo 
or 
0.02% PHMB + 0.1% Propamidine 
Inclusion in the study 
Culture and PCR 
Not enrolled 
(out of the study) 
Negative  
Culture and PCR 
For all randomized 
subjects  
YES 
Enter follow-up treatment plan  
NO 


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Note: this is being done for study quality, for correlation with confocal microscopy findings, and 
to identify patients with fungal and viral infection. Because culturing and PCR will take 2 to 8 
days, during which time subjects thought clinically to have Acanthamoeba keratitis would be 
starting treatment before randomization, this assessment is not part of the inclusion criteria. 
Subjects who have been proven to have fungal and viral infections, if randomized on the basis of 
clinical and confocal microscope findings, will be removed from the study, even though they may 
have concomitant Acanthamoeba keratitis. 
Subjects with a negative confocal microscopy evaluation, but a positive PCR/Culture (confocal 
false negative) will not be enrolled in the study but will be treated with the best clinically available 
treatment outside the study.  
 Subjects meeting the inclusion criteria, including findings on slit lamp examination and confocal 
microscopy consistent with Acanthamoeba keratitis are entered into the study and randomized to 
one of the two treatment arms. They will then enter the Treatment Plan and Follow-up protocol as 
presented in figure 2: Schematic Presentation of the Treatment and Follow-up Protocol as 
presented in 5.3. 
3.1.3. 
Treatment Period 
On Day 0, subjects will arrive at the clinical research center for the baseline assessments and will 
receive instructions on how to apply the eye drops. Subjects will be randomized to 1 of the 2 
treatment groups.  
Subjects will receive multiple doses of 0.08% PHMB + placebo or 0.02% PHMB + 0.1% 
propamidine combination therapy in the affected eye until resolution according to the treatment 
schedule presented in Section 5.2 Drug dosage and Administration.  
On Day 0, the first application of study medication will be done at the research center after 
completion of all baseline assessments and randomization. Thereafter, subjects will leave the 
clinical research center and study drug will be self-administered at home. Subjects will return to 
the clinical research center for ambulant visits on Days 7, 14, 21, 30 and every 30 days until 
resolution.  
Assessments will be performed during the treatment period as presented in the schedule of 
assessments.  
Follow-up medical examinations will be performed 30 and 90 days after end of treatment. The 
specific procedures that will be performed are presented in the schedule of assessments. 
3.2. 
Study Design Rationale 
The active control for this study is combination therapy of 0.02% PHMB and 0.1% propamidine, 
which is the most common therapy used for treatment of patients affected by Acanthamoeba 
keratitis.5,17 Randomization will be used to minimize bias in the assignment of subjects to treatment 
groups, to increase the likelihood that known and unknown subject attributes (e.g., demographic 
and baseline characteristics) are evenly balanced across treatment groups, and to enhance the 


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validity of comparisons across treatment groups. The study is double-masked to reduce potential 
bias during data collection and evaluation of endpoints. Since each subject will receive clinical 
supplies that cannot be made identical (single-use vials and multi-use containers), the study is also 
double dummy. To maintain the masking, subjects in each arm will use single-use vials (0.08% 
PHMB or 0.02% PHMB) and multi-use containers (placebo or 0.1% propamidine). 
4. 
SUBJECT POPULATION 
The study will be performed in subjects affected by Acanthamoeba keratitis. 
Screening will be performed on the day of the first drug administration (Day 0), or up to two days 
before study drug administration.  
4.1. 
Inclusion Criteria 
Each potential subject must satisfy all of the following criteria to be enrolled in the study:  
1. 
Subject must be able and willing to give informed consent. 
2. 
Subject must be a man or woman of any race and ≥12 years of age, inclusive. Subjects <18 
years will only be enrolled in selected study sites. 
3. 
Subject must be able to understand and willing to comply with study procedures, 
restrictions and requirements, as judged by the investigator. 
4. 
Clinical findings consistent with Acanthamoeba keratitis. 
Clinical findings include the following: 
 
Epithelial lesions: epithelial punctate keratopathy, epithelial infiltrates, epithelial 
defects, dendritiform epithelial ulcers.  
 
Extracorneal 
lesions: 
limbal 
inflammation 
(limbitis), 
anterior 
scleral 
inflammation, diffuse or nodular.  
 
Stromal lesions: perineural infiltrates, anterior stromal infiltrates, disciform 
corneal swelling, stromal ulceration, ring abscess.  
 
Anterior chamber lesions: keratic precipitates, hypopyon.  
 
Late findings: fixed dilated pupil, mature cataract. 
5. 
Confocal microscopy findings consistent with Acanthamoeba keratitis (performed within 
7 days prior to study entry or as part of screening procedures) 
 
Confocal microscopy findings include: cysts are round or ovoid, may show a 
double wall and are 15-30 µm in size. 
6. 
Subjects using the following previous treatments for Acanthamoeba keratitis are eligible 
for the study: 


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 Antibiotics: subjects who have an ocular bacterial infection at baseline are eligible 
for the study. However, only topical moxifloxacin is permitted, unless resistant  or 
contraindicated. 
Note: subjects who develop intercurrent bacterial infections will be retained in the 
study and treated with topical moxifloxacin. 
 Antiviral drugs and antifungal drugs: subjects are often misdiagnosed as having 
these infections when they have Acanthamoeba keratitis. Subjects taking antivirals 
and antifungal agents (except for any using PHMB or Chlorhexidine) for a 
misdiagnosis can be included, but must discontinue these drugs after entry into the 
study. 
Note: subjects who are thought to have combined Acanthamoeba keratitis with 
herpes or fungal keratitis are excluded from the study. 
 Anti-inflammatory drugs: Subjects using topical steroids and/or oral NSAIDs 
before the diagnosis of Acanthamoeba keratitis are eligible for the study. 
However, these subjects must agree to change therapy to the topical steroids and 
oral NSAIDs that are specified for use in the study (as described in section 4.3 
Concomitant Medication and Other Restrictions during Study). 
 
7. 
Females of childbearing potential will be included if they are either sexually inactive 
(sexually abstinent for 14 days prior to the first study drug dose continuing through 28 days 
after the last study drug dose, or using one of the following highly effective contraceptive 
(i.e. results in <1% failure rate when used consistently and correctly) methods in this study: 
a. 
intrauterine device (IUD); 
b. 
surgical sterilization of the partner (vasectomy for 6 months minimum); 
c. 
combined (estrogen or progestogen containing) hormonal contraception associated 
with the inhibition of ovulation (either oral, intravaginal, or transdermal); 
d. 
progestogen only hormonal contraception associated with the inhibition of ovulation 
(either oral, injectable, or implantable); 
e. 
intrauterine hormone releasing system (IUS);  
f. 
bilateral tubal occlusion. 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the study 
treatments. In this study, abstinence is only acceptable if in line with the subjects preferred 
and usual lifestyle. 
Periodic abstinence (calendar, symptothermal, post-ovulation methods), withdrawal 
(coitus interruptus), spermicides only, and lactational amenorrhoea method (LAM) are not 


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acceptable methods of contraception.  As well, a female condom and a male condom should 
not be used together.  
8. 
Females of childbearing potential agree to remain sexually inactive or to keep the same 
birth control method for at least 28 days following the last study drug dose. 
9. 
A female of non-childbearing potential must have undergone one of the following 
sterilization procedures at least 6 months prior to the first study drug dose: 
a. 
hysteroscopic sterilization; 
b. 
bilateral tubal ligation or bilateral salpingectomy; 
c. 
hysterectomy; 
d. 
bilateral oophorectomy; 
or be postmenopausal with amenorrhea for at least 1 year prior to the first study drug dose 
and follicle stimulating hormone (FSH) serum levels consistent with postmenopausal status 
10. 
A non-vasectomized male subject agrees to use a condom with spermicide or abstain from 
sexual intercourse during the study until 90 days beyond the last dose of study drug and 
the female partner agrees to comply with inclusion 7 or 8. For a vasectomized male who 
has had his vasectomy 6 months or more prior to study start, it is required that they use a 
condom during sexual intercourse. A male who has been vasectomized less than 6 months 
prior to study start must follow the same restrictions as a non-vasectomized male. 
Periodic abstinence (calendar, symptothermal, post-ovulation methods), withdrawal 
(coitus interruptus), spermicides only, and LAM are not acceptable methods of 
contraception. As well, a female condom and a male condom should not be used together. 
11. 
If male, they must agree not to donate sperm from the first study drug dose until 90 days 
after dosing. 
4.2. 
Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from participation in the study: 
1. 
Subject with documented history and/or clinical signs of concomitant presence of an ocular 
infection caused by viruses (herpes simplex virus [HSV]) or fungi.  
2. 
Subject treated with drugs having effects on Acanthamoeba cysts prior to study entry, 
including biguanides (PHMB, chlorhexidine) and diamidines (propamidine, hexamidine). 
3. 
Subjects requiring systemic immunosuppression for Acanthamoeba associated scleritis. 
4. 
Subjects requiring urgent surgical intervention for advanced Acanthamoeba keratitis in 
either eye (e.g., for advanced corneal thinning/melting etc.). 


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5. 
Subject with known or suspected allergy to biguanides, diamidines or intolerance to any 
other ingredient of the investigational treatments. 
6. 
Subject affected by immunodeficiency diseases or taking systemic immunosuppressive 
therapy. 
7. 
Subject with a major systemic disease or other illness that would, in the opinion of the 
investigator, compromise subject’s safety or interfere with the collection or interpretation 
of study results. 
8. 
If female, pregnancy, planned pregnancy, or breast-feeding. 
9. 
Subject is participating in another interventional clinical study with an experimental or 
unapproved/unlicensed therapy or has participated in another interventional clinical study 
within 4 weeks prior to this study. 
The investigator must ensure that all study enrolment criteria have been met at randomization. If 
a subject's status changes after randomization, but before the first dose of study drug is given such 
that he or she no longer meets all eligibility criteria, then the subject should be excluded from 
participation in the study. 
4.3. 
Concomitant Medication and Other Restrictions during Study 
Potential subjects must be willing and able to adhere to the following prohibitions and restrictions 
during the course of the study to be eligible for participation: 
1. Female subjects of childbearing potential have to be sexually inactive (abstinent) or have to 
use an acceptable birth control method as described in Section 4.1 during the study and 
continuing through 28 days after the last dose of study medication. 
2. Non-vasectomized male subjects have to use an acceptable birth control method as described 
in Section 4.1 during the study continuing through 90 days after the last dose of study 
medication. Female partners should also use an acceptable birth control method as described 
in Section 4.1 during the study and continuing through 28 days after the last dose of study 
medication. 
3. Subjects are allowed to use the following concomitant medications during the study: 
Antibiotics: Topical moxifloxacin is permitted for the treatment of intercurrent bacterial 
infections (unless culture and sensitivity, or clinical progress demands a change). Topical 
moxifloxacin is not permitted for use as a prophylactic antibiotic in patients with corneal 
ulcers; PHMB is a good broad spectrum anti-bacterial and an additional antibacterial is not 
needed for this. The value of prophylaxis is unproven. 
Antiviral drugs and antifungal drugs: The use of these drugs is not allowed during the study. 
If subjects are using antiviral or antifungal drugs at study entry, they must be discontinued.  


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Anti-inflammatory drugs: For subjects on steroids at study entry (already using topical 
steroids for e.g. mistaken diagnosis of HSV keratitis or as adjunctive treatment for bacterial 
keratitis), there are the following options: 
a. Either stop steroids, OR maintain, OR reduce the doses (at the investigator’s 
discretion). Unpreserved Dexamethasone (0.1% or 0.15%)  is the only topical steroid 
permitted for use in this trial.  Patients using any other topical steroids on trial entry 
should be changed to this at the appropriate frequency.  Diclofenac is the only oral 
NSAID permitted in the trial and will be ADDED at the appropriate dose (75 mg to 
150 mg daily, divided in two or three  doses) and continued at any level while topical 
steroids are in use during the study. 
 
b. Subjects using topical NSAIDs and ciclosporin at study entry should have these 
discontinued after randomization. 
c. Subjects using no topical steroids at study entry can have these started together with 
oral NSAIDs (recommended diclofenac, 75 mg to 150 mg daily, divided in two or three  
doses) during the study as specified in the schematic overview of the Treatment and 
Follow-up Protocol. 
i. 
Dosing frequency of topical dexamethasone (0.1% or 0.15%) : at the discretion 
of the principal investigator between 1 drop every second day to hourly 
depending on the degree of inflammation. 
ii. 
Dose of diclofenac: at the discretion of the principal investigator. The 
recommended dosing is 75 mg to 150 mg daily, divided in two or three doses 
depending on the degree of inflammation 
Other permitted topical mediations: Unpreserved lubricants, mydriatics (cyclopentolate, 
homatropine or atropine) and glaucoma medications are permitted. For glaucoma medications 
a timolol preparation is recommended as first line (for its low toxicity) and prostaglandin 
analogues as last resort because of their potential to increase inflammation. 
All concomitant medication used in relation to the current eye infection will be reported. For the 
remaining concomitant medication, only medication in use within 30 days before signing the ICF 
will be reported in the case report form (CRF). The name of the drug, start and stop date, the 
indication for use, the total daily dose and route of administration will be recorded. Medications 
will be coded by standardized dictionary. 
4.4. 
Subject Completion/Withdrawal 
Any subject may voluntarily discontinue the study at any time without prejudice. The investigator 
may elect to discontinue a subject for reasons unrelated to the study drug (e.g. failure to comply 
with the study protocol, missed visits etc.), for reasons related to the study drug ([serious] adverse 
event ([S]AE])) or in case of treatment failure. In either event, reason(s) for discontinuation should 
be recorded on the CRF. Possible reasons for study discontinuation include the following: 
 
Ocular intolerance. 


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 
(S)AEs necessitating discontinuation from the study. 
 
Treatment failure. 
 
The subject is lost to follow-up. 
 
Subject decision not related to an (S)AE. 
 
Investigator decision (specify). 
 
Other reason (specify). 
Subjects who miss up to 1 full day of treatment within the first 5 days after starting treatment or 
up to 2 full days of treatment after the first 5 days of treatment should not be discontinued, but will 
be classified as non-compliant. Subjects will be discontinued from the study if in the opinion of 
the investigator drug compliance is insufficient.   
If the subject discontinues, all the end-of-study visit assessments must be performed, if possible. 
Moreover, the investigator should ensure (as far as possible) that all used and unused study drug 
has been collected from the subject. The subject will exit the study and be treated at the 
investigator’s discretion. Discontinued subjects will not be replaced. 
Subjects discontinued for (S)AE(s) will be followed-up after subject’s discontinuation until the 
event is resolved or considered medically stable by the investigator. If necessary, the investigator 
may schedule further visits at his/her discretion. Data collected in the period up to the end-of-study 
visit will be recorded in the CRF. Data collected after Day 90 following the end of treatment, and 
related to the (S)AE that led to discontinuation will be recorded in the subject’s medical record, 
and in the sponsor’s Safety Database. 
In case a subject is lost-to-follow-up, the investigator will attempt to contact the subject (by phone, 
letter or e-mail) at least twice. 
A subject will be considered to have completed the study if all required assessments up to and 
including the 90 days after end of treatment and end-of-study assessments have been completed.  
5. 
TREATMENTS 
5.1. 
Treatment Assignment  
After obtaining oral and written informed consent, subjects meeting the eligibility criteria will be 
randomized to 1 of the treatment groups in a 1:1 ratio. The randomization schedule will be 
generated using a computer program and verified for accuracy using strict quality control 
procedures. Eligible patients will receive a masked treatment assignment with a unique 
randomization code based on the randomization list. The assigned randomization code will be 
captured in the electronic Case Report Form (eCRF). This randomization code does not disclose 
any treatment assignment.  


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Protocol for bilateral disease: if both eyes are affected, only one eye will be treated with the study 
treatment and will be considered for the study. This will be the right eye, unless the severity of the 
infection differs in both eyes, in which case the worst affected eye should be selected. The fellow 
eye should be treated with the best treatment according to the clinical practice. 
Subjects will be assigned to the following treatment groups: 
Group 1: 0.08% PHMB + placebo  
Group 2: 0.02% PHMB + 0.1% propamidine combination therapy 
5.2. 
Drug dosage and Administration 
On Day 0, subjects will receive instructions on how to apply the eye drops. Subjects will receive 
the assigned treatment to be administered in the affected eye until resolution. Subjects will be 
treated for a maximum of 1 year after randomization and subjects meeting the criteria for clinical 
resolution at or before 12 months will be followed up for 90 days before completing the study. 
Subjects will receive an initial intensive course of 0.08% PHMB + placebo or 0.02% PHMB + 
0.1% propamidine combination therapy. 
Treatment throughout the study will be given using 1 drop of each ophthalmic solution in the study 
eye at different frequencies during the daytime only. At study entry, an intensive 19-day 
antiamoebic treatment protocol will be initiated as follows: 
Day 0 to 5* 
1-hourly drops (16 drops in a day) for 5 days 
Day 6 to 12 
2-hourly drops (8 drops in a day) for 7 days 
Day 13 to 19 
3-hourly drops (6 drops in a day) for 7 days 
Day 20  
Then 4x daily thereafter 
 
* on the first day of treatment (after randomization) 16 drops might not be feasible, in this case the 
treatment should be applied as many times as possible but not exceeding the hourly regimen. 
Notes on treatment 
i. 
The frequency will be maintained at 4x daily unless there is a relapse (see Box XI, Relapse 
on page 42). In the event of a relapse, culture negative subjects will receive the same 
intensive 19-day treatment course as given at study entry.  
ii. 
Antiamoebic drops will be discontinued when clinical resolution is reached (see Figure 2 
and Legend IV Discontinue treatment).  
iii. 
Clinical resolution is a clinical definition, resulting from a slit lamp examination, with the 
following findings:  


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 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and 
oedema) that requires treatment, with a healed corneal epithelium and minimal punctate 
staining (10 dots or less equivalent to Grade 1 on the Oxford Scale).   
 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, 
tarsal hyperemia): mild conjunctival inflammation is acceptable if related to other 
concurrent conditions such as blepharitis. 
 
No limbitis, scleritis or anterior chamber inflammation.  
For subjects on steroids or oral NSAIDs at study entry (already using topical steroids for e.g. 
mistaken diagnosis of HSV keratitis or as adjunctive treatment for bacterial keratitis), the same 
intensive 19-day antiamoebic treatment protocol will be used. For their steroid use there are the 
following options: 
 Either stop steroids, OR maintain, OR reduce the doses (at the investigator’s discretion). 
Unpreserved Dexamethasone (0.1% or 0.15%)  is the only topical steroid permitted for use 
in this trial.  Patients using any other topical steroids on trial entry should be changed to 
this at the appropriate frequency.  Diclofenac is the only oral NSAID permitted in the trial 
and will be ADDED at the appropriate dose (usually 75 mg to 150 mg three times daily) 
and continued at any level while topical steroids are in use during the study. 
 
Detailed instructions about the use of concomitant medication and adjunctive therapy are 
given in Section 4.3 “Concomitant medication and other restrictions during study”  and also in 
Section 5.3 “Treatment and Follow up Protocol” 
The study drug(s) will be applied to the affected eye. In case both eyes are affected, only 1 eye 
will be treated with study treatment. This will be the right eye, unless the severity of the infection 
differs between eyes, in which case the worst affected eye should be selected. The fellow eye 
should be treated with the best treatment according to the clinical practice.  
One drop of 0.1% propamidine or placebo (multiple-use containers) will be administered first, 
followed after a minimum of 5 minutes by 0.08% or 0.02% PHMB (single-use vials).  
The single-use vials should be used immediately after opening and the remaining solution should 
be discarded. The multiple-use containers should be closed after each use and kept for up to 
28 days after opening. 
On Day 0, study drugs will be administered after completion of all baseline assessments and 
randomization. The first application will be done at the clinical research center by the subject under 
supervision of a member of the study team. Thereafter, study drugs will be applied at home. 
Before administration, both single-use vials and the multiple-use containers should be shaken. 
If the subject misses a dose of study drug, the subject should wait and take the next dose according 
to the treatment schedule. Subjects who miss up to 1 full day of treatment within the first 5 days 


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after starting treatment, or up to 2 full days of treatment after the first 5 days following the start of 
treatment should not be discontinued, but will be classified as non-compliant. These subjects will 
be included in the Full Analysis Set and excluded from the analysis of the Per-Protocol Analysis 
Set (see Section 7.17.1).  
The vials and containers should be stored at a temperature lower than 25°C (or at room 
temperature) and protected from freezing. Research center staff will instruct subjects on how to 
store study drug for at-home use. 
 
5.3. 
Treatment and Follow-up Protocol 
A schematic overview of the treatment and follow-up period is presented in figure 2.  
 


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. 
 
 
 
Figure 2: Schematic Presentation of the Treatment and Follow-up Protocol  
 
 
 


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Legend for Figure 2 - The text boxes and lines in the figure are color coded:  
 
 
Pale blue text boxes and lines for responding disease or treatment success and STUDY 
END POINT 
 
Red text boxes and lines for poorly responsive and recurrent / relapsing disease presumed to 
be due to replication of Acanthamoeba  
 
Pink text box for exacerbations of inflammation presumed to be due to flare ups of 
inflammation secondary to the immune response to non-viable Acanthamoeba 
 
Black text boxes for treatment failure endpoints ALL leading to TRIAL EXIT 
 
 
This protocol defines the treatment of patients. Treatment options are defined as far as possible to 
reduce variations, which will complicate or invalidate the analysis of the results, whilst 
maintaining optimal standards of care based on current evidence and expert opinion. The changes 
of therapy in this protocol, and therapeutic failures, are determined by both the development and 
resolution of clinical signs of inflammation and ulceration, as well as the identification of 
Acanthamoeba by culture during disease relapses.  
Each box is labelled with a Roman numeral I to XVIII. The legend below describes the rationale for each 
step: 
 
BOX I. 
FOLLOW UP PROTOCOL from day zero at TRIAL ENTRY. 
Treatment throughout the study is given using 1 drop of each ophthalmic solution in the study eye 
at different frequencies during the daytime only.  
At study entry initiate an intensive 19-day antiamoebic treatment protocol: 
Day 0 to 5 
1-hourly drops (16 drops in a day) for 5 days 
Day 6 to 12 
2-hourly drops (8 drops in a day) for 7 days 
Day 13 to 19 
3-hourly drops (6 drops in a day) for 7 days 
Day 20  
then 4x daily thereafter  
 
a. This frequency is maintained at 4x daily unless a relapse (BOX XI - RELAPSE) occurs. In 
case of a relapse, culture negative subjects receive the same intensive 19-day treatment 
course as given at study entry.  
b. Antiamoebic drops are discontinued when IV - DISCONTINUE TREATMENT is reached.  
In subjects on steroids at TRIAL ENTRY: (already using topical steroids for e.g. mistaken 
diagnosis of HSV keratitis or as adjunctive treatment for bacterial keratitis), the same intensive 19-
day antiamoebic treatment protocol is used. For their steroid use there are these options: 
a. Either stop steroids, OR maintain steroids, OR reduce the doses of steroids (at the 
investigator’s discretion). Change steroid therapy to unpreserved topical 
dexamethasone (0.1% or 0.15%) if not using this already. When steroid is to be 
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(NSAID). The oral NSAID used in the trial is diclofenac; change therapy to 
diclofenac if patient is on alternative NSAID at study entry.  
Dexamethasone (0.1% or 0.15%) drops and oral diclofenac are permitted in the study and may be 
discontinued, tapered or continued on inclusion into the study at the discretion of the investigator: 
 Dosing frequency of topical dexamethasone (0.1% or 0.15%): between 1 drop every second 
day to hourly depending on the degree of inflammation. 
 Dose of diclofenac: The recommended dosing is 75 mg to 150 mg daily, divided in two or 
three doses depending on the degree of inflammation 
For subjects using antifungals or antivirals on study entry: discontinue antifungals or 
antivirals. 
For subjects using topical antibiotics on study entry for concurrent bacterial keratitis: alter 
antibiotic to moxifloxacin unless there is evidence from microbiology studies that the bacteria are 
resistant to moxifloxacin.  
Prophylactic antibiotics for patients with ulceration: discontinue prophylactic antibiotics. 
Other permitted topical mediations: Unpreserved lubricants, mydriatics (cyclopentolate, 
homatropine or atropine) and glaucoma medications are permitted. For glaucoma medications a 
timolol preparation is recommended as first line (for its low toxicity) and prostaglandin analogues 
as last resort because of their potential to increase inflammation.   
After the 20 day treatment in Box I patients will either stabilize or improve and move to the 
treatment pathway in Box II or deteriorate and move to the pathway in Box VIII.  
 
5.3.1. 
Responding Disease or Treatment Success 
BOX II  
STABLE 
Stable at 3 weeks is defined as no deterioration but not improving. These STABLE subjects may 
improve or not over the next few weeks. If no improvement, then these patients may be started 
on anti-inflammatory treatment as in Box IX (no re-culture needed. Alternatively the 
inflammation may deteriorate (this may occur at any stage of treatment) and the rationale for 
managing this problem is described here: 
DETERIORATION of inflammation (after excluding co-infection with other organisms). 
 
The two possibilities which the PI needs to differentiate between are:  
 


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a. Relapse of infection (Box XI) presumed or proven to be due to continued 
replication of Acanthamoeba) OR  
b. Exacerbation of inflammation (Box XII) presumed to be due to inflammation 
secondary to the immune response to non-viable Acanthamoeba 
XI RELAPSE of infection is identified by a positive culture, unfortunately very insensitive die to 
the persistence of deep organisms, supported by an increase in cysts on confocal (also insensitive 
in severe disease) or using clinical criteria: development of more severe corneal inflammation, 
melting, ulceration, hypopyon, development of ring abscess necessitating another intensive 
course of therapy. This is as opposed to:  
XII EXACERBATION of inflammation: which is accompanied by mild conjunctival or corneal 
inflammation (sometimes coarse anterior stromal infiltrates like those following adenovirus 
keratitis) and/or mild scleritis. XV SCLERITIS unresponsive to topical steroids and oral 
NSAIDS is another cause of this: these patients are complex to manage and as a result we 
decided that these should exit the trial.  
Once a decision has been made between these two the appropriate protocol is followed for each 
scenario as outlined in XI and XII.  
Note: if this occurs after 11 month (335 Days) it is a treatment failure XVII (as patients relapsing 
at this point will not have time to respond to treatment) and the patient exits the trial. 
 
BOX III 
IMPROVEMENT  
Reducing inflammation and some healing of any ulcers and/or improving symptoms. These 
subjects may continue to improve to the point they go to IV Discontinue treatment. 
Deterioration of inflammation: Patients in this subset may deteriorate due to: XI Relapse of 
Infection or XII Exacerbation of inflammation in which case they are managed as described in 
paragraph II a, b for these issues.   
 
 
BOX IV 
DISCONTINUE TREATMENT  
DISCONTINUE TREATMENT (includes topical steroids, oral NSAIDs and topical 
antiamoebics/microbials) is when any ulcers have healed and all signs of Acanthamoeba keratitis 
related inflammation have resolved (see detailed description in the protocol; mild conjunctival 
inflammation related to drugs or other condition such as blepharitis are acceptable) at or before 12 
months (365 days) from study entry. Clinical resolution (CR) after 365 days is a failure (see Box 
XVI - RELAPSE AFTER 11 MONTHS). It is recommended that topical steroids are discontinued 
2-4 weeks before discontinuing topical anti-amoebics. 
BOX V 
Follow-up visit (Day 30 after last treatment) 


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Deterioration of inflammation: Patients in this subset may deteriorate due to: XI Relapse of 
Infection or XII Exacerbation of inflammation in which case they are managed as described in 
paragraph II a, b for these issues.   
 
BOX VI 
Follow-up visit (Day 90 after last treatment) 
Follow-up visit 90 days (D90) after last treatment has two outcomes: if there are no signs of a 
relapse by 15 months (Day 455) at latest, the pathway goes to Box VII - TREATMENT SUCCESS 
maintained STUDY-END-POINT. In the event of signs of a RELAPSE, the pathway is the same 
as for relapses in  box XI or XIIP).  
BOX VII  
Treatment success maintained study end point. EXIT TRIAL 
 
5.3.2. 
Poor Response at 3 weeks or Treatment Failure 
BOX VIII 
POOR RESPONSE at 3 weeks. 
Poor response at 3 weeks from I Trial entry or XIII RESTART: patients are managed using the 
guidelines in IX. Criteria for this include enlarging ulcer, stromal thinning, deteriorating corneal,  
inflammation (infiltrate including ring abscess), intraocular inflammation (uveitis and hypopyon) 
and/or scleritis.  
BOX IX 
INTRODUCE ANTI-INFLAMMATORY TREATMENT 
a. For subjects NOT using topical steroids start these WITH oral NSAIDs. The topical steroid 
that is permitted in the Trial is unpreserved dexamethasone (0.1% or 0.15%). The oral 
NSAID used in the trial is diclofenac. The frequency of the drops and the dose of oral 
NSAID that is used is at the discretion of the Investigator.   
b. For subjects already on unpreserved topical dexamethasone (0.1% or 0.15%) and oral 
diclofenac, increase the frequency of the steroid drops and dosage of oral diclofenac when 
possible.  
This is expected to result in an improvement for most cases and they move to BOX II.1  STABLE 
or BOX III IMPROVEMENT. 
BOX X 
 
CONTINUED DETERIORATION 
Alternatively, if continued deterioration occurs, cases move to Box XVIII  - TREATMENT 
FAILURE and EXIT Trial. 
5.3.3. 
Relapse  
BOX XI 
RELAPSE WITHIN 11 MONTHS  
Relapse within 11 months, after an initial response is common. Cultures for Acanthamoeba and 
other organisms (fungi, bacteria and herpes) must be repeated, deriving from a repeated corneal 
biopsy, if the investigator thinks this is appropriate.  


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c. If the CULTURES are NEGATIVE patients this may mean that the anti-amoebic 
therapy is having some effect although we have no way of being certain about this 
(confocal and PCR don’t help as these can be positive when the organisms are non-
viable). These patients are managed as described in XIII below with another 19 day 
course of intensive therapy, with or without anti-inflammatory therapy depending 
on status.  
d. If CULTURES are POSITIVE patients have TREATMENT FAILURE and 
managed as described in XIV and XVII below. 
 
Box XII. 
EXACERBATION of inflammation is presumed to be due to inflammation 
secondary to the immune response to non-viable Acanthamoeba and anti-inflammatory treatment 
can be re-started or increased at the discretion of the PI without the need for re-culture and a further 
course of intensive anti-amoebic therapy. IMPORTANT: if anti-amoebic treatment had been 
stopped restart this when anti-inflammatory treatment is re-started.  
Distinguishing between XI and XII is often difficult and guidelines for this are given in II a, b 
above. 
Box XIII. 
Restart with another 19 day intensive course of anti-amoebic therapy with or 
without topical steroids and oral NSAIDS:  
a. 
If patients were not on these at the time of the relapse of infection we recommend not using 
these until after the 19 day course of intensive treatment and depending on response move to as 
above. 
b. 
If they were on them at the time of the relapse continue at a dose determined by the PI and 
follow pathways II, III, IX or X. 
Box XIV. 
CULTURE POSITIVE these patients are Exit Trial and are managed as described 
in XVII below.  
Box XV. 
SCLERITIS UNRESPONSIVE to treatment in IX. This is a failure (see XVII 
below). 
Box XVI. 
RELAPSE after day 335 or ingoing inflammation and treatment at day 365: this is 
a treatment failure and is managed as described in XVII below 
Box XVII. 
TREATMENT FAILURE and EXIT TRIAL. The treatment randomization 
allocation will be opened by the Sponsor at this point. Trial drug (PHMB 0.08%) will be available 
to the patient, if requested by the Investigator, providing the patient continues trial follow up for 
safety evaluation. 
XVIII. Summary of Study end points. 
 


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5.3.4. 
End of Study 
XIX. 
Summary of Study end points. 
End-of-study is defined as the last follow-up visit at 90 days after end of treatment, or the day of 
study discontinuation. Discontinuation because of clinical failure is defined as follows: 
1. 
Clinical resolution not achieved after 1 year of study treatment 
2. 
Clinical relapse within 3 months after end-of-treatment, if occurring more than 12 months 
after randomization 
 


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5.4. 
Identity of Investigational Product 
Test treatment: 0.08% PHMB + placebo  
Name 
: PHMB 
Active compound 
: polyhexamethylene biguanide 
Activity 
: Cationic surface-active antimicrobial agents 
Indication 
: Not applicable 
Strength 
: 0.08% 
Dosage form 
: eye-drops solution provided in 0.3 mL single-use vials 
Manufacturer 
: SIFI SpA, Italy 
Name 
: placebo  
Substance 
: sodium chloride, sodium phosphate buffer, benzalkonium chloride and 
water for injections 
Active compound 
: not applicable 
Dosage form 
: eye-drops solution provided in 10 mL multiple-use containers, visually 
identical to test reference treatment (propamidine multiple-use containers) 
Manufacturer 
: SIFI SpA, Italy 
Reference treatment: 0.02% PHMB + 0.1% propamidine combination therapy 
Name 
: PHMB 
Active compound 
: polyhexamethylene biguanide 
Activity 
: Cationic surface-active antimicrobial agents 
Indication 
: not applicable 
Strength 
: 0.02% 
Dosage form 
: eye-drops solution provided in 0.3 mL single-use vials 
Manufacturer 
: SIFI SpA, Italy 
Name 
: propamidine (Brolene) 
Active compound  : propamidine isethionate 
Activity 
: bacteriostatic properties against a wide range of organisms 
Indication 
: minor eye infections 
Strength 
: 0.1% 
Dosage form 
: eye-drops solution provided in 10 mL multiple-use containers 
Manufacturer 
: Sanofi, United Kingdom 
Excipients of PHMB are sodium chloride, sodium phosphate buffer and water for injections. 
Excipients of propamidine are ammonium chloride, sodium chloride, benzalkonium chloride, 
sodium hydroxide, water for injections. 
Excipients of placebo are sodium chloride, sodium phosphate buffer, benzalkonium chloride and 
water for injections. 


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5.5. 
Selection of Doses in the Study 
There is consensus that a combination of 0.02% PHMB and 0.1% propamidine eye drops is 
effective for treatment of Acanthamoeba keratitis. However, cases with very poor clinical response 
have been reported. These failures are possibly related to poor drug penetration into the deep 
cornea. Therefore, the bioavailability of PHMB in the corneal stroma is likely to be increased by 
increasing the concentration of PHMB above 0.02%. A randomized, double-masked, placebo-
controlled Phase 1 study was conducted to establish the ocular safety and tolerability, and systemic 
safety of 3 different concentrations of PHMB in healthy subjects (0.04%, 0.06% and 0.08% 
administered 12 times daily (1 drop every hour, daytime only) for 7 days followed by 6 times daily 
(1 drop every 2 hours, daytime only) for an additional 7 days). This study showed that intensive 
administration of 0.08% PHMB is safe and well tolerated in healthy subjects. Therefore, consistent 
with the CHMP advice from the EMA provided in July 2014, the highest tolerated concentration 
tested in the Phase 1 study (0.08%) will be used in this Phase III study in subjects with 
Acanthamoeba keratitis. 
5.6. 
Masking 
This study is designed as double-masked, double-dummy study. However, the bottle of the dummy 
(placebo), although very similar, is not identical to the bottle of the study product, as it was not 
possible to obtain an identical bottle. Because unmasking is allowed per protocol in case of lack 
of efficacy, there is a small risk of disclosing of the treatment assignment for subjects continuing 
to participate in the study. To reduce this risk, and to protect the integrity of the data, efficacy 
assessments should be performed by study personnel not having access to the study treatment, 
dispensing logs, accountability forms, the eCRF, or other sources of treatment assignment 
information (assessor-masked design). In particular the records of unmasked subjects should not 
be seen by the doctors carrying out the study. The investigator and other study staff, the subjects, 
the monitors and the sponsor will only have access to masked treatment assignment information 
on a ‘need to know basis’ until data collection has been completed, the database is locked and the 
protocol deviations and the primary reason for discontinuation from the study for each subject are 
determined. The exact requirements and process will be outlined in the Study Operations Manual. 
The randomization code does not disclose any treatment information. Until the moment of 
unmasking, the treatment assignments linked to the codes are accessible only by the individuals 
appointed by the Sponsor as Qualified Persons responsible for Pharmacovigilance. Emergency 
Unmasking should only be done if knowledge of treatment assignment is considered relevant for 
medical care of the patient. Unmasking is an Investigator’s responsibility and, if in doubt, can be 
discussed with the Medical Monitor.  Details will be provided in the Study Operations Manual. 
Subjects will be treated for a maximum of 1 year after randomization. Study treatment may be 
unmasked for serious safety reasons.  These unmasked subjects will be discontinued from the study 
The randomization code will be broken when all subjects have finished the study. 


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5.7. 
Drug Accountability 
The investigational site will maintain an inventory of study drug supplied by the Sponsor. All drug 
supplies must be kept in a locked room that can be accessed only by the pharmacist, the 
investigator, or another duly designated person. Specifically study staff performing efficacy 
assessments must not have access to the study drug. This study drug must not be used outside the 
context of this protocol. Under no circumstances should the investigator or site personnel supply 
study drug to other investigators or clinics, or allow the supplies to be used other than as directed 
by this protocol without prior authorization from the sponsor.  
Adequate records on receipt, use, return, loss, or other disposition of medication must be 
maintained. A specific drug accountability form or computer records used by the pharmacy at the 
investigational center, can be used to provide drug accountability information. In either case, 
information describing study drug supplies and the dispensing record, subject by subject, must be 
provided, signed by the investigator (or the pharmacist or other person who dispensed the drug) 
and collected by the local study monitor. Requisite data includes relevant dates, quantities, batches 
or code numbers, and subject identification for subjects who received study drug. 
The investigator or designated study-site personnel will maintain a log of all study drug dispensed 
and returned. Drug supplies for each subject will be inventoried and accounted for throughout the 
study. When study drug is self-administered by subjects, the number of single-use vials and 
multiple-use containers with study drug dispensed will be recorded and compared with the number 
of used and unused vials returned.  
At the end of the study, following authorization by study management and on agreement of the 
sponsor, drug may be destroyed on site as dictated by the appropriate standard procedures at the 
participating institution. Destruction must be documented. Alternatively, all unused products will 
be collected by the local monitor and returned for destruction. 
 
6. 
STUDY EVALUATIONS 
6.1. 
Study Procedures 
In the case of both eyes being affected, all study procedures will be performed in the ‘study eye’ 
only. 
6.1.1. 
Overview 
The schedule of assessments, presented on page 22, summarizes the frequency and timing of 
assessments applicable to this study. 
6.1.2. 
Screening Measurements 
Identification of Acanthamoeba by Confocal Microscopy 


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The subject will undergo confocal microscopy before any corneal scrapings. Confocal microscopy 
will be performed according to Standard Operating Procedures and applying a standard set of 
evaluation criteria, the Investigator is responsible for complying to this. Details will be provided 
in the Study Operations Manual. For each subject, 1 full-thickness scan that include endothelial, 
stromal, and epithelial images will be taken of the central cornea, regardless of infiltrate location. 
In addition, at least 1 additional full-thickness scan will be taken in the area of the infiltrate, by 
instructing the subject to look in the appropriate direction. Readers will also comment on the 
presence or absence of trophozoite-like and cyst-like structures (presumed to be Acanthamoeba), 
perineural inflammation, filaments or hyphae (presumed to be fungus), and inflammatory 
infiltrate, as well as the anatomic location of each of these findings (endothelial, posterior stroma, 
anterior stroma, or epithelial). Cysts will be defined as high contrast, hyper-reflective, spherical 
structures measuring 15 to 30 µm. Graders will record whether any cysts are present in the group 
of images. A positive confocal test will have at least 1 cyst. 
Images will be sent to a central reading center, however, the investigator’s decision will be 
accepted for inclusion in the study. 
6.1.3. 
Measurements for Routine Clinical Management 
Identification of Acanthamoeba by microbiological culture or cytological smear 
Corneal scrapings will be obtained under topical anesthesia using an aseptic technique. 
Cytopathology techniques 
To ensure homogeneity smears should be examined using preferably calcafluor white stain at all 
centers. Laboratories may use any additional techniques according to their local protocols.  
Culture techniques 
The study specimen (corneal epithelium, a corneal ulcer scrape or a corneal biopsy) should be 
cultured on non-nutrient agar plates inoculated with E. coli and processed according to standard 
local routine microbiological procedures. Any changes in culture techniques during the course of 
the study should be reported.  
 
Identification of Acanthamoeba DNA by PCR 
PCR will be performed locally according to standard local routine procedures.  
6.1.4. 
Baseline Measurements 
Vital Signs  
Blood pressure, pulse rate and body temperature will be assessed with the subject in supine position 
after 5 minutes of rest.  


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6.1.5. 
Efficacy Measurements 
All efficacy measurements should be performed by a staff member not having access to treatment 
assignment.  
Efficacy evaluations will consist of assessment of BCVA, pupil test (swinging light test), slit lamp 
examination, EQ-5D questionnaire and VFQ25 questionnaire. Assessments will be performed in 
accordance with the schedule of assessments. When multiple assessments are scheduled at the 
same time, completion of questionnaires by the subject will be done before ocular assessments to 
be performed by the investigator. 
6.1.5.1. 
Ocular Assessments by Investigator 
Best corrected visual acuity (BCVA) 
BCVA will be determined using pinhole with or without spectacles, soft contact lenses or rigid 
contact lenses. Detailed information about the procedure is provided in the Study Operation 
Manual.  
Pupil test (swinging light test) 
A pupil test (swinging light test also known as a test for a relative afferent pupillary defect) will 
be used to detect retinal or optic nerve involvement when the retina cannot be examined due to the 
presence of corneal opacity or cataract. Detailed information is in the Study Operation Manual. 
Slit lamp examination  
Slit lamp biomicroscopy will be performed by the examining ophthalmologist, using a slit lamp 
biomicroscope. Detailed information about the procedure is provided in the Study Operation 
Manual. 
 
 


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6.1.5.2. 
Subject-reported outcomes 
Subjects will complete quality of life questionnaires. If the subject is unable to complete the 
questionnaires without assistance, the questionnaires may be verbally answered and the subject’s 
responses recorded by site personnel or a chaperone. 
EQ-5D 
The EQ-5D questionnaire will be completed by all subjects. Subjects being ≤15 years of age will 
complete the youth version of this questionnaire. The EQ-5D questionnaire is a standardized self-
administered instrument for use as a measure of health outcome. An example of the questionnaire 
is provided in Appendix 3. 
EQ-5D questionnaire scores will be calculated according to the standard procedure of the 
questionnaire.  
Visual Functioning Questionnaire (VFQ25) 
The VFQ25 questionnaire will be completed, in addition to the EQ-5D,  by all subjects over 18 
years of age. An example of the questionnaire is provided in Appendix 3. VFQ25 questionnaire 
scores will be calculated according to the standard procedure of the questionnaire.  
6.1.6. 
Safety and Tolerability Measurements 
Safety and tolerability evaluations will consist of AE reporting, clinical laboratory (hematology, 
biochemistry and urinalysis), IOP, ophthalmoscopy, and photography of affected cornea. 
Assessments will be performed in accordance with the schedule of assessments. Between clinical 
research centers, assessments will be standardized as much as possible.  
Adverse Events 
Recording of AEs will commence with signing of the ICF. AEs will be captured in the CRF. AEs 
will be followed up by the investigator as specified in Section 0, Adverse Event Reporting. 
Clinical Laboratory Tests 
Blood samples for serum chemistry and hematology and a mid-stream urine sample for urinalysis 
will be collected at the time points indicated in the schedule of assessments. The local clinical 
laboratory will clearly mark all laboratory test values that are outside the normal range and the 
investigator will indicate which of these deviations are clinically significant or not. The 
investigator must record all clinically significant abnormal values during the study in the AE 
section of the CRF.  
Clinical significance is defined as any variation in signs, symptoms, or testing that has medical 
relevance according to the investigator and may result in an alteration in medical care. 
The following tests will be performed: 
 
Hematology: full blood count (total red blood cell count / white blood cell count with 
differential), hematocrit, hemoglobin, platelets. 


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 
Blood biochemistry: serum creatinine, aspartate aminotransferase, alanine aminotransferase, 
glucose, serum electrolytes (sodium, potassium), total protein, total bilirubin, alkaline 
phosphatase, gamma-glutamyl transferase, cholesterol and triglycerides. 
 
Urinalysis (by dipstick): specific gravity, pH, protein, glucose, ketones, urobilinogen, blood 
(erythrocytes, leucocytes). 
 
Pregnancy testing in urine (by dipstick) (for women of childbearing potential only). 
Intraocular Pressure 
At each visit, IOP measurement will be done using any CE marked tonometer (e.g., Goldmann, 
Icare or non-contact tonometers). For consistency, subjects should preferably be tested using the 
same equipment at each visit.  
Ophthalmoscopy  
Ophthalmoscopy will be performed after pupil dilatation to examine the vitreous, retina, macula 
and choroid at screening and at the final visit AND at any time if the vision deteriorates or either 
the pupil tests or IOP are abnormal. 
6.2. 
Appropriateness of Measurements 
The assessments which will be performed in this study are standard, and generally recognized as 
reliable, accurate and relevant. 
6.2.1. 
Timing of Assessments 
The date of all assessments will be recorded in the CRF. 
When multiple assessments are scheduled at the same time, completion of questionnaires by the 
subject will be done before ocular assessments to be performed by the investigator. 
6.3. 
Primary Efficacy Variables 
The primary efficacy variable chosen to assess drug efficacy is the CRR_12 (the clinical resolution 
rate at 12 months from randomization, defined as the percentage of subjects cured 30 days after 
discontinuing all study therapies, within 12 months of randomization). 
Criteria for clinical resolution: 
A subject will be considered cured if resolution of all the following clinical signs are observed, 
resulting from a slit lamp examination: 
 No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and 
oedema) that requires treatment, with a healed corneal epithelium and minimal punctate 
staining (10 dots or less equivalent to Grade 1 on the Oxford Scale).   
 No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal 
hyperemia): mild conjunctival inflammation is acceptable if related to other concurrent 
conditions such as blepharitis. 
 No limbitis, scleritis or anterior chamber inflammation.  


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6.4. 
Secondary Variables 
The secondary efficacy variables are: 
 BCVA 
 Corneal scarring as identified by slit lamp examination 
 Ulceration severity as identified by slit lamp examination using a 2 grade scoring 
procedure; i.e. present or absent 
 Anterior chamber inflammation as identified by ophthalmoscopy using a 3 grade scoring 
procedure  
 EQ-5D questionnaire 
 VFQ25 questionnaire 
 
The secondary safety variables are: 
 Adverse events 
 Clinical laboratory tests 
 Intraocular pressure (IOP) 
 Ophthalmoscopy 
 Worsening of the corneal epithelial defect and definable inflammatory signs (development 
of ring abscess and hypopyon) despite >30 days of treatment with the study drug) 
 Rate of subjects with a relapse 
 Rate of subjects requiring surgery, including amniotic membrane transplants, superficial 
keratectomy, application of cyanoacrylate glue, therapeutic penetrating, lamellar 
keratoplasty, cataract surgery, evisceration, or enucleation 
 Rate of subjects requiring non-study therapies, e.g., topical steroids and NSAIDs 
 Rate of subject discontinuation from study: to permit alteration of anti-amoebic therapy or 
for other unrelated specified reasons.   
 Incidence of secondary complications, such as significant corneal neovascularization, 
corneal scarring, corneal perforation, scleritis, secondary glaucoma, cataract, retinopathy. 
 
 
 


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7. 
STATISTICAL METHODS 
All statistical analyses will be performed using the STATA or SAS® System, latest version 
available. 
7.1. 
Study Analysis Sets 
7.1.1. 
Analysis Data Sets for the Efficacy Analyses 
Full Analysis Set (FAS)  
All subjects who have been randomized and for whom the primary efficacy variable is assessed 
will be included in the FAS. The FAS will be used in all efficacy analyses. 
Per-Protocol Analysis Set (PPAS) 
All subjects in the FAS without any relevant protocol deviations. The criteria for inclusion in the 
PPAS will be described in the SAP and documented before code breaking. The PPAS will be used 
in the analysis of the primary efficacy variable only. 
7.1.2. 
Analysis Data Set for the Safety Analysis 
Safety Analysis Set 
All subjects who have received at least 1 dose of study medication will be included in the Safety 
Analysis Set. This set will be used in all analyses of safety data. 
7.2. 
Statistical Analytical Plan (SAP) for Efficacy, Safety and Tolerability 
Evaluation 
A SAP with more technical and detailed elaboration of the principal features of the proposed 
statistical analyses, presentations, and the way in which anticipated analysis problems will be 
handled, will be written before the code is broken. 
7.3. 
Demographic Variables and Baseline Characteristics 
Demographic variables and baseline characteristics will be summarized in tabular form showing 
absolute and relative frequencies for categorical variables, and mean, standard deviation, median, 
25th and 75th percentiles, minimum and maximum for continuous variables. 
7.4. 
Evaluation of Efficacy Parameters 
All efficacy evaluations will be based on the FAS. Moreover, the primary efficacy variable, CRR, 
will be evaluated also for the PPAS. 
7.4.1. 
Primary Efficacy Variable 
Since it is also possible to test for non-inferiority if the superiority hypothesis is not met, additional 
conditions are specified (see list below) to meet the strict requirements of a non-inferiority study, 
making it feasible to test for non-inferiority using the results from the superiority analysis, without 
a further separate analysis. The 90% confidence interval for the difference between the treatments 


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obtained from the superiority analysis gives the necessary information. Position of the lower end 
of the confidence interval relative to a pre-defined agreed 0.20 non-inferiority margin (Δ) provides 
the key information for making decisions (conclusions) about non-inferiority. 
The additional conditions specified to make testing for non-inferiority feasible are the following: 
 Pre-defined non-inferiority margin (Δ=0.20) based on both clinical and statistical 
considerations, i.e., chosen on the basis of external information and not chosen to fit the 
data. 
 Analysis of the FAS and the PPAS, giving similar results with respect to p-values and 
confidence intervals for the efficacy measure at issue. 
 Study design and implementation directed at minimizing deviation from protocol (a 
particularly important source of bias in non-inferiority studies), e.g., special care to 
minimize violations of: entry criteria; non-compliance; withdrawals; loss; missing data; 
etc. This should help ensure the second bullet point.  
 Evidence that the standard treatment is showing its usual (expected) level of efficacy. 
These specifications are in accord with the recommendations by the Committee for Proprietary 
Medicinal Products (CPMP) 482/99. 
Null hypothesis superiority: 
  No difference between test treatment and reference treatment 
Non-inferiority clinical studies aim to demonstrate that the test treatment is no worse than the 
reference by more than a pre-specified small amount. 
Null hypothesis non-inferiority: 
  Test treatment is inferior to the reference by Δ or more 
CRR_12 will be analyzed with a logistic regression model and model-based estimates of the 
difference between treatments, odds ratio with corresponding confidence intervals and p-values 
will be provided. Time-to-event (clinical resolution) analysis will also be performed for the 
comparison of CRR_12s between treatments, including the Cox Proportional Hazards regression 
(subject to validity of the 'proportional hazards' assumption) and Kaplan-Meier survival plots. If 
the proportional hazard assumption is not fulfilled a logrank test will be performed. 
7.4.1.1. 
Justification for choice of the non-inferiority margin  
7.4.1.1.1. 
Justification on Statistical grounds 
Our pre-defined non-inferiority margin of =0.20 is based on previous studies (historical data), 
i.e., the study referred to in the protocol showing 1/20 cured without treatment being the only data 
available that can be used to estimate the cure with placebo, and the results of the reference 
(standard) treatment from the SIFI / Moorfields observational studies20, adjusted for inclusion of 


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advanced disease cases in the study. The table below demonstrates the statistical grounds for the 
choice of . The chosen  of 0.20 satisfies the condition that "the test treatment is expected to 
retain at least 50% of the standard treatment effect over placebo, in order to be considered as non-
inferior". 
Table 1 
Selection of Non-Inferiority Margin () based on statistical issues 
Proportion
A 
Cured with no treatment: Historic Data
0.05
B 
Cured with Standard Treatment: SIFI Study Data
0.67
Ba 
B adjusted for inclusion of stage-3 cases  (63/100)
0.63
Ba(lower) 
Lower 95% confidence interval (CI) bound for Ba (Binomial 
exact)
0.53
M1 
Ba(lower) - A: the standard treatment effect over placebo
0.48
 
Non-inferiority Margin
0.20
Proportion of M1 retained by the Test Treatment: (M1 - Delta) / M1
58%
The Test treatment  retains well over  50% of the Standard treatment effect over placebo 
 
7.4.1.1.2. 
Justification on Clinical grounds   
The Sponsor proposes as clinically acceptable a non-inferiority margin of 0.20: this is within the 
accepted level for a study of this kind in view of the following: 
 A poor response to therapy is common in this disease. Disease progression is slow in this 
disease (over weeks and months) and clinicians have to assess the effect of treatments every 
1-2 weeks, in cases not progressing well, and modify therapies to optimize outcomes. 
Because of the disease chronicity the use of an ineffective treatment for these periods  
results in a delayed response, but no serious short term morbidity.  The study results may 
indicate non-inferiority, when at worst the true difference in proportion 'cured' (Combined 
- PHMB alone) is 0.20. The most likely impact of such a finding on clinical practice will 
be to encourage the use of PHMB monotherapy as a first line treatment.  
 However, if PHMB monotherapy fails in clinical practice during the early stages of 
treatment, which is to be expected in some subjects, the clinician is unlikely to abandon 
the good clinical practice of monitoring the disease progress closely, and adding other anti-
amoebics to the therapy when necessary.  
 At worst, the impact of the study finding might result in a little delay (matter of days) in 
starting a combined treatment in some patients, whilst giving a chance for the biguanide 
mono-therapy to act. This scenario is unlikely to result in blindness or serious morbidity. 
It may cause some delay in the clinical resolution.  


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 By contrast, the possibility that the true difference may be much smaller than 0.2, or even 
in favor of PHMB mono-therapy, will have important beneficial consequences for patients 
and ophthalmic services. 
7.4.2. 
Secondary Efficacy Variable 
Which test to be used in the analyses of secondary efficacy variables depends on the type of 
variable, e.g., logistic regression for binary variables, proportional odds model for an ordinary 
scaled variable, analysis of variance (ANOVA) or covariance (ANCOVA) for continuous 
variables as appropriate. If the underlying assumptions are not fulfilled, data transformation or 
non-parametric test will be performed. For comparison of proportions, Chi square tests, Fisher's 
exact test, or Mantel-Haenszel procedures, as appropriate, will be considered. For continuous 
variables, the Wilcoxon rank-sum test or rank ANOVA/ANCOVA will be used. The STATA or 
SAS statistical software will be used for the analysis. 
EQ-5D and VFQ25 will be evaluated according to their respective analysis protocols. 
7.5. 
Evaluation of Safety and Tolerability Parameters 
7.5.1. 
Adverse events 
AEs data will be captured and encoded by system organ class (SOC) and the lowest level term 
(Medical Dictionary for Regulatory Activities, current version). A listing of AEs will be created. 
This listing, at minimum, will contain a description of AEs as to seriousness, severity, onset date 
and end date, duration, action taken (if any), outcome and likelihood of drug causation (“relation"). 
A frequency table will be compiled for AEs by treatment group, SOC, and lowest level term. 
Frequency tables will be compiled showing the number of subjects per treatment group affected 
by one or more (related) AEs, including percentages, the total number of AEs per treatment group 
and the average number of AEs per subject exposed to the respective treatment. 
7.5.2. 
Other Safety Parameters 
Clinical laboratory tests will be presented with summary statistics. In addition, the tests will be 
analyzed with ANCOVA (baseline value of respective laboratory test will be included in the 
model).  
All results will be regarded as descriptive only. The p-values will be used to flag safety and 
tolerability variables worthy of further attention.  
Null hypotheses:  
 No difference between test treatment and reference treatment 
7.6. 
Sample Size Determination 
From the results of the sponsor’s observational, case series retrospective study 038/SI, a CRR_12 
of 67% for the conventional combination therapy of PHMB 0.02% and propamidine 0.1% is 
expected.20 This figure is in the range of cure rates described in the literature.13,22,23 If the true 


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difference in CRR_12 is 0.20 (Δ) (or more) in favor of PHMB monotherapy (0.08% PHMB), a 
total sample size of 116 subjects (allowing for 10% loss to follow-up) should give the study at 
least 80% power to detect superiority, with 2-sided alpha=0.10 (or equivalently 1-sided 
alpha=0.05). 
Assuming a prevalence of late stage disease, and worse outcomes, in 38% of subjects, the expected 
CRR_12 in the control group will be reduced from 67% to 63%. To account for the inclusion of 
this group, the sample size has been adjusted to 130 subjects (allowing for 10% loss to follow-up). 
7.7. 
Interim Analysis 
No interim analysis is planned.  
8. 
ADVERSE EVENT REPORTING 
8.1. 
Definitions 
8.1.1. 
Adverse Event 
An AE is any untoward medical occurrence in a study subject administered a pharmaceutical 
product; the event does not necessarily have a causal relationship with the treatment or usage. 
AEs include the following: 
 Abnormal test findings, as specified below 
 Clinically significant signs and symptoms 
In addition, signs and symptoms resulting from the following should also be handled according to 
the same principles as AEs: 
 Overdose 
 Interactions 
 Abuse 
 Misuse 
8.1.2. 
Abnormal test findings 
The investigator must record all clinically significant abnormal clinical laboratory analysis values 
during the study in the AE section of the CRF.  
Clinical significance is defined as any variation in signs, symptoms, or testing that has medical 
relevance according to the investigator and may result in an alteration in medical care. 
Note, that the symptom, not the test result, should be recorded as an AE. 


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8.1.3. 
Pre-existing conditions 
Disease signs, symptoms, and/or laboratory abnormalities already existing prior to the start of the 
study (signing the informed consent) are not considered AEs, unless they re-occur after the subject 
has recovered from the pre-existing condition or in the opinion of the investigator they represent a 
clinically significant exacerbation in intensity or frequency. 
8.1.4. 
Procedures 
Diagnostic and/or therapeutic non-invasive and invasive procedures, such as surgery, should not 
be reported as AEs. However, the medical condition for which the procedure was performed 
should be reported if it meets the definition of an AE. For example, an acute appendicitis that 
begins during the AE reporting period should be reported as the AE and the resulting 
appendectomy should be entered in the CRF. 
8.1.5. 
Serious Adverse Events 
An AE that meets 1 or more of the following criteria/outcomes is classified as serious: 
 Results in death. 
 Is life-threatening (i.e., at immediate risk of death). 
 Requires in-patient hospitalization or prolongation of existing hospitalization. 
 Results in persistent or significant disability/incapacity. 
 Is a congenital anomaly/birth defect (i.e., in an offspring to the study subject). 
Other medically important AEs that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the subject or may require medical or surgical intervention to prevent one of the 
outcomes listed above. Examples of such events are intensive treatment in an emergency room or 
at home for allergic bronchospasm, that does not result in hospitalization. Any suspected 
transmission of an infectious agent via the study drug shall also be considered serious. 
Serious also includes any other event that the investigator or Sponsor judges to be serious. Some 
events (such as, for example, corneal vascularization, corneal ulceration, corneal perforation, 
corneal scarring, non-infectious scleritis) are known to be a consequence of Acanthamoeba 
keratitis and therefore are expected to occur during the study. The investigator is recommended to 
consult the Investigator’s Brochure10. 
8.1.6. 
Hospitalization 
Hospitalization includes transfers within a hospital and also includes admissions of less than 24 
hours. The following situations are not considered hospitalizations (although other SAE criteria 
may still apply): 


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 Outpatient procedures / ambulatory care. 
 Emergency department visits. 
Hospitalization in the absence of an AE occurring during the study should not be considered an 
SAE. This includes: 
 Hospitalization due to a pre-existing condition not associated with a worsening of the pre-
existing condition. 
 Protocol specified admission. 
 Elective admission, e.g., due to cosmetic surgery. 
 Pre-planned admission for a condition specified at baseline for the subject. 
8.2. 
Adverse Event Reporting Period 
The period for recording AEs, including SAEs, on the CRF begins immediately after the subject 
has signed the informed consent (Visit 1) and ends at the last study visit. SAEs that are related to 
the study drug and continue beyond the normal collection period (i.e., are ongoing) will be 
followed until the SAE is resolved or considered medically stable by the investigator. Furthermore, 
any SAE should be reported irrespective of the time of occurrence if a causal relationship between 
the event and the study drug is suspected. Data collected after the end-of-study visit will be 
recorded in the subject’s medical record, and in the sponsor’s Safety Database Eliciting and 
Recording Adverse Event Information. 
The investigator is to record all directly observed AEs, and all AEs spontaneously reported by the 
subject, in the CRF using concise medical terminology. In addition, each subject will be questioned 
about AEs at each clinic visit following initiation of treatment. The question asked will be “Have 
you had any health problems since your last clinic visit?” 
When possible and appropriate, a diagnosis rather than individual signs and symptoms shall be 
recorded. The investigator is responsible for obtaining sufficient information to determine 
seriousness, causality and outcome of each AE. 
8.2.1. 
Severity Assessment 
The intensity of AEs will be graded using the following scale: 
 
Mild (Grade I): Asymptomatic or mild symptoms not interfering with the normal activity 
of the subject: clinical or diagnostic observations only; intervention not indicated. 
 
Moderate (Grade II): Minimal, local or non-invasive intervention indicated; limited age-
appropriate instrumental activities of daily living. 


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 
Severe (Grade III): Severe or medically significant but not immediately life-threatening: 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
activities of daily living; incapacitating with inability to work or perform normal daily 
activity. 
 
Life-threatening (Grade IV): consequences: urgent intervention indicated.  
 
Death (Grade V) related to AE. 
If an AE has multiple aspects, the aspect with the highest severity will be graded. 
Note the distinction between the gravity (seriousness) and the intensity (severity) of an AE. Severe 
is a measure of intensity; thus, a severe reaction is not necessarily a serious reaction. For example, 
a headache may be severe in intensity, but would not be classified as serious unless it met one of 
the criteria for serious events listed above. 
8.2.2. 
Causality Assessment 
For each AE, the investigator must make a causality assessment to determine if there is a 
reasonable possibility that the study drug caused the AE. 
The relationship of any AE to the study drug will be assessed and graded on a five point scale.  
Unrelated:  Sufficient information exists to indicate that causality is unrelated to drug i.e. the 
event is due to extraneous causes (an underlying medical condition, concurrent drugs, 
environmental factors etc.) 
Unlikely:  
The AE is likely to have been produced by the subject's clinical state, environmental 
or toxic factors or other therapeutic interventions but an effect of drug cannot be ruled 
out. 
Possible:  
The AE follows a reasonable temporal sequence from drug administration; is unlikely 
to have been produced by the subject's clinical state, environmental or toxic factors 
or other therapeutic interventions. 
Probable: 
The AE follows a reasonable temporal sequence from drug administration, or is 
associated with established drug concentration in body tissues; improves on stopping 
or reducing drug dosage (de-challenge); and could not reasonably be explained by 
the study subject's clinical state environmental or toxic factors, or other therapeutic 
interventions. (Note: - There are exceptions when an AE does not disappear upon 
discontinuation of the drug. For example: bone marrow depression, fixed drug 
eruptions, tardive dyskinesias, etc.).  
Related:  
As for "probable" - but on the basis of the observation of objective clinical signs any 
other cause may be excluded.  


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8.3. 
Serious Adverse Event Reporting 
Both serious and non-serious AEs are to be reported on the AE page of the CRF as specified in the 
CRF instructions. 
If an SAE occurs, the investigator or clinical site personnel should notify the Monitor and the 
sponsor’s Qualified Person Responsible for Pharmacovigilance (QPPV) regardless of relationship 
to the investigational drug, using the designated Serious Adverse Event Form within 24 hours of 
clinical site personnel becoming aware of the event.  
Where the same data are collected in the CRF and on the SAE form, these must be completed in a 
consistent manner. For example, the same AE term should be used on both forms.  
All new information obtained (follow-up information), relevant to an SAE report, should be 
forwarded to the Monitor and the sponsor’s QPPV within the same timeframe as the initial 
information (within 24 hours from clinical site personnel becoming aware of the event). 
The investigator shall provide the Monitor and the sponsor’s QPPV with sufficient information to 
enable a complete medical assessment of the reported event. Best efforts shall be made by the 
investigator to provide the Monitor and the sponsor’s QPPV with additional information related to 
any SAE as requested. 
All SAE reports should be submitted by e-mail or fax to the following contacts: 
Daria Rasà 
Qualified Person Responsible for Pharmacovigilance, SIFI SpA (ITALY) 
Tel +39 095 79 22 204 
Mob +39 346 8399737 
Fax +39 095 78 93 451 
E-mail:  
daria.rasa@sifigroup.com   
AND: pharmacovigilance@sifigroup.com 
or 
Michele Puglia 
Deputy QPPV, SIFI SpA (ITALY) 
Tel: +39 095 79 22 356 
Mob: +39 335 60 77 919 
E-mail:  
michele.puglia@sifigroup.com 
AND: pharmacovigilance@sifigroup.com 
 
Where required by local regulations, the Institutional Review Board (IRB)/(Independent Ethics 
Committee (IEC) and/or regulatory authorities will be informed of SAEs in accordance with local 
regulation timeframes and reporting requirements. 


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8.4. 
Exposure during Pregnancy 
All events of exposure to the study during pregnancy (female subject) shall be reported to the 
Monitor and the sponsor’s QPPV within 24 hours of awareness by any study personnel, whether 
the exposure is associated with an AE or not. This includes all situations where a female is or has 
been found to be pregnant after being exposed to the study drug; directly, indirectly or via her 
partner (paternal exposure). 
In all reported situations of exposure during pregnancy, the sponsor’s QPPV will provide the 
investigator with a Pregnancy Report Form which shall be completed and returned by the 
investigator. The investigator is responsible for monitoring the outcome of the pregnancy and to 
inform the Monitor and the sponsor’s QPPV of relevant information and any information requested 
related to the outcome of the pregnancy. 
Any AEs and SAEs observed during and in relation to pregnancy or delivery should be recorded 
in the CRF and as applicable be reported to the Monitor and the sponsor’s QPPV as described 
previously in this section. 
8.5. 
Follow-up of Adverse Events 
All AEs should be followed-up until they are resolved or the investigator assesses them as 
persistent. In particular, all AEs assessed by the investigator as related to the study drug will 
continue to be followed-up until 28th day after the last application of study drug, even after the 
subject's participation in the study is completed, or in case of withdrawal of the subject from the 
clinical trial or of anticipated conclusion of the study. 
8.6. 
Recording 
All AEs reported by the subjects or apparent from their physical appearance during the clinical 
study will be reported in the CRF. 
This will include the following information:  
 
Description of the AE 
 
Date and time (SAEs only) of onset – End date and time (SAEs only) 
 
Date and time of last study drug dose for drug related AEs 
 
Severity of symptoms 
 
Action taken with study drug 
 
Action taken (medication) 
 
AE outcome 
 
Assessment of relation to study medication 
 
Optional comments by investigator 
In addition, clinically significant abnormal clinical laboratory analysis values should also be 
recorded as AEs. Reporting as an AE should not be triggered by: 
 
Merely repeating an abnormal test or  


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 
Any abnormal test result that is determined to be an error. 
9. 
QUALITY CONTROL AND QUALITY ASSURANCE 
Monitoring visits to the clinical research center will be made periodically, according to the 
standard operating procedure of the contract research organization, during the study to ensure that 
Good Clinical Practice (GCP) and all aspects of the protocol are followed. Source documents will 
be reviewed for verification of agreement with data on CRFs. The investigator/institution 
guarantees direct access to source documents by the sponsor and appropriate regulatory authorities. 
The clinical research center may also be subject to review by an IEC, to quality assurance audits 
performed by the sponsor, and/or to inspection by appropriate regulatory authorities. 
It is important that the investigator(s) and their relevant personnel are available during the 
monitoring visits and possible audits or inspections and that sufficient time is devoted to the 
process. 
10. 
ETHICAL ASPECTS 
10.1. 
Independent Ethics Committee/Institutional Review Board 
Prospective approval of the study protocol, protocol amendments, informed consent forms, and 
other relevant documents (e.g., advertisements), if applicable, will be obtained from the IEC/IRB. 
All correspondence with the IEC/IRB should be retained in the Investigator File. Copies of 
IEC/IRB approvals should be forwarded to the sponsor. 
The only circumstance in which an amendment may be initiated prior to IEC/IRB approval is 
where the change is necessary to eliminate apparent immediate hazards to the subjects. In that 
event, the investigator must notify the IEC/IRB and the local site personnel in writing within 
5 working days after the implementation. 
10.2. 
Ethical Conduct of the Study 
The study will be performed in accordance with International Conference on Harmonization GCP 
guidelines, the Declaration of Helsinki (adopted by the 18th World Medical Association General 
Assembly, Helsinki, Finland, June 1964, and subsequent amendments24), and applicable local 
regulatory requirements and laws. 
10.3. 
Subject Information and Consent 
All subjects will be given full and adequate verbal and written information regarding the objective 
and procedures of the study and the possible risks involved. This information must be provided to 
the subject prior to undertaking any study-related procedure. The subjects must be informed about 
their right to withdraw from the study at any time. Written subject information, approved by the 
IEC/IRB, must be given to each subject before any study-related procedure is undertaken. The 
written subject information must not be changed without prior approval by the sponsor and the 
IEC/IRB. Furthermore, it is the responsibility of the investigator to obtain signed informed 
consent. 


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10.4. 
Privacy of Personal Data 
All personal details will be treated as confidential by the investigator and staff of the research 
center and handling of personal data will be in compliance with the applicable local laws and 
regulations. 
11. 
STUDY ADMINISTRATIVE STRUCTURE 
11.1. 
Documentation 
11.1.1. 
Archiving 
All documents concerning the study will be kept on file in the Central Archives of the sponsor for 
at least 25 years after study completion and completion of the Clinical Study Report. The sponsor 
will receive the completed CRFs and the final database in SAS. 
11.1.2. 
Case report form 
A CRF is required and should be completed for each included subject. In this study an electronic 
CRF will be used, which is fully ICH-GCP/EU Clinical Trial Directive compliant..  
It is the responsibility of the investigator to ensure completion and to review and approve all CRFs. 
CRFs must be signed by the investigator, using an electronic signature. These signatures serve to 
attest that the information contained on these CRFs is correct. At all times, the investigator has 
final responsibility for the accuracy and authenticity of all data entered on the CRFs.  
11.1.3. 
Source data 
Subject source documents are the physician’s subject records maintained at the clinical research 
center. In most cases, the source documents will be the hospital’s or the physician’s chart. In those 
cases, the information collected on the CRFs must match those charts. All reports and printouts 
should be stored in the subject’s medical record.  
12. 
CONFIDENTIALITY AND PUBLICATION POLICY 
By signing this protocol the investigator reaffirms to the sponsor that he/she will maintain in 
confidence all information furnished to him/her, or resulting from this study. He/she will only 
divulge such information as may be necessary to the IEC and the members of the staff and the 
subjects who are involved in this study. 
The results of this study may be published or presented at scientific meetings. If this is envisaged, 
both parties agree to inform each other and to submit all manuscripts or abstracts to the other party 
prior to publication or presentation. 
Prior notice of any planned publication shall be given to the other parties concerned at least 45 days 
before the publication. Any objection to the planned publication shall be made in writing to the 
sponsor and to any party concerned within 30 days after receipt of the notice. If no objection is 
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(a) 
The objecting Party's legitimate academic or commercial interests are compromised by the 
publication; or   
(b) 
The protection of the objecting Party's Foreground or Background is adversely affected. 
The objection has to include a precise request for necessary modifications. 
If an objection has been raised the involved parties shall discuss how to overcome the justified 
grounds for the objection on a timely basis (for example by amendment to the planned publication 
and/or by protecting information before publication) and the objecting Party shall not unreasonably 
continue the opposition if appropriate actions are performed following the discussion. 
This allows the parties concerned to protect proprietary information and to provide comments 
based on information that may not yet be available to the other party.  
Authorship of any formal publication of the study will be determined by mutual agreement 
between the investigator and the sponsor. 
 
 


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REFERENCES 
1 
Gilbert P, Pemberton D, Wilkinson DE. Barrier properties of the gram-negative cell envelope 
towards high molecular weight polyhexamethylene biguanides. J Appl Bacteriol 1990; 69:585-92. 
 
2  
Ikeda T, Ledwith A, Bamford CH, Hann RA. Interaction of a polymeric biguanide biocide with 
phospholipid membranes. Biochim Biophys Acta 1984; 769:57-66. 
 
3  
Broxton P, Woodcock PM, Heatley F, Gilbert P. Interaction of some polyhexamethylene 
biguanides nd membrane phospholipids in Escherichia coli. J Apl Bacteriol. 1984;57:115-24. 
 
4  
Seal DV. Acanthamoeba keratitis update—incidence, molecular epidemiology, and new drugs for 
treatment. Eye 2003; 17:893–905. 
 
5 
Dart JK, Saw VP, Kilvington S. Acanthamoeba keratitis: diagnosis and treatment update. Am J 
Ophthalmol. 2009;148(4):487-499. 
 
6  
Mathers W. Use of higher medication concentrations in the treatment of Acanthamoeba keratitis. 
Arch Ophthalmol. 2006;124:923. 
 
7  
Scientific Committee on Consumer Safety, Bernauer U. Opinion of the scientific committee on 
consumer safety (SCCS)--2nd Revision of the safety of the use of poly(hexamethylene) biguanide 
hydrochloride or polyaminopropyl biguanide (PHMB) in cosmetic products. Regul Toxicol 
Pharmacol. 2015;73(3):885-886. 
 
8  
Kramer A, Behrens-Baumann W. Prophylactic use of topical anti-infectives in ophthalmology. 
Ophthalmologica. 1997;211 Suppl 1:68-76. 
 
9  
Research Toxicology Center (RTC). PHMB ophthalmic solution at 0.08%. 26-week ocular 
tolerance/toxicity study in rabbits (Study A2018), 2016 
 
10  
Investigator’s Brochure Polyhexamethylene biguanide eye-drops solution. September 2017. 
 
11  
Duguid IG, Dart JK, Morlet N, Allan BD, Matheson M, Ficker L, Tuft S. Outcome of 
Acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine. Ophthalmology. 
1997 Oct;104(10):1587-92. 
 
12  
Alkharashi M, Lindsley K, Law HA, Sikder S. Medical interventions for Acanthamoeba keratitis. 
Cochrane Database Syst Rev. 2015;2:CD010792. 
 
13  
Lim N, Goh D, Bunce C, Xing W, Fraenkel G, Poole TR, Ficker L. Comparison of 
polyhexamethylene biguanide and chlorhexidine as monotherapy agents in the treatment of 
Acanthamoeba keratitis. Am J Ophthalmol 2008; 145:130–135. 2008 by Elsevier 
 
14  
Alizadeh H, Silvany RE, Meyer DR, Dougherty JM, McCulley JP. In vitro amoebicidal activity of 
propamidine and pentamidine isethionate against Acanthamoeba species and toxicity to corneal 
tissues. Cornea. 1997;16(1):94-100. 
 
 
                                                 
 
 


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15  
Johns KJ, Head WS, O'Day DM. Corneal toxicity of propamidine. Arch Ophthalmol. 1988 
Jan;106(1):68-9. 
 
16  
Dart JK, Summary of outcomes of Acanthamoeba keratitis from 1975-1985. 2014 
 
17  
Oldenburg CE, Acharya NR, Tu EY, Zegans ME, Mannis MJ, Gaynor BD, Whitcher JP, Lietman 
TM, Keenan JD. Practice patterns and opinions in the treatment of Acanthamoeba keratitis. Cornea. 
2011;30(12):1363-8.  
 
18  
Pérez-Santonja JJ, Kilvington S, Hughes R, Tufail A, Matheson M, Dart JK. Persistently culture positive 
Acanthamoeba keratitis: in vivo resistance and in vitro sensitivity. Ophthalmology. 2003;110(8):1593-600. 
 
19  
Vontobel SF, Kaufman C, Zinkernagel AS, Hauser PC, Thiel MA. Corneal penetration of 
Polyhexamethylene Bigaunide and Chlorhexidine Digluconate. Clinical and Experimental 
Ophthalmology. 2015;6(3):52015. 
 
20  
Study 038/SI. Retrospective evaluation of the clinical management of patients affected by 
Acanthamoeba keratitis, SIFI 2013. 
 
21  
The European Agency for the Evaluation of Medicinal Products Evaluation of Medicines for 
Human Use, CPMP/EWP/482/99, available at 
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC50000
3658.pdf, accessed at 23 December 2016. 
 
22  
Butler TK, Males JJ, Robinson LP, Wechsler AW, Sutton GL, Cheng J, Taylor P, McClellan K. 
Six-year review of Acanthamoeba keratitis in New South Wales, Australia: 1997-2002. Clin 
Experiment Ophthalmol. 2005;33(1):41-46. 
 
23  
Larkin DF, Kilvington S, Dart JK. Treatment of Acanthamoeba keratitis with polyhexamethylene 
biguanide. Ophthalmology. 1992;99(2):185-191. 
 
24 
WMA Declaration of Helsinki (18th WMA General Assembly 1964), revised at 64th World 
Medical Association General Assembly. Fortaleza. October 2013. 
 
 
 


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APPENDIX 1  
TYPICAL CLINICAL FINDINGS OF AN ACANTHAMOEBA 
CORNEAL INFECTION 
 
 
1. epithelial lesions: epithelial punctate keratopathy, epithelial infiltrates, epithelial 
defects, dendritiform epithelial ulcers  
2. extracorneal lesions: limbal inflammation (limbitis), anterior scleral inflammation, 
diffuse or nodular  
3. stromal lesions: perineural infiltrates, anterior stromal infiltrates, disciform corneal 
swelling, stromal ulceration, ring abscess  
4. anterior chamber lesions: keratic precipitates, hypopyon  
5. iris lesions: fixed dilated pupil  
6. lens: mature cataract 
 
 
 
 
 


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APPENDIX 2  
EUROQOL FIVE-DIMENSION SCALE (EQ-5D) QUESTIONNAIRE 
 
 
 
 


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APPENDIX 3  
VISUAL FUNCTIONING QUESTIONNAIRE 25 (VFQ25) 
 
 


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LAST PAGE 

SAP_001.pdf: 
 
 
 
 
 
 
 
 
 
Statistical Analysis Plan 
 
 
 
Study Information: 
Sponsor Study Code: 
043-SI 
Study Title: 
Randomized, Assessor-Masked, Active-Controlled, Phase 3 Study to Evaluate 
Efficacy, Safety and Tolerability of 0.08% Polyhexamethylene Biguanide 
(PHMB) Ophthalmic Solution in Comparison with 0.02% PHMB + 0.1% 
Propamidine Combination Therapy in Subjects Affected by Acanthamoeba 
keratitis. 
Protocol Version and Date: 
2.0, 08-Oct-2018 
LINK Medical Study ID: 
PSR012 
 
 
Document Control: 
SAP Version and Date: 
3.0, 01-Jul-2021 
Status: 
Final 
Author: 
Christina Ehrenkrona 
LINK Medical AS 
Jörgen Kocksgatan 4 
SE-211 20 Malmö, Sweden 
e-mail: Christina.Ehrenkrona@linkmedical.eu 
 
Mobile: +46 706 040 010  
 
 
 
Transaction 09222115557449620377
Signed CE, VP, MS


 
Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
Document History 
 
Version Modified by (name and role) 
Date 
Description of Changes 
0.1 
Trond Haider 
03-Sep-2019 
First internal draft 
0.2 
Trond Haider 
30-Sep-2019 
Input from Malin Schollin 
0.3 
Trond Haider 
01-Nov-2019 
Input from Malin Schollin and Kerstin Wiklund 
0.8 
Trond Haider 
22-Nov-2019 
Further input: This version is Sponsor review draft 
0.9 
Trond Haider 
09-Jan-2020 
Input from Sponsor 
0.95 
Trond Haider 
15-Jan-2020 
T-con with Sponsor 
0.98 
Trond Haider 
04-Aug-2020 
Info from Sponsor: No interim analysis 
0.99 
Trond Haider 
15-Oct-2020 
Input from PI 
1.0 
Trond Haider 
25-Nov-2020 
Teams meeting: PI/Sponsor 
1.1 
Christina Ehrenkrona 
18-Feb-2021 
Input from PI 
2.0 
Christina Ehrenkrona 
26-Apr-2021 
Teams meeting: Sponsor 
3.0 
Christina Ehrenkrona 
1-Jul-2021 
Clean File meeting 
 
 
 
Transaction 09222115557449620377
Signed CE, VP, MS


 
Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
Table of Content 
 
1 
Acronyms and Abbreviations used in the Document ........................................................................... 5 
2 
Introduction ............................................................................................................................................... 6 
3 
Study Objectives ...................................................................................................................................... 6 
3.1 
Definitions ...................................................................................................................................... 6 
3.2 
Primary Objective .......................................................................................................................... 6 
3.2.1 
Primary Variables ....................................................................................................................... 6 
3.3 
Secondary Objectives ................................................................................................................... 7 
3.3.1 
Secondary Efficacy Variables .................................................................................................... 7 
3.3.2 
Secondary Safety Variables ....................................................................................................... 7 
3.4 
Hypothesis ..................................................................................................................................... 8 
4 
Study Design ............................................................................................................................................. 8 
5 
Study Population ...................................................................................................................................... 8 
5.1 
Sample Size .................................................................................................................................. 8 
5.2 
Randomisation .............................................................................................................................. 9 
6 
Assessments ............................................................................................................................................ 9 
6.1 
Primary efficacy endpoint criteria for assessing clinical resolution ............................................... 9 
6.2 
Secondary efficacy endpoints assessment ................................................................................... 9 
6.2.1 
Assessment of BCVA ................................................................................................................. 9 
6.2.2 
Assessment of corneal scarring ............................................................................................... 10 
6.2.3 
Assessment of ulceration severity ........................................................................................... 10 
6.2.4 
Pupil test .................................................................................................................................. 10 
6.2.5 
Assessment of anterior chamber inflammation ........................................................................ 10 
6.2.6 
EQ-5D-5L and VFQ25 assessments ....................................................................................... 10 
6.3 
Secondary safety endpoints assessment ................................................................................... 10 
6.3.1 
AEs ........................................................................................................................................... 10 
6.3.2 
Clinical laboratory tests assessment ........................................................................................ 10 
7 
Methods of Analysis ............................................................................................................................... 10 
7.1 
General ........................................................................................................................................ 10 
7.1.1 
Presentation of Results ............................................................................................................ 10 
7.1.2 
Baseline ................................................................................................................................... 11 
7.1.3 
Analysis Relative Day .............................................................................................................. 11 
7.1.4 
Analysis Visit ............................................................................................................................ 11 
7.1.5 
Handling of Missing Data ......................................................................................................... 12 
7.1.6 
Interim Analyses ....................................................................................................................... 12 
7.1.7 
Multiplicity ................................................................................................................................. 12 
7.1.8 
Subgroups ................................................................................................................................ 12 
7.2 
Analysis Sets ............................................................................................................................... 12 
7.2.1 
Full Analysis Set (FAS) ............................................................................................................ 13 
7.2.2 
Per-Protocol Analysis Set (PPAS) ........................................................................................... 13 
7.2.3 
Safety Analysis Set .................................................................................................................. 13 
7.3 
Disposition of Subjects ................................................................................................................ 13 
7.4 
Protocol Deviations ..................................................................................................................... 13 
7.5 
Demographics and Baseline Characteristics .............................................................................. 14 
7.6 
Medical History and Concurrent Diseases .................................................................................. 14 
Transaction 09222115557449620377
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Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
7.7 
Prior and Concomitant Medication .............................................................................................. 14 
7.8 
Compliance ................................................................................................................................. 15 
7.9 
Efficacy Evaluation ...................................................................................................................... 15 
7.9.1 
Primary Efficacy Variable ......................................................................................................... 15 
7.9.2 
Justification for choice of the non-inferiority margin ................................................................. 16 
7.9.3 
Secondary Efficacy Variables .................................................................................................. 17 
7.10 
Safety Evaluation ........................................................................................................................ 20 
7.10.1 
Extent of Exposure ................................................................................................................... 20 
7.10.2 
Adverse Events ........................................................................................................................ 21 
7.10.3 
Clinical laboratory ..................................................................................................................... 21 
7.10.4 
Vital Signs ................................................................................................................................ 23 
7.10.5 
Intraocular Pressure Measurement (mmHg)............................................................................ 23 
7.10.6 
Ophthalmoscopy ...................................................................................................................... 23 
7.10.7 
Worsening of corneal inflammation .......................................................................................... 23 
7.10.8 
Rate of subjects with a relapse ................................................................................................ 23 
7.10.9 
Rate of subjects requiring surgery ........................................................................................... 23 
7.10.10 Rate of subjects requiring non-study therapies ....................................................................... 23 
7.10.11 Rate of subjects’ discontinuation from study ........................................................................... 23 
7.10.12 Incidence of secondary complication ....................................................................................... 23 
7.10.13 Cure rate of subjects with risk factor ........................................................................................ 24 
7.10.14 Rate of subjects with Adjunctive therapy ................................................................................. 24 
7.11 
Changes to Planned Analysis ..................................................................................................... 24 
8 
Derived Variables ................................................................................................................................... 24 
8.1 
Disposition of Subjects ................................................................................................................ 24 
8.2 
Demographics and Baseline Characteristics .............................................................................. 24 
8.3 
Time to Event .............................................................................................................................. 25 
8.4 
Compliance ................................................................................................................................. 25 
8.5 
Duration ....................................................................................................................................... 25 
9 
References .............................................................................................................................................. 25 
10 Signoff ..................................................................................................................................................... 26 
 
 
 
Transaction 09222115557449620377
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Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
1 
Acronyms and Abbreviations used in the Document 
 
Acronyms & Abbreviations 
AE 
Adverse event 
ANOCVA 
Analysis of covariance 
ANOVA 
Analysis of variance 
ATC 
Anatomical Therapeutic Chemical (classification system) 
BCVA 
Best corrected visual acuity 
CI 
Confidence interval 
CDISC 
Clinical Data Interchange Standards Consortium 
CPMP 
Committee for Proprietary Medicinal Products 
CRF 
Case report form 
CRR 
Clinical resolution rate 
CRR_12 
Clinical resolution rate at 12 months after randomization 
CS 
Clinically significant 
DDP 
Data Display Plan 
dL 
Decilitre 
EMA 
European Agency for the Evaluation of Medicinal Products 
EoT 
End of treatment 
EQ-5D-5L 
EuroQol 5 dimensional, 5 levels standardized measure of health status 
EU 
European Union 
FAS 
Full analysis set 
G 
grams 
IMP 
Investigational medicinal product 
ICH 
International Conference on Harmonisation 
IMP 
Investigational medicinal product 
IOP 
Intralocular pressure 
ITT 
Intention to treat 
L 
Litre 
LINK 
LINK Medical Research AS 
LogMAR 
Logarithm of the Minimum Angle of Resolution 
Mg 
Milligrams 
MedDRA 
Medical Dictionary for Regulatory Activities 
Mmol 
Millimole 
NCS 
Not clinically significant 
NSAID 
Non-steroidal  
ODAK 
Orphan Drug for Acanthamoeba keratitis 
PCT 
Propamidine combination therapy 
PHMB 
Polyhexamethylene biguanide 
PP 
Per protocol 
PPAS 
Per-protocol analysis set 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SOC 
System organ class 
SDTM 
Study Data Tabulation Model 
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Statistical Analysis Plan 
LINK Medical Study ID: 
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Acronyms & Abbreviations 
U 
units 
VAMS 
Visual Acuity Measurement Standard 
VFQ25 
25-item visual functioning questionnaire 
WHO 
World Health Organization 
2 
Introduction 
The Orphan Drug for Acanthamoeba keratitis (ODAK) is a project aiming to investigate the potential of 
polyhexamethylene biguanide (PHMB) as a safe and effective drug for the treatment of the rare eye disease 
Acanthamoeba keratitis. This debilitating infectious disease is caused by a commonly occurring protozoan and 
in the absence of treatment can result in blindness. There are currently no approved drugs to treat this disease. 
PHMB has received the orphan drug designation (EU/3/07/498) according to EC regulations 141/2000. 
The Statistical Analysis Plan (SAP) is a complementary document to the Clinical Study Protocol and includes 
a more technical and detailed elaboration of the principal features of the proposed statistical analysis and 
presentations, and the way in which anticipated analysis problems will be handled. 
If the SAP suggests changes to the principal features stated in the protocol, these should also be documented 
in a protocol amendment. Otherwise, it will suffice to record the changes in the SAP. 
3 
Study Objectives 
3.1 
Definitions 
Clinical resolution is a clinical definition, resulting from slit lamp examination, with the following findings: 
• 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and oedema) that 
requires treatment, with a healed corneal epithelium and minimal punctate staining (10 dots or less 
equivalent to Grade 1 on the Oxford Scale). 
• 
No or mild conjunctival inflammation (including bulbar injections, bulbar oedema, tarsal hyperemia): 
mild conjunctival inflammation is acceptable if related to other concurrent conditions such as 
blepharitis. 
• 
No limbitis, scleritis or anterior chamber inflammation. 
• 
No relapse within 30 days of discontinuing all topical and systemic therapy given for Acanthamoeba 
keratitis. 
For regulatory and verification reasons there is an additional 60-day follow-up to exclude late relapses. 
The CRR_12 is the clinical resolution rate at 12 months from randomisation and is defined as the percentage 
of subjects cured 30 days after discontinuing all study therapies, with 12 months of randomisation.  This is the 
primary outcome measure.  Subjects whose disease has not resolved at 12 months are considered treatment 
failures per protocol. 
3.2 
Primary Objective 
The primary objective of the study is to compare the Clinical Resolution Rate (CRR) at 12 months from 
randomization (CRR_12) of 0.08% PHMB + placebo (Group 1), with that of 0.02% PHMB + 0.1% propamidine 
combination therapy (Group 2) estimating the difference in CRR_12 together with the surrounding degree of 
uncertainty, and to test for therapeutic superiority or non-inferiority of 0.08% PHMB monotherapy. 
3.2.1 
Primary Variables 
The primary efficacy variable is the CRR_12. 
The CRR_12 is the clinical resolution rate at 12 months from randomization and is defined as the percentage 
of subjects cured 30 days after discontinuing all study therapies, within 12 months of randomization. 
Criteria for clinical resolution: 
A subject will be considered cured if resolution or absence of all the following clinical signs is confirmed by a 
slit lamp examination: 
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Statistical Analysis Plan 
LINK Medical Study ID: 
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• 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and oedema) that 
requires treatment, with a healed corneal epithelium and minimal punctate staining (10 dots or less 
equivalent to Grade 1 on the Oxford Scale). 
• 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal hyperemia): 
mild conjunctival inflammation is acceptable if related to other concurrent conditions such as 
blepharitis. 
• 
No limbitis, scleritis or anterior chamber inflammation. No relapse within 30 days of discontinuing all 
topical and systemic therapy given for Acanthamoeba keratitis. 
For regulatory and verification reasons, there is an additional follow-up at Day 90 after last treatment to exclude 
late relapses.  Subjects whose disease has not resolved at 12 months are considered treatment failures per 
protocol. 
3.3 
Secondary Objectives 
Secondary objectives of this study are to obtain additional safety information on 0.08% PHMB ophthalmic 
solution. 
Two secondary hypotheses have been specified in the protocol: 
• 
That adverse events (AEs), and those relating to toxicity in particular, are less with PHMB 0.08% 
monotherapy compared to the comparator 
• 
Time-to-cure is shorter in subjects receiving PHMB 0.08% monotherapy compared to the comparator 
3.3.1 
Secondary Efficacy Variables 
The following secondary variables were defined in the protocol: 
• 
Best corrected visual acuity (BCVA), i.e. the eyesight test 20/20 (6/6 in metric units; the Snellen 
notation) 
• 
Corneal scarring as identified by slit lamp examination 
• 
Ulceration severity as identified by slit lamp examination using a 2-grade scoring procedure, i.e. 
present or absent 
• 
Anterior chamber inflammation as identified by ophthalmoscopy using a 4-grade scoring procedure 
• 
EQ-5D-5L questionnaire 
• 
VFQ25 (self-administered format) questionnaire version 2000, without optional items 
In addition, Time-to-cure will be assessed.  Other variables may be analysed in an explorative approach.  
Some disease aspects will be recorded in fields of type Any other abnormalities? If Yes, Specify.  Such 
information will be assessed, converted to Absent/Present variables and analysed if sufficient numbers of 
occurrence are seen. Such findings will be tabulated. 
3.3.2 
Secondary Safety Variables 
The following safety variables were defined in the protocol: 
• 
AEs 
• 
Clinical laboratory tests 
• 
Intraocular pressure (IOP) 
• 
Ophthalmoscopy 
• 
Worsening of the corneal epithelial defect and definable inflammatory signs (development of ring 
abscess and hypopyon) despite >30 days of treatment with the study drug) 
• 
Rate of subjects with a relapse of infection 
• 
Rate of subjects requiring surgery, including amniotic membrane transplants, superficial keratectomy, 
application of cyanoacrylate glue, therapeutic penetrating, lamellar keratoplasty, cataract surgery, 
evisceration, or enucleation 
• 
Rate of subjects requiring non-study therapies, e.g., topical steroids and NSAIDs 
• 
Rate of subject discontinuation from study: to permit alteration of anti-amoebic therapy or for other 
unrelated specified reasons 
• 
Incidence of secondary complications, such as significant corneal neovascularization, corneal 
scarring, corneal perforation, scleritis, secondary glaucoma, cataract, retinopathy 
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Statistical Analysis Plan 
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3.4 
Hypothesis 
The primary hypothesis to be tested is that the CRR_12 of subjects treated with 0.08% PHMB monotherapy, 
is superior, or worse by no more than an acceptable pre-defined 0.20 non-inferiority margin (Δ), compared to 
the CRR_12 of a 0.20 PHMB + 0.1% propamidine combination therapy, administered according to the 
Treatment and Follow-up Protocol described in Section 5.3, which is based on a consensus of currently clinical 
guidelines. 
Secondary hypotheses are: 
• 
That adverse events, and those relating to toxicity in particular, are less with PHMB 0.08% 
monotherapy compared to the comparator. 
• 
That time to a cure is shorter in subjects receiving PHMB 0.08% monotherapy compared to the 
comparator. 
Note: The estimated CRR_12 from the start of treatment is 67% for the conventional (0.02% PHMB + 0.1% 
propamidine) combination therapy (from the sponsor’s observational, case series retrospective study 
038/SI20) (63% when assuming a prevalence of late stage diseases in 38% of subjects). 
4 
Study Design 
This is a randomised, assessor-masked, active-controlled, multiple centre, parallel-group Phase 3 study to 
evaluate the efficacy, safety and tolerability of 0.08% PHMB ophthalmic solution compared to the conventional 
0.02% PHMB + 0.1% propamidine combination therapy in male and female subjects affected by 
Acanthamoeba keratitis. 
The study is designed as a superiority study with the possibility to test for non-inferiority if the superiority 
hypothesis is not met, according to the requirements of the guidance from the European Agency for the 
Evaluation of Medicinal Products (EMA) (CPMP/EWP/482/99).21 The specific conditions for this outcome are 
described in section 7.4 of the protocol. 
The study consists of an eligibility screening visit, a treatment period including short ambulant visits, and follow-
up visits. 
Group 1: 0.08% PHMB + placebo 
Group 2: 0.02% PHMB + 0.1% propamidine combination therapy (PCT) 
The study will be performed in male and female subjects affected by Acanthamoeba keratitis, ≥12 years of 
age, inclusive.  The inclusion- and exclusion criteria are presented in Chapter 4 of the protocol. 
5 
Study Population 
The study will be performed in subjects affected by Acanthamoeba keratitis. 
Screening will be performed on the day of the first drug administration (Day 0), or up to two days before study 
drug administration.  The specific inclusion and exclusion criteria can be seen in sections 4.1 and 4.2 of the 
protocol. 
5.1 
Sample Size 
A total of 130 subjects affected by Acanthamoeba keratitis will be assigned to one of the above two treatment 
groups in a ratio of 1:1. 
The protocol states, in section 7.6: 
“From the results of the sponsor’s observational, case series retrospective study 038/SI, a CRR_12 of 67% for 
the conventional combination therapy of PHMB 0.02% and propamidine 0.1% is expected. This figure is in the 
range of cure rates described in the literature. If the true difference in CRR_12 is 0.20 (Δ) (or more) in favour 
of PHMB monotherapy (0.08% PHMB), a total sample size of 116 subjects (allowing for 10% loss to follow-up) 
should give the study at least 80% power to detect superiority, with 2-sided alpha=0.10 (or equivalently 1-sided 
alpha=0.05). 
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Statistical Analysis Plan 
LINK Medical Study ID: 
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Assuming a prevalence of late stage disease, and worse outcomes, in 38% of subjects, the expected CRR_12 
in the control group will be reduced from 67% to 63%. To account for the inclusion of this group, the sample 
size has been adjusted to 130 subjects (allowing for 10% loss to follow-up, i.e. 116 evaluable subjects). 
5.2 
Randomisation 
After obtaining oral and written informed consent, subjects meeting the eligibility criteria were randomized to 
one of the treatment groups in a 1:1 ratio. The randomization schedule was generated using a computer 
program and verified for accuracy using strict quality control procedures. Eligible patients received a masked 
treatment assignment with a unique randomization code based on the randomization list. The assigned 
randomization code was captured in the electronic Case Report Form (eCRF). This randomization code was 
designed as not to disclose any treatment assignment, see section 5.1 of the protocol. 
Until the moment of unmasking, the treatment assignments linked to the codes should only be accessible for 
the individuals appointed by the Sponsor as Qualified Persons responsible for Pharmacovigilance. Emergency 
Unmasking should only be done if knowledge of treatment assignment was considered relevant for medical 
care of the patient.  The randomization code will be broken when all subjects have finished the study at the 
formal request from the study statistician after the Clean file report has been signed, any early unblinding has 
been accounted for and the database has been locked. 
6 
Assessments 
6.1 
Primary efficacy endpoint criteria for assessing clinical resolution 
A subject will be considered cured if resolution of all the following clinical signs are observed, resulting from 
a slit lamp examination: 
• 
No corneal inflammation (including subepithelial infiltrates, stromal infiltrates and oedema) that 
requires treatment, with a healed corneal epithelium and minimal punctate staining (10 dots or less 
equivalent to Grade 1 on the Oxford Scale). 
• 
No or mild conjunctival inflammation (including bulbar injection, bulbar oedema, tarsal hyperemia): 
mild conjunctival inflammation is acceptable if related to other concurrent conditions such as 
blepharitis. 
• 
No limbitis, scleritis or anterior chamber inflammation. 
• 
No relapse within 30 days of discontinuing all topical and systemic therapy given for Acanthamoeba 
keratitis. 
For regulatory and verification reasons, there is an additional 60-day follow-up to exclude late relapses. 
Relapse of infection is identified by a positive culture, unfortunately very insensitive due to the persistence of 
deep organisms, supported by an increase in cysts on confocal (also insensitive in severe disease) or using 
clinical criteria: development of more severe corneal inflammation, melting, ulceration hypopyon, development 
of ring abscess necessitating another intensive course of therapy. 
The primary hypothesis to be tested is that the CRR_12 of subjects treated with 0.08% PHMB monotherapy, 
is superior, or worse by no more than an acceptable pre-defined 0.20 non-inferiority margin (Δ), compared to 
the CRR_12 of 0.02% PHMB + 0.1% propamidine combination therapy, administered according to the 
Treatment and Follow-up Protocol presented on page 16 of the Protocol. 
6.2 
Secondary efficacy endpoints assessment 
6.2.1 
Assessment of BCVA 
BCVA is being determined using pinhole with or without spectacles, soft contact lenses or rigid contact lenses. 
Detailed information about the procedure is provided in the Study Operation Manual.  International Council of 
Ophthalmology approved a Visual Acuity Measurement Standard (VAMS) in 1984 (3). In Table II in the VAMS 
different types of notations of visual acuity are presented, one being the Snellen notation, such as 20/16 when 
measured in feet or 6/4.8 when measured in metres.  Other notations are the decimal notation, 20 divided by 
16 (6 divided by 4.8) which equals 1.25, the visual angle (the inverse of the decimal notation, 0.80) or the 
logarithm (base 10) of the visual angle, referred to as LogMAR. 
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In addition, refraction will be assessed, together with any potential relationship between BCVA and refraction. 
6.2.2 
Assessment of corneal scarring 
Corneal scarring is being assessed as Present or Absent, using the slit lamp biomicroscopy performed by the 
examining ophthalmologist. Detailed information about the procedure is provided in the Study Operation 
Manual. 
6.2.3 
Assessment of ulceration severity 
Ulceration severity is being assessed as Present or Absent by the examining ophthalmologist using the slit 
lamp biomicroscope. 
6.2.4 
Pupil test 
Pupil test (swinging light test) is reported as Normal; Abnormal, NCS; Abnormal CS.  The specific abnormalities 
will be listed. 
6.2.5 
Assessment of anterior chamber inflammation 
Anterior chamber inflammation is being assessed using a 3-grade scoring procedure using the slit lamp 
biomicroscope: Abnormal Clinically significant (CS), Abnormal Not CS (NCS), and Normal.  Clinically 
significant events are defined as “any variation, symptoms, or testing that has medical relevance according to 
the investigator and may result in an alteration in in medical care". 
6.2.6 
EQ-5D-5L and VFQ25 assessments 
EQ-5D-5L and VFQ5 are two instruments for subject-reported outcomes; the former addressing the overall 
health status of the subjects and the latter their visual function status.  More detailed information can be seen 
in appendices 2 and 3 of the protocol. 
6.3 
Secondary safety endpoints assessment 
6.3.1 
AEs 
Recording of AEs will commence with signing of the ICF. AEs will be captured in the CRF. AEs will be followed 
up by the investigator as specified in section 8 of the protocol, AE Reporting.  Standard CDISC STDM 
annotation is being used for AE data. 
6.3.2 
Clinical laboratory tests assessment 
Clinical laboratory results (haematology, biochemistry and urinalysis) were conducted at the Screening or 
Baseline visit and at the End-of-study visit.  These are annotated using standard SDTM names.  Clinical 
laboratory tests will be presented with summary statistics. In addition, the tests will be analysed with ANCOVA 
(baseline value of respective laboratory test and treatment group will be included in the model). All results will 
be regarded as descriptive only. The p-values will be used to flag safety and tolerability variables worthy of 
further attention. 
7 
Methods of Analysis 
7.1 
General 
All statistical analyses will be performed in accordance with the ICH E9 guideline for Statistical Principles for 
Clinical Trials (1), using SAS® (Version 9.4 or higher, SAS Institute Inc., Cary, NC, USA). 
7.1.1 
Presentation of Results 
All results will be presented for each visit by treatment group and in total, unless stated otherwise. It should be 
clearly stated which unit applies to each presented variable. 
Continuous data will be summarised using descriptive statistics, and the following parameters will be reported: 
• 
number of subjects with evaluable observations and missing observations 
• 
arithmetic mean and change from baseline with standard deviations 
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• 
median 
• 
first and third quartiles 
• 
minimum and maximum 
Categorical data will be presented using absolute frequency and percentage. When the absolute frequency is 
zero, the percentage will not be presented. Unless stated otherwise, the denominator for percentage 
calculations will be the total number of subjects in the applicable analysis set, including subjects with missing 
data. For variables with missing values, the number and percentage of subjects with missing values will be 
presented. 
Significance tests will be two-sided and performed at the 5% significance level, unless stated otherwise. When 
reporting the results of significance tests, p-values will be presented. 
All confidence intervals (CIs) presented will be two-sided with a nominal confidence level of 95%, unless stated 
otherwise. 
Data will be presented using an appropriate number of decimal places, to ensure that undue precision is not 
implied (e.g. the number of decimals should not exceed the accuracy of the measuring instrument). Raw data 
will be presented with the same number of decimals as collected, and derived data with an appropriate number 
of decimals based on general practice, mathematical rationale or scientific rationale. 
Minimum and maximum values will be presented with the same number of decimals as the analysed variable 
and the other descriptive statistics will be presented with one decimal more. Percentages and proportions will 
be presented with one decimal. Odds ratios and hazard ratios will be presented with 3 decimals, and small 
and large values will be presented as ‘<0.001’ and ‘>999.999’ respectively. CI bounds will be presented with 
the same number of decimals as the corresponding point estimate, and p-values will be presented with 4 
decimals or as ‘<.0001’. 
Mock tables and graphs are presented in the Data Display Plan (DDP), which is a supplementary document 
to this analysis plan. Individual subject data listings will be presented according to the ICH E3 guideline for 
Structure and Content of Clinical Study Reports (2), unless stated otherwise. 
7.1.2 
Baseline 
Unless stated otherwise, the baseline value for a parameter is defined as the last non-missing value before 
the first dose of the investigational medicinal product (IMP). 
7.1.3 
Analysis Relative Day 
The analysis relative day for an assessment/value is defined as the time in days from the date of randomization 
to the date of the assessment. The date of randomization is considered as day 1, and earlier dates will 
correspond to a negative day.  In the Schedules of assessments table on page 22 and 23 of the protocol, the 
day of randomisation, which also encompass some baseline measurements and drug dispensing, is called 
Day 0.  As CDISC does not use 0 for day numbering, Day 1 will be used for the day of randomisation.  Hence, 
the day numbering after the baseline day in the Schedules table will be increased by one. 
7.1.4 
Analysis Visit 
An analysis visit is defined as a categorical variable used to classify values within an analysis variable into 
temporal or conceptual groups used for analyses. 
The visits as defined in the case report form, CRF, will be used as analysis visits.  The Schedule of 
Assessments table provide acceptable deviations from the specified study day at which study visits should 
occur.  These are ± 2 days up to study day 22, called study day 21 in the Schedules table, ref. section 7.1.3 
above, ± 3 days from study day 31 to 30 days after end of treatment (EoT), and ± 5 days at the 90 day after 
end of treatment visit.  A review of assessment times will be conducted before the clean file meeting.  
Necessary revisions will be documented in the Clean file report.  Results tables will present statistics by visit 
number.  Where deemed necessary, longitudinal analyses will use the observed durations from baseline.  The 
mean duration in days between visits will be presented. 
 
 
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Statistical Analysis Plan 
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Study day windows 
Study visit day 
Target study day of visit 
Window of study day visit 
0 
0 
± 0 days 
7 
7 
± 2 days 
14 
14 
± 2 days 
21 
21 
± 2 days 
30 
30 
± 3 days 
60 
60 
± 3 days 
30 days after previous visit§ until resolution 
30 after previous visit 
± 5 days 
30 days after EoT  
30 days after EoT 
± 3 days 
90 days after EoT  
90 days after EoT 
± 5 days 
§ The protocol states “and every 30 ± 5 days until resolution”. This is interpreted as 30 days after the previous visit, and not 
a multiplicity of 30 days based on a fixed setup from baseline, i.e. not 31+60+n*30. 
 
In general, data from unscheduled visits will be presented in data listings only and not included in analysis or 
summary tables. An exception to this is data used to confirm eligibility in association with screening or 
randomisation where the last assessment will be considered in summaries of screening data. 
7.1.5 
Handling of Missing Data 
Sensitivity analyses performed for a single variable are described in detail in the corresponding section below. 
Data listings will include the observed values. For derived variables, values based on imputed data will be 
presented in listings. 
7.1.6 
Interim Analyses 
No interim analysis is planned. 
7.1.7 
Multiplicity 
Three hypotheses are specified in the Protocol, one primary and two secondary hypotheses.  Time-to-
resolution using the visit day at which resolution was first seen, will be considered statistically significant if the 
sum of the p-value for the primary variable and the p-value from the time-to-resolution analysis is less than the 
specified overall significant level.  The use of a non-inferiority alternative if the difference alternative should not 
be obtained, does not affect the overall p-value level of the primary hypothesis. 
Hypotheses tested for other secondary variables will all be analysed using significance elves of 0.05. 
7.1.8 
Subgroups 
No subgroup analyses will be performed. 
7.2 
Analysis Sets 
The decision on the classification of subjects to each analysis set will be taken at the clean file meeting, prior 
to breaking the blind, and documented in the clean file report together with the reasons for excluding subjects 
from analysis sets.  Subjects who have not attended any visits where disease status is assessed, i.e. those 
who did not reach the day 30 visit, the first visit where disease status is recorded, will be classified as dropouts 
from the efficacy analysis sets. Any 30dayFU visit that is under the window, i.e. visit occurred too soon, will 
not be removed from the analysis population as long as the 90DayFU visit also proved that the patient was 
cured. 
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7.2.1 
Full Analysis Set (FAS) 
All subjects who have been randomized and has a confirmed diagnosis of Acanthamoeba keratitis and for 
whom the primary efficacy variable has been assessed will be included in the FAS. The FAS will be used in 
all efficacy analyses.  The last disease status assessment will be used for disease status assessment. 
Analysis using the FAS will be based on the planned treatment (i.e. subjects will be analysed ‘as randomised’). 
7.2.2 
Per-Protocol Analysis Set (PPAS) 
The per-protocol analysis set is defined as the subset of subjects in the FAS for whom no major protocol 
deviation judged as having an impact on the primary efficacy analysis was reported or identified. The last 
disease status assessment within 12 months of randomisation will be used for disease status assessment.  
For subjects with disease resolution at that last visit, the 30-day and 90-day follow-up assessments should 
confirm disease resolution. Otherwise, the disease will be regarded as not having been resolved. 
The decision as to which protocol deviations should be considered as reason for exclusion from the PPAS 
should be made at the clean file meeting and documented in the clean file report. 
Analysis on the PPAS will be based on the actual treatment (i.e. subjects will be analysed ‘as treated’).  The 
primary efficacy variable, CRR_12, will also be evaluated using the PPAS. 
7.2.3 
Safety Analysis Set 
All subjects who have received at least one dose of study medication will be included in the Safety Analysis 
Set. This set will be used in all analyses of safety data. 
Analysis on the safety analysis set will be based on the actual treatment (i.e. subjects will be analysed ‘as 
treated’). 
7.3 
Disposition of Subjects 
The following will be presented: 
• 
Number of screened subjects, in total, if available. 
• 
Number of screening failures, in total, if available. 
• 
Number of randomised subjects, by treatment group and in total. 
Based on the number of randomised subjects, the following will also be presented, by treatment group and in 
total: 
• 
Number and percentage of subjects who did not meet randomisation inclusion/exclusion criteria  
• 
Number and percentage of subjects who did not receive any dose of IMP. 
• 
Number and percentage of subjects who received at least one dose of IMP. 
• 
Number and percentage of subjects who completed the required treatment. 
• 
Number and percentage of subjects who withdrew prematurely from the study, i.e. withdrew before 
completing the follow-up visits. 
• 
Number and percentage of subjects in each of the analysis sets. 
In addition, a frequency table on the primary reason for premature withdrawal from the study will be presented 
by treatment group and in total. Percentages for this table will be based on the number of prematurely 
withdrawn subjects. 
The number of subjects attending each study visit will also be summarised. 
7.4 
Protocol Deviations 
Protocol deviations will be presented in a data listing. 
The number and percentage of randomized subjects with at least one major protocol deviation leading to 
exclusion from an analysis set will be presented. 
Major deviations are those deviations that could affect the scientific integrity of the study: 
• 
Did not comply with the allocated treatment 
• 
Did not attend the two follow-up visits 
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7.5 
Demographics and Baseline Characteristics 
Summary statistics and frequencies on demographic data (age and sex) will be presented for all analysis sets. 
In addition, the summary of Best sphere (dioptres) will be summarised for the following categories: 
Hypermetropes (Refraction greater than 0) 
Emmetropes (Refraction equal to 0) 
Myopes (Refraction less than 0) 
7.6 
Medical History and Concurrent Diseases 
Medical history and concurrent diseases will be coded according to the latest available version of Medical 
Dictionary for Regulatory Activities (MedDRA). 
For each system organ class and preferred term, the number and percentage of subjects with at least one 
condition in that system organ class or preferred term will be presented. Medical history and concurrent 
diseases will be presented in separate tables, based on the safety analysis set. 
Medical history is defined as events stopped prior to study inclusion. Concurrent diseases are defined as 
ongoing events and events stopped on or after baseline. If the start and/or stop date is partially unknown, the 
following imputation rules will be used for the purpose of classifying the events: 
Table: Imputation of missing parts of dates 
 
Imputed start date 
Imputed end date 
Unknown year 
Missing 
Missing 
Unknown month 
1 January 
31 December 
Unknown day 
First of month 
Last of month 
If it is not possible to classify the condition based on the reported and/or imputed start and end dates, it will be 
considered as concurrent. In data listings, the dates will be presented as reported. 
7.7 
Prior and Concomitant Medication 
Medications will be coded according to the World Health Organization (WHO) anatomical therapeutic chemical 
(ATC) classification system and summarized by therapeutic subgroup (ATC level 2) and preferred name. 
For each therapeutic subgroup and preferred name, the number and percentage of subjects who used at least 
one medication of that therapeutic subgroup or preferred name will be presented. Prior and concomitant 
medications will be summarized in separate tables, based on the safety analysis set. 
If a reported medication cannot be coded with a preferred name, the lowest available higher-level dictionary 
term will be used instead in the summary tables. If a medication cannot be coded on a lower level than the 
therapeutic subgroup or the anatomical main group (ATC level 1), that medication will be presented as ‘Not 
codable’ under that therapeutic subgroup/anatomical main group. 
Prior medication is defined as medication stopped prior to baseline. Concomitant medication is defined as 
ongoing medication or medication stopped on or after baseline. If the start and/or stop date is partially 
unknown, the following imputation rules will be used for the purpose of classifying the medication: 
 
Imputed start date 
Imputed end date 
Unknown year 
Missing 
Missing 
Unknown month 
1 January 
31 December 
Unknown day 
First of month 
Last of month 
If it is not possible to classify a medication based on the reported and/or imputed start and end dates, it will be 
considered as concomitant. In data listings, the dates will be presented as reported. 
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Statistical Analysis Plan 
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7.8 
Compliance 
Subjects who miss up to 1 full day of treatment within the first 5 days after starting treatment or up to 2 full 
days of treatment after the first 5 days of treatment should not be discontinued but will be classified as non-
compliant. Subjects will be discontinued from the study if in the opinion of the investigator drug compliance is 
insufficient. 
The rate of compliance with the IMP will be summarised for the FAS.  The reasons will be listed. 
7.9 
Efficacy Evaluation 
All efficacy evaluations will be based on the FAS.  The PPAS will be used for supportive sensitivity analysis of 
the primary efficacy variable. 
7.9.1 
Primary Efficacy Variable 
The main endpoint disease resolution 12 months after baseline will be assessed at each visit up to study day 
365. 
Clinical resolution 12 months after baseline for a patient is defined as: 
The percentage of subjects cured 30 days after discontinuing all study therapies, within 12 months of 
randomization. A clinical resolution classification requires a Yes response to the eCRF variable CRYN 
question: “Was clinical resolution obtained?” at one of the study visits from day 1 up to study day 365 in 
combination with a response No to the eCRF variable RELAPYN question “Did the subject experience relapse 
since the last visit?” at the pursuing visits, up to the last recording of this information.  There should be no 
relapses after (the last) clinical resolution classification. Discontinued subjects will be regarded as not cured. 
For patients defined as fulfilling disease resolution within 12 months, the date of the visit at which annotated 
CRYN is first answered with a Yes will be used as the date of resolution.  The time-to-cure secondary variable 
will be the number of days from randomisation to the visit where resolution was recorded. 
Since it is also possible to test for non-inferiority if the superiority hypothesis is not met, additional conditions 
are specified (see list below) to meet the strict requirements of a non-inferiority study, making it feasible to test 
for non-inferiority using the results from the superiority analysis, without a further separate analysis. The 90% 
CI for the difference between the treatments obtained from the superiority analysis gives the necessary 
information. Position of the lower end of the CI relative to a pre-defined agreed 0.20 non-inferiority margin (Δ) 
provides the key information for making decisions (conclusions) about non-inferiority. 
PHMB has received the orphan drug designation. The use of 90% CI for the difference between the two 
treatments is to provide a 95% lower CI limit for non-inferiority. 
The additional conditions specified to make testing for non-inferiority feasible are the following: 
• 
Pre-defined non-inferiority margin (Δ=0.20) based on both clinical and statistical considerations, i.e., 
chosen on the basis of external information and not chosen to fit the data. 
• 
Analysis of the FAS and the PPAS, giving similar results with respect to p-values and CIs for the 
efficacy measure at issue. 
• 
Study design and implementation directed at minimizing deviation from protocol (a particularly 
important source of bias in non-inferiority studies), e.g. special care to minimize violations of: entry 
criteria; non-compliance; withdrawals; loss; missing data; etc. This should help ensure the second 
bullet point. 
• 
Evidence that the standard treatment is showing its usual (expected) level of efficacy. 
These specifications are in accord with the recommendations by the Committee for Proprietary Medicinal 
Products, in document Points to consider on switching between superiority and non-inferiority (CPMP) 482/99.  
The recommendation regarding similar results with respect to p-values and CIs is founded on that the FAS “is 
generally not conservative and its role should be considered very carefully” in non-inferiority trials. 
Null hypothesis difference: 
• 
No difference between test treatment and reference treatment 
Non-inferiority clinical studies aim to demonstrate that the test treatment is no worse than the reference by 
more than a pre-specified small amount. 
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Statistical Analysis Plan 
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Null hypothesis non-inferiority: 
• 
Test treatment is inferior to the reference by Δ or more 
CRR_12 will be analysed using a general linear model (GLM) approach with treatment as factor to estimate 
the difference between success rates for the treatments, and test for difference between treatments.  This will 
be the primary test of efficacy.  The new treatment will be declared as non-inferior to the 0.02% PHMB + 0.1% 
propamidine combination therapy, if the lower limit of the 95% CI satisfies the requirement of a non-inferiority 
margin of Δ=0.20.  The effects of covariate variables on the success rate will also be assessed using the GLM 
model with treatment, age and sex as explanatory variables.  Further explorative analyses will be conducted 
by adding other, selected variables such as steroid use before treatment start, to the GLM model. 
7.9.2 
Justification for choice of the non-inferiority margin 
On statistical grounds: 
Our pre-defined non-inferiority margin of Δ=0.20 is based on previous studies (historical data), i.e., the study 
referred to in the protocol showing 1/20 cured without treatment being the only data available that can be used 
to estimate the cure with placebo, and the results of the reference (standard) treatment from the SIFI / 
Moorfields observational studies, adjusted for inclusion of advanced disease cases in the study. The table 
below demonstrates the statistical grounds for the choice of Δ. The chosen Δ of 0.20 satisfies the condition 
that "the test treatment is expected to retain at least 50% of the standard treatment effect over placebo, in 
order to be considered as noninferior". 
 
Table 1:  Selection of Non-Inferiority Margin (Δ) based on statistical issues 
Proportion 
A 
Cured with no treatment: Historic Data 
0.05 
B 
Cured with Standard Treatment: SIFI Study Data 
0.67 
Ba 
B adjusted for inclusion of stage-3 cases (63/100) 
0.63 
Ba (lower) 
Lower 95% CI bound for Ba (Binomial exact) 
0.53 
M1 
Ba (lower) – A: the standard treatment effect over placebo 
0.48 
Δ 
Non-inferiority margin 
0.20 
Proportion of M1 retained by the Test treatment; (M1 – Δ)/M1 
58% 
The Test Treatment retains well over 50% or the Standard treatment effect over placebo 
 
On clinical grounds: 
The Sponsor proposes as clinically acceptable a non-inferiority margin of 0.20: this is within the accepted level 
for a study of this kind in view of the following: 
• 
A poor response to therapy is common in this disease. Disease progression is slow in this disease 
(over weeks and months) and clinicians have to assess the effect of treatments every 1-2 weeks, in 
cases not progressing well, and modify therapies to optimize outcomes. Because of the disease 
chronicity the use of an ineffective treatment for these periods results in a delayed response, but no 
serious short term morbidity. The study results may indicate non-inferiority, when at worst the true 
difference in proportion 'cured' (Combined - PHMB alone) is 0.20. The most likely impact of such a 
finding on clinical practice will be to encourage the use of PHMB monotherapy as a first line treatment. 
• 
However, if PHMB monotherapy fails in clinical practice during the early stages of treatment, which is 
to be expected in some subjects, the clinician is unlikely to abandon the good clinical practice of 
monitoring the disease progress closely and adding other antiamoebics to the therapy when 
necessary. 
• 
At worst, the impact of the study finding might result in a little delay (matter of days) in starting a 
combined treatment in some patients, whilst giving a chance for the biguanide mono-therapy to act. 
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Statistical Analysis Plan 
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This scenario is unlikely to result in blindness or serious morbidity. It may cause some delay in the 
clinical resolution. 
• 
By contrast, the possibility that the true difference may be much smaller than 0.2, or even in favour of 
PHMB monotherapy, will have important beneficial consequences for patients and ophthalmic 
services. 
7.9.3 
Secondary Efficacy Variables1  
Time-to-cure comparison between the treatment groups is specified as efficacy hypothesis and will be 
subjected to formal statistical testing.  Other analyses of secondary efficacy variables will be done on a 
hypothesis-generating basis, i.e. no p-value adjustments will be conducted, and conclusions regarding the 
analysis results will be commented on such a basis. 
Time-to-cure 
The aim of the hypothesis test is to show that the time is less among the subjects receiving PHBM 0.08% 
monotherapy compared to the comparator.  Patients will either be classified as Cured at some point of time 
while in the study or leave the study without being classified as Cured. As a secondary efficacy variable, time-
to-cure (clinical resolution) will be analysed.   The Cox Proportional Hazards regression (subject to validity of 
the 'proportional hazards' assumption) and Kaplan-Meier survival plots. If the proportional hazard assumption 
is not fulfilled a logrank test will be performed.  Not all patients may have information regarding when they were 
cured, e.g. due to death before being cured, or completed the study without being cured.  Both the Cox 
Proportional Hazards regression and the Kaplan-Meier plot uses the censoring timepoint in their calculations.  
The censoring timepoint will be defined based on the timepoint of the last observation when cure status was 
assessed.  The null hypothesis is that the two treatment arms have equal hazard functions.  Relevant data will 
be presented at the Clean file meeting for discussion and acceptance. 
BCVA 
The VAMS states that: “Depending on the problem, this notation can be most useful when analysing or 
graphically plotting visual acuity scores because equal linear steps on the LogMAR scale represent equal 
ratios in the standard size sequence.”  Hence, BCVA will be presented and analysed using LogMAR values, 
where 
𝐿𝑜𝑔𝑀𝐴𝑅= log⁡(
𝑂𝑅𝑅𝐸𝑆𝐵𝐶𝐴2
𝑂𝑅𝑅𝐸𝑆𝐵𝐶𝐴1), 
where ORRES_BCA1 and ORRES_BCA2 are the annotated values from the BCVA test.  These values are 
positive number with up to one decimal value.  ORRES_BCA1 values recorded in the eCRF, the distance from 
the subject to the chart, varies at least from 0.6 to 10 metres. Even if different scales are used (Decimal, US, 
6m), the LogMAR is not affected since the ratio will be the same, therefore no converting to unified units is 
needed2. BCV A, as a secondary efficacy variable, will be analysed.  ANCOVA will be used with treatment and 
the Baseline value of LogMAR as predictors.  Refraction at Baseline may also be used as a predictor. 
Drug compliance 
Drug compliance is recorded as Yes or No answer to the question “Was the study drug administration 
compliant with the protocol?”, annotated as PROTCOMYN.  Drug compliance will be analysed with a logistic 
regression model and model-based estimates of the difference between treatments, odds ratio with 
corresponding CIs and p-values will be provided. Covariate variables such as treatment, age and sex will be 
included.  Any available information regarding drug use will also be presented, such as number of vials 
returned. 
EQ-5D-5L3 & EQ VAS, at all visits 
EQ-5D-5L consists of 5 dimensions, Mobility, Self-care, Usual activities, Pain/discomfort, and 
Anxiety/depression, each answered with one of five different answers: 
Mobility: 
I have No problems/Slight problems/Moderate problems/Severe problems in walking about/I am 
unable to walk about 
 
1 For safety variables, see Section 7.10.2 
2 RR.2 Reference Tables For Equivalent Visual Acuity Notations 
3 One subject, aged 15 years, used the EQ-5D-Y version.  At site 31, Silesia, all subjects at one or more visits used the 
EQ-5D-3L version 
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Statistical Analysis Plan 
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Self-care: 
I have No problems/Slight problems/Moderate problems/Severe problems in washing or dressing 
myself/I am unable to wash or dress myself 
Usual activities (e.g. work, study, housework, family or leisure activities): 
I have No problems/Slight problems/Moderate problems/Severe problems doing my usual activities/I 
am unable to do my usual activities 
Pain/discomfort: 
I have No pain or discomfort/Slight pain or discomfort /Moderate pain or discomfort /Severe pain or 
discomfort/I have extreme pain or discomfort 
Anxiety/depression: 
 
I am not/slightly/moderately/severely/extremely anxious or depressed. 
The EQ-5D will be summarised for each dimension.  A table presenting the number of subjects reporting a 
problem, i.e. a score higher than 1, will also be presented.  A repeated measures ANCOVA for each dimension 
will be conducted using treatment and the baseline value as predictors. 
The EQ VAS score, annotated as ORRES_EQ5D0206, is being recorded at all visits, including Baseline.  
Integer values range from 0 to 100.  A repeated measures ANCOVA will be used to compare potential 
differences in trends over the treatment period using treatment and the Baseline VAS value as predictors. 
Slit-Lamp Examination, at all visits 
Below can be seen the different efficacy variables based on the Slit lamp examination.  These will be presented 
in tables, as can be seen in the Data Display Plan (DDP).  Some of these variables will only be recorded in 
Specify types of eCRF fields and will be converted to a Yes/No variable before tabulation and possible analysis. 
Corneal scarring 
Corneal scarring is being assessed as Present or Absent, using the slit lamp biomicroscopy, and is annotated 
as SLECOSCAR.  This is being performed by the examining ophthalmologist, using a slit lamp biomicroscope. 
Detailed information about the procedure is provided in the Study Operation Manual.  As a stated secondary 
variable, corneal scarring at EoT will be analysed with logistic regression using treatment group and corneal 
scarring at baseline as predictors. 
Ulceration severity 
Ulceration severity is being assessed as Present or Absent by the examining ophthalmologist using the slit 
lamp biomicroscope and is annotated as SLECOUL.  As a stated secondary variable, ulceration severity will 
be analysed with logistic regression using treatment group and ulceration severity at Baseline as predictors.   
Conjunctiva 
 
Erythema grade SLECER (using grades 0, 1, 2, 3) 
 
Oedema grade SLECED (using grades 0, 1, 2, 3, 4) 
 
Discharge SLECOND (Present, Absent) 
 
Papillae SLECONP (Present, Absent) 
 
Follicles SLECONF (Present, Absent) 
Limbitis 
Scleritis 
Corneal Abnormalities4 
Dendritiform epithelial lesions 
Perineural infiltrates 
Corneal epithelial defects (punctate erosions), 
Corneal epithelial ulcer (confluent erosions) 
Corneal epithelial defects SLECOED (Present, Absent) 
Corneal ulceration SLECOUL (Present, Absent) 
Corneal epithelial opacity SLECOEO (Present, Absent) 
Corneal stromal opacity SLECOSO (Present, Absent) 
 
4 Some of these may appear an Any other abnormality and will be taken from the Specify variable 
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Corneal scarring SLECOSCAR (Present, Absent) 
Corneal ring abscess 
Any other abnormalities SLECOTHSP (Yes, No), Yes-> Specify 
Lens 
 
Lens SLELEN (Normal, Abnormal, NCS, Abnormal, CS) 
Anterior chamber inflammation 
Anterior chamber inflammation is being assessed using a 4-grade scoring procedure.  The annotation is 
SLEACI with values 0, 1, 2 and 3, signifying None (no Tyndall effect), Mild (Tyndall effect barely discernible), 
Moderate (Tyndall beam in the anterior chamber is moderately intense), and Severe (Tyndall beam in the 
anterior chamber is severely intense).  As a secondary efficacy variable, the anterior chamber inflammation 4-
grade scores will be analysed using an ordinal logistic regression approach with treatment and the Baseline 
anterior chamber inflammation as predictors.  
Pupil test, at all visits 
Result: PUPTORRES (Normal/Abnormal, NCS/Abnormal, CS) 
VFQ-25, at all visits 
The Visual Functioning Questionnaire consists of 25 questions, two with one or three sub-questions designed 
to elucidate the answer to the main question, see Tables 2and 3 below. 
 
Table2: Scoring Key for VFQ-25 
Item numbers 
Change original 
response category(a) 
To recoded value of: 
1, 3, 4, 15c(b) 
1 
2 
3 
4 
5 
100 
75 
50 
25 
0 
2 
1 
2 
3 
4 
5 
6 
100 
80 
60 
40 
20 
0 
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16a 
1 
2 
3 
4 
5 
6 
100 
75 
50 
25 
0 
* 
17, 18, 19, 20, 21, 22, 23, 24, 25 
1 
2 
3 
4 
5 
0 
35 
50 
75 
100 
(a)  Pre-coded response choices as printed in the questionnaire 
(b) Item 15c has four-response levels, but is expanded to a five-levels using item 15b 
Note: If 15b=1, then 15c should be recoded to “0”. 
 
If 15b=2, then 15c should be recoded to missing. 
 
If 15b=3, then 15c should be recoded to missing. 
 * 
Response choice “6” indicates that the person does not perform the activity because 
 
non-vision related problems. If this choice is selected, the item is coded as “missing”. 
 
 
Table 3: Averaging of items to generate VFQ-25 sub-scales 
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Scale 
Number of items 
Items to be averages (after 
recoding per Table 2) 
Annotated variable names, 
prefix=ORRES_VFQ2 
General health 
1 
1 
01 
General vision 
1 
2 
02 
Ocular vision 
2 
4, 19 
04, 19 
Near activities 
3 
5, 6, 7 
05, 06, 07 
Distance activities 
3 
8, 9, 14 
08, 09, 14 
Vision specific: 
 
 
 
 
Social functioning 
2 
11, 13 
11, 13 
 
Mental health 
4 
3, 21, 22, 25 
03, 21, 22, 25 
 
Role difficulties 
2 
17, 18 
17, 18 
 
Dependency 
3 
20, 23, 24 
20, 23, 24 
Driving 
3 
15c, 16, 16a 
15C, 16, 16A 
Colour vision 
1 
12 
12 
Peripheral vision 
1 
10 
10 
 
Averaging the means for each scale for each patient, excluding the General health score, provides a composite 
score for each patient, which can then be used to calculate the composite score for each treatment group. 
The mean for each sub-scale per patient is calculated as; 
𝑀𝑒𝑎𝑛=⁡(𝑆𝑢𝑚⁡𝑜𝑓⁡𝑠𝑐𝑜𝑟𝑒⁡𝑓𝑜𝑟⁡𝑒𝑎𝑐ℎ⁡𝑖𝑡𝑒𝑚⁡𝑤𝑖𝑡ℎ⁡𝑛𝑜𝑛−𝑚𝑖𝑠𝑠𝑖𝑛𝑔⁡𝑎𝑛𝑠𝑤𝑒𝑟)
(𝑇𝑜𝑡𝑎𝑙⁡𝑛𝑢𝑚𝑏𝑒𝑟⁡𝑜𝑓⁡𝑖𝑡𝑒𝑚𝑠⁡𝑤𝑖𝑡ℎ⁡𝑛𝑜𝑛−𝑚𝑖𝑠𝑠𝑖𝑛𝑔⁡𝑎𝑛𝑠𝑤𝑒𝑟𝑠)
 
 
The Visual Functioning Questionnaire consists of 25 questions, two with one or three sub-questions designed 
to elucidate the answer to the main question.  The main questions are annotated as ORRES_VFQ201 to 
ORRES_VFQ225.  The sub-questions are annotated as ORRES_VFQ215A to ORRES_VFQ215C and 
ORRES_VFQ216A.  The values are integer numbers from 1 to 2, 3, 4, 5 or 6 depending on the number of 
response categories, 
7.10 
Safety Evaluation 
All evaluations of safety data will be performed using the safety analysis set.  Some information may only be 
found in text fields of the eCRF.  Evaluations incorporating information not available from the eCRF will thus 
not be possible. 
7.10.1 
Extent of Exposure 
The protocol specifies the following dosing regimen.  On Day 0, the first study drug application will be at the 
clinical research centre. Thereafter, study drug will be self-administered at home. When the subject is at the 
clinical research centre for assessments, subjects will apply the study drug themselves at the clinical research 
centre. On Days 0 to 5, subjects will apply study drug every hour daytime only (1 drop of each ophthalmic 
solution in the affected eye). On Days 6 to 12, (1 week), subjects will apply study drug every 2 hours daytime 
only. On Days 13 to 19, (1 week), subjects will apply study drug every 3 hours daytime only. On Day 20 until 
resolution, subjects will apply study drug 4 times a day at daytime only. Subjects will be treated for the 
maximum of 1 year after randomization. 
Summary statistics on the duration of exposure will be presented. 
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Statistical Analysis Plan 
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7.10.2 
Adverse Events 
AEs data will be captured and encoded by system organ class (SOC) and the lowest level term (Medical 
Dictionary for Regulatory Activities, current version). A listing of AEs will be created. This listing, at minimum, 
will contain a description of AEs as to seriousness, severity, onset date and end date, duration, action taken 
(if any), outcome and likelihood of drug causation (“relation"). A frequency table will be compiled for AEs by 
treatment group, SOC, and lowest level term. Frequency tables will be compiled showing the number of 
subjects per treatment group affected by one or more (related) AEs, including percentages, the total number 
of AEs per treatment group and the average number of AEs per subject exposed to the respective treatment 
for the following: 
• 
AEs. 
• 
Serious AEs. 
• 
AEs leading to withdrawal of the IMP. 
• 
Fatal AEs. 
• 
AEs, broken down by severity. 
• 
AEs, broken down by causality assessment. 
There will also be tables on the most frequently reported AEs, on system organ class level and on preferred 
term level. The decision on the frequency cut-off for these tables will be taken during the analysis of the AEs 
data in consultation with the author of the clinical study report and could be influenced by factors such as the 
overall number of AEs, study design, and the nature of the indication. The frequency cut-off should be 
mentioned in a table note. 
The second secondary hypothesis, as specified in the protocol, is that AEs, and those relating to toxicity in 
particular, are less with PHMB 0.08% monotherapy compared to the comparator.  This translates into: 
Hypothesis: Rate of AEs is equal in the PHMB 0.08% monotherapy is equal to that of the 
comparator.  In particular: 
Hypothesis: Rate of toxicity-related AEs is equal in the PHMB 0.08% monotherapy is equal to that of 
the comparator 
The null hypothesis is that rate of occurrence of AEs and toxicity-related AEs are equal for the two treatment 
arms. 
If these are rejected, the conclusion is that the rates are different, and less with PHMB 0.08% monotherapy 
than for the comparator if the latter rate is higher.  The AETOXGR will be used to assess toxicity rates. 
These will be tested using ordinal logistic regression model and model-based estimates of the difference 
between treatments.  Odds ratio with corresponding CIs and p-values will be provided. Covariate variables 
such as treatment, age and sex will be included. 
7.10.3 
Clinical laboratory 
Haematology, biochemistry and urinalysis are collected at the baseline and end-of-study visits only. For the 
purpose of summary tables on laboratory test results, any value reported as below the lower limit of 
quantification or as undetectable will be considered as missing, and any value reported as above the upper 
limit of quantification will be considered as being equal to the upper limit. In data listings, the reported value 
will be presented. 
For continuous laboratory parameters, summary statistics on the laboratory test results will be presented by 
visit. For the end-of-study visit, summary statistics on the change from baseline will also be presented. 
For categorical laboratory parameters, frequency tables on the laboratory test results will be presented by visit. 
For the end-of-study visits, the shift from baseline will also be presented. 
For all laboratory parameters, frequency tables on the clinical interpretation of the laboratory test results (e.g. 
normal, abnormal but not clinically significant, abnormal and clinically significant) will be presented by visit. For 
the end-of-study visit, the shift from baseline will also be presented. 
Each parameter will be presented in a separate table. A list of all laboratory parameters (including specification 
of the standardised units to be used for analysis) is presented below: 
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Clinical laboratory tests will be presented with summary statistics. In addition, the tests will be analysed with 
ANCOVA (baseline value of respective laboratory test will be included in the model). 
All results will be regarded as descriptive only. The p-values will be used to flag safety and tolerability variables 
worthy of further attention. 
Null hypothesis: 
• 
No difference between test treatment and reference treatment 
Clinical laboratory tests conducted at planned visits for which laboratory sampling was not planned, as well as 
unplanned visits with laboratory sampling that are reported through the eCRF, will be included in the listings 
and, if deemed necessary, commented in the Statistical report. 
Laboratory class 
Parameter* 
Unit** 
Haematology 
Total red blood cell count 
x10^12/L; x10^6/microlitre 
 
White blood cell count 
x10^9/L; x10^3/microlitre ; /microlitre 
 
Neutrophils 
x10^9/L; %; /microlitre 
 
Lymphocytes 
x10^9/L; %; /microlitre 
 
Monocytes 
x10^9/L; %; /microlitre 
 
Eosinophils 
x10^9/L; %; /microlitre 
 
Basophils 
x10^9/L; %; /microlitre 
 
Haematocrit 
L/L; % 
 
Haemoglobin 
Mmol/L; mmolFe/L; g/dL; g/L 
 
Platelets 
x10^9/L; x10^3/microlitre 
 
 
 
Biochemistry 
Serum creatinine 
Micromol/L; mg/dL 
 
Aspartate aminotransferase 
U/L 
 
Alanine aminotransferase 
U/L 
 
Glucose 
Mmol/L; mg/dL 
 
Serum sodium 
Mmol/L; mEg/L 
 
Serum potassium 
Mmol/L; mEg/L 
 
Total protein 
g/L; g/dL 
 
Total bilirubin 
Micromol/L; mg/dL 
 
Alkaline phosphatase 
U/L 
 
Gamma-glutamyl transferase 
U/L 
 
Cholesterol 
Mmol/L; mg/dL 
 
Triglycerides 
Mmol/L; mg/dL 
 
 
 
Urinalysis 
Specific gravity 
 
 
pH 
 
 
Protein 
Positive/Negative  
 
Glucose 
Positive/Negative 
 
Ketones 
Positive/Negative 
 
Urobilinogen 
Positive/Negative 
 
Erythrocytes 
Positive/Negative 
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Leucocytes 
Positive/Negative 
*All measured values are coded as either NCS=Not clinically significant or CS=Clinically significant 
** The results of the analyses and data presented in tables will be in SI units. 
 
Test results reported in other units will be converted from their original units to the standardised units specified 
in the table above before analysis, using appropriate conversion factors. Data listings will include test results 
in both SDTM, SI and original units, wherever they differ, by using the LBSCAT variable in SDTM. 
7.10.4 
Vital Signs 
Diastolic and systolic blood pressure, pulse rate and body temperature are only measured at baseline, which 
will be presented in a table.  These are annotated as ORRES_DIABP, ORRES_SYSBP, ORRES_PULSE and 
ORRES_TEMP. 
7.10.5 
Intraocular Pressure Measurement (mmHg) 
IOP is measured at all visits as integer numbers with unit mmHg.  The annotation is IOPRES. 
7.10.6 
Ophthalmoscopy 
Ophthalmoscopic assessments are conducted for the vitreous, retina, macula and choroid.  Clinically 
significant findings, reported as Abnormal, CS, are to be recorded in the AE log.  The annotations are 
ORRES_VIT, ORRES_RET, ORRES_MAC and ORRES_CHD.  The tables can be seen in the DDP. 
7.10.7 
Worsening of corneal inflammation  
This is defined as development of ring abscess and/or hypopyon despite >30 days of treatment with the study 
drug.  The table is presented in the DDP. 
7.10.8 
Rate of subjects with a relapse 
The rate of subjects with relapse will be tabulated, see the DDP.  Relapse is recorded in the annotated variable 
RELAPSYN.   
7.10.9 
Rate of subjects requiring surgery 
Rate of subjects requiring surgery, including amniotic membrane transplants, superficial keratectomy, 
application of cyanoacrylate glue, therapeutic penetrating, lamellar keratoplasty, cataract surgery, 
evisceration, or enucleation, will be tabulated, see the DDP.  Such surgery will be seen in the annotated 
variable CMTRT, Name of (prior or concomitant) drug/medication/therapy. 
7.10.10 Rate of subjects requiring non-study therapies 
Rate of subjects requiring non-study therapies will be tabulated or listed, see DDP.  The annotated variable 
CMTRT will be used as basis. 
7.10.11 Rate of subjects’ discontinuation from study 
The rate of subjects discontinuing the study will be presented, see the DDP.  The annotated variable DSCOMP 
with values Yes/No as answer to the question “Did the subject complete the study as per protocol?” will be 
used.  The primary reason for study discontinuation, annotated variable DSDECOD, will also be presented. 
The rate of subjects discontinuing the treatment will be presented in a similar fashion, using the DSTRTYN 
annotated variable, which answers the question “Did the subject complete the treatment as per protocol?”.  
The primary reason for discontinuation, annotated variable DSDECOD, will also be presented.  The primary 
reason for treatment discontinuation, annotated variable DSDECOD_TRT, will also be presented. 
7.10.12 Incidence of secondary complication 
Incidence of secondary complications, such as significant corneal neovascularization, corneal scarring, 
corneal perforation, scleritis, secondary glaucoma, cataract, retinopathy will be presented, see the DDP.  
Such complications will be retrieved from the AE data. 
Transaction 09222115557449620377
Signed CE, VP, MS


 
Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
7.10.13 Cure rate of subjects with risk factor  
The cure rate of subjects with risk factor defined in section 8.2 will be presented in a frequency table. 
7.10.14 Rate of subjects with Adjunctive therapy 
The rate of subjects that need Adjunctive therapy will be presented in a frequency table. 
Adjunctive therapy is defined as new use after baseline or any increase of at least one of following 
medication with ATC5: 
Topical steroid: dexamethasone, prednisolone, fluorometholone, loteprednol 
Oral non-steroidal anti-inflammatories (NSAIDS): diclofenac, ibuprofen, flurbiprofen, naproxen 
7.11 
Changes to Planned Analysis 
At this time, no changes to the planned analyses are being considered. 
However, the definition of the PPAS have been altered compared to the protocol due to one wrong sentence.   
The use of normal-distribution test assumptions will be the primary choice.  If such assumptions should be 
found incorrect during the analysis of the data, an assessment of which type of analysis that should be used, 
will be conducted. This will be described in detail in the clinical study report. 
8 
Derived Variables 
8.1 
Disposition of Subjects 
A screening failure is defined as a screened but not randomised subject. 
8.2 
Demographics and Baseline Characteristics 
Age and sex will be presented using counts, means and rates.  Frequency of risk factors present at baseline 
will also be tabulated. Risk factor is defined as any use of at least one of the following drugs given before 
baseline with ATC5: 
Topical antifungals: amphotericin, voriconazole, chlorhexidine 0.2%, natamycin 
Oral antifungals: voriconazole, itraconazole, fluconazole 
Topical antivirals: aciclovir, gangciclovir 
Oral antivirals: aciclovir, famciclovir, valaciclovir 
Topical antibiotics: chloramphenicol, moxifloxacin, levofloxacin, ofloxacin, gatifloxacin, gentamicin, neomycin  
Topical steroid potent: dexamathasone 0.1% or Prednisolone forte 1%)  
Topical steroid mild: prednisolone 0.5%, FML (fluromethalone), loteprednol 
 
Diagnostic test criteria will be categorised into four (4) groups and be presented in a frequency table: 
• 
Patients diagnosed by IVCM alone: variables CFMPERF and CFMORRES with value Yes  
• 
Patients diagnosed by IVCM with culture: variables CFMPERF and CFMORRES with value Yes and 
CSORRES with value Positive 
• 
Patients diagnosed by IVCM with PCR: variables CFMPERF and CFMORRES with value Yes and 
PCRORRES with value Positive 
• 
Patients diagnosed by IVCM with PCR and culture: variables CFMPERF and CFMORRES with 
value Yes and CSORRES and CFMORRES with value Positive 
 
Change from Baseline 
Transaction 09222115557449620377
Signed CE, VP, MS


 
Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
Change from baseline will be computed as the difference between a post-baseline value and the corresponding 
baseline value. 
Percentage change from baseline will be computed as 100 times the change from baseline divided by the 
baseline value. 
8.3 
Time to Event 
The time to clinical resolution in days will be computed as the time in number of days from the start of treatment 
to the date of resolution or censoring. 
For subjects completing the study or being prematurely withdrawn from the study without experiencing clinical 
resolution, the time to event will be right-censored at the date of completion/withdrawal. 
8.4 
Compliance 
The rate of compliance will be assessed using the Yes / No answer to the eCRF question: Was the study drug 
administration compliant with the protocol? 
8.5 
Duration 
The time to resolution in days will be computed as the time in days from randomisation to the day of resolution 
plus 1 day. 
9 
References 
1. ICH Harmonised Tripartite Guideline for Statistical Principles for Clinical Trials E9. February 1998. 
2. ICH Harmonised Tripartite Guideline for Structure and Content of Clinical Trial Reports E3. November 1995. 
3. Visual acuity measured standard. International Council of Ophthalmolgy, Italian Journal of Ophthalmology 
II / I 1988, pp 1 / 15. 
 
 
Transaction 09222115557449620377
Signed CE, VP, MS


 
Statistical Analysis Plan 
LINK Medical Study ID: 
PSR012 
 
10 Signoff 
We have read this SAP for the 043-SI study and confirm that, to the best of our knowledge, the statistical 
analyses to be performed in this study are accurately described. 
SIFI SpA Approver: Vincenzo Papa MD Ph.D., Medical Affairs Manager 
SIGNATURE AND DATE 
LINK Medical SAP Author: Christina Ehrenkrona, Sr. Statistician 
SIGNATURE AND DATE 
LINK Medical SAP Approver: Malin Schollin, Associate Director Biostatistics 
SIGNATURE AND DATE 
 
 
Transaction 09222115557449620377
Signed CE, VP, MS


Document
Odak-sifi_43-
si_statisticalanalysisplan_v3.0_jul_01_2021_clean
Main document
26 pages
Initiated on 2021-07-01 14:42:19 CEST (+0200) by Intility 
eSign (Ie)
Finalised on 2021-07-02 10:29:44 CEST (+0200)
Initiator
Intility eSign (Ie)
esign@intility.no
Signing parties
Christina Ehrenkrona (CE)
On behalf of Link Medical Research
christina.ehrenkrona@linkmedical.eu
706040010
Signed 2021-07-01 14:42:42 CEST (+0200)
Vincenzo Papa (VP)
vincenzo.papa@sifigroup.com
Signed 2021-07-02 09:44:50 CEST (+0200)
Malin Schollin (MS)
On behalf of LINK Medical Research AB
malin.schollin@linkmedical.eu
761636014
Signed 2021-07-02 10:29:44 CEST (+0200)
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