A Multi-national, Multi-center, Prospective, Randomized, Double Blinded, Placebo-controlled Trial to Evaluate the Efficacy of HyperBox Cyclical Topical Wound Oxygen Therapy (TWO2) in the Treatment of Chronic Diabetic Foot Ulcers
Phase: NA
Conditions: Diabetic Foot Ulcer
Sponsor: AOTI Ltd.

 
 
 
  
STUDY PROTOCOL 
 
A Multi-national, Multi-center, Prospective, Randomized, Double Blinded, Placebo-controlled 
Trial to Evaluate the Efficacy of HyperBox Cyclical Topical Wound Oxygen Therapy (TWO2) 
in the Treatment of Chronic Diabetic Foot Ulcers 
 
 
Device 
 
HyperBox Cyclical Topical Wound Oxygen Therapy (TWO2) 
System  
 
 
Indication 
Chronic, diabetic foot ulcers 
 
 
Sponsor 
AOTI Ltd.  
Qualtech House  
Parkmore Business Park West  
Galway, Ireland  
 
 
 
Principal Investigator 
Robert Frykberg, DPM MPH 
Chief of Podiatry & Residency Director 
Phoenix VA Healthcare System 
650 E. Indian School Road 
Phoenix, AZ 85012, USA 
 
 
 
 
 
 
Version 013.0 (September 24, 2016), Amendment 8.  
 
 
THIS INVESTIGATIONAL PROTOCOL CONTAINS CONFIDENTIAL INFORMATION FOR 
THE USE BY THE INVESTIGATORS AND THEIR DESIGNATED REPRESENTATIVES 
PARTICIPATING IN THE EVALUATION OF THE TWO2 SYSTEM. IT SHOULD BE HELD 
CONFIDENTIAL AND MAINTAINED IN A SECURE LOCATION. IT SHOULD NOT BE 
COPIED OR MADE AVAILABLE FOR REVIEW BY ANY UNAUTHORIZED PERSON OR 
FIRM.  
 
 
 
AOTI Ltd 
Qualtech House 
Parkmore West Business 
Park, 
Ballybrit, Galway, Ireland 
 
Telephone   +353 91 660310 
Fax              +353 91 752418 

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1 
PROTOCOL APPROVAL 
 
Signature Page  
 
By signing the protocol, we the undersigned confirm our agreement with the contents of the 
protocol and our commitment to comply with the procedures contained in the study manual, 
with the conditions and principles of Good Clinical Practice in accordance with the 
International Conference on Harmonisation (ICH) E6: “Good Clinical Practice: Consolidated 
Guideline,” 62 Federal Register 25,692 (1997) where and as adopted by the FDA and with all 
requirements of the relevant Regulatory Authorities and IRB/Ethics Committees.  
 
 
 
Lead Principal Investigator  
Robert Frykberg, DPM MPH 
____________________________________ 
Chief of Podiatry & Residency Director 
Phoenix VA Healthcare System 
Robert Frykberg 
650 E. Indian School Road 
Phoenix, AZ 85012 
____________________________________ 
United States of America  
Date 
 
 
 
Sponsor  
Mike Griffiths, DProf, DMS, CRT, FCMI 
____________________________________  
AOTI Ltd.  
 
 
 
 
Mike Griffiths  
3512 Seagate Way, Suite 100 
 
 
Oceanside, CA 92056  
 
 
____________________________________ 
United States of America  
Date 
 
 
 
Statistician 
Prof. Peter Franks 
 
Centre for Research & Implementation 
____________________________________ 
of Clinical Practice  
Peter Franks 
St Luke's Crypt 
Sydney Street  
____________________________________ 
London SW3 6NH  
Date 
United Kingdom  
 
 
 

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Investigator Signature Page  
 
By signing the protocol, we the undersigned confirm our agreement with the contents of the 
protocol and study manual and our commitment to comply with the procedures contained  
within, with the conditions and principles of Good Clinical Practice in accordance with the 
International Conference on Harmonisation (ICH) E6: “Good Clinical Practice: Consolidated 
Guideline,” 62 Federal Register 25,692 (1997) where and as adopted by the FDA and with all 
requirements of the relevant Regulatory Authorities and IRB/Ethics Committees.  
 
 
 
 
Principal Investigator 
 
_____________________  
 
_____________________  
________ 
PRINT NAME 
 
 
 
 
SIGNATURE 
 
 
 
DATE 
 
_____________________________ 
INSTITUTION NAME 
 
_____________________________ 
 
_____________________________ 
 
_____________________________ 
 
_____________________________ 
ADDRESS 
 
  
 

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Contents 
 
1. 
SYNOPSIS ................................................................................................................................................. 6 
2. 
INTRODUCTION........................................................................................................................................ 7 
2.1. 
BACKGROUND AND RATIONALE ................................................................................................................ 7 
2.2. 
MEDICAL DEVICE .................................................................................................................................. 8 
2.3. 
RISK-BENEFIT ASSESSMENT ..................................................................................................................... 8 
2.4. 
COMPLIANCE STATEMENT ....................................................................................................................... 8 
3. 
OVERALL STUDY DESIGN AND PLAN ......................................................................................................... 9 
3.1. 
OVERVIEW .......................................................................................................................................... 9 
3.2. 
FLOWCHART OF SUBJECT FLOW .............................................................................................................. 11 
3.3. 
ENDPOINTS ....................................................................................................................................... 12 
3.3.1. 
Primary Study Endpoints ........................................................................................................ 12 
3.3.2. 
Secondary Study Endpoints .................................................................................................... 12 
3.3.3. 
Safety Endpoints .................................................................................................................... 12 
3.3.4. 
Justification for Trial Design ................................................................................................... 13 
4. 
SELECTION OF TRIAL POPULATION ......................................................................................................... 14 
4.1. 
INCLUSION CRITERIA ............................................................................................................................ 14 
4.2. 
EXCLUSION CRITERIA:........................................................................................................................... 15 
4.3. 
REMOVAL OF SUBJECTS FROM THERAPY OR ASSESSMENT .............................................................................. 15 
5. 
RANDOMIZATION .................................................................................................................................. 16 
5.1. 
IDENTIFICATION NUMBERS ..................................................................................................................... 16 
5.2. 
ALLOCATION CONCEALMENT .................................................................................................................. 16 
6. 
TRIAL TREATMENT MODALITIES............................................................................................................. 17 
6.1. 
TRIAL TREATMENT............................................................................................................................... 17 
6.2. 
CONTROL TREATMENT ......................................................................................................................... 18 
6.3. 
STANDARD CARE TREATMENT ................................................................................................................ 18 
6.3.1. 
Debridement .......................................................................................................................... 18 
6.3.2. 
Off-Loading ............................................................................................................................ 19 
6.3.3. 
Dressing Changes and Wound Dressing .................................................................................. 20 
6.3.4. 
Monitoring and treatment of wound infection ........................................................................ 21 
6.3.5. 
Education............................................................................................................................... 21 
6.4. 
TREATMENT MODALITIES NOT ALLOWED IN THE STUDY.................................................................................. 21 
7. 
ASSESSED VARIABLES ............................................................................................................................. 22 
7.1. 
EFFICACY ASSESSMENT ......................................................................................................................... 22 
7.1.1. 
Primary Efficacy Assessment .................................................................................................. 22 
7.1.2. 
Secondary Efficacy Assessments ............................................................................................. 22 
7.2. 
SAFETY ENDPOINTS ............................................................................................................................. 23 
7.2.1. 
Definition of Adverse Event .................................................................................................... 23 
7.2.2. 
Definition of Adverse Device Effects ........................................................................................ 24 
7.2.3. 
Definition of Unanticipated Adverse Device Effects ................................................................. 24 
7.2.4. 
Serious Adverse Device Effect ................................................................................................. 24 
7.2.5. 
Definition of Serious Adverse Events ....................................................................................... 24 
7.2.6. 
Relationship of Events to Study Treatment ............................................................................. 24 
7.2.7. 
Severity of Events and Effects ................................................................................................. 25 
7.2.8. 
Anticipated Adverse Device Effects ......................................................................................... 25 
7.2.9. 
Reporting Serious Adverse Effects ........................................................................................... 25 
7.2.10. 
Follow-up of Adverse Events ................................................................................................... 26 
7.2.11. 
Reporting Safety Information ................................................................................................. 26 
8. 
TRIAL CONDUCT ..................................................................................................................................... 27 

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8.1. 
OVERVIEW OF SCREENING TREATMENT AND FOLLOW-UP REQUIREMENTS ........................................................ 27 
8.2. 
ASSESSMENT BY TIME POINT ................................................................................................................. 28 
8.2.1. 
Screening Assessment ............................................................................................................ 28 
8.2.2. 
Baseline Procedures (after 2 week run-in period) .................................................................... 29 
8.2.3. 
Randomization Procedures ..................................................................................................... 29 
8.2.4. 
Weekly procedures during treatment period ........................................................................... 29 
8.2.5 
Punch biopsies ........................................................................................................................... 30 
8.2.6 
Premature Withdrawal of a Subject from the Trial ...................................................................... 30 
8.2.7 
Procedures for confirmation of wound closure ............................................................................ 31 
8.2.8 
12 Week Follow up ..................................................................................................................... 32 
8.2.9 
38 Week Follow up ..................................................................................................................... 32 
8.2.10 
Definition of the End of Trial ................................................................................................... 32 
8.2.11 
Premature Termination of the Trial ........................................................................................ 32 
8.2.12 
Subjects Lost-to-follow-up ...................................................................................................... 33 
9 
STATISTICAL ANALYSIS ........................................................................................................................... 34 
9.1 
SAMPLE SIZE AND POWER ANALYSIS ........................................................................................................ 34 
9.2 
GENERAL STATISTICAL METHODS ............................................................................................................ 35 
9.3 
PRIMARY OUTCOME ANALYSIS ................................................................................................................ 35 
9.4 
SECONDARY OUTCOME ANALYSES ............................................................................................................ 35 
9.4.1 
Time to complete wound closure ................................................................................................ 35 
9.4.2 
Change in wound size ................................................................................................................. 36 
9.4.3 
Wound recurrence rate. ............................................................................................................. 36 
9.4.4 
Incidence of amputation ............................................................................................................. 36 
9.4.5 
Safety and Tolerability ................................................................................................................ 36 
9.5 
MISSING AND SPURIOUS DATA ............................................................................................................... 36 
9.6 
INDEPENDENT DATA MONITORING COMMITTEE ......................................................................................... 37 
10 
DATA HANDLING .................................................................................................................................... 38 
10.1 
QUALITY ASSURANCE AND QUALITY CONTROL OF DATA................................................................................ 38 
10.2 
CASE REPORT FORMS AND SOURCE DOCUMENTS ........................................................................................ 38 
10.3 
DIRECT ACCESS TO SOURCE DATA ........................................................................................................... 38 
10.4 
DOCUMENTATION OF SUBJECT PARTICIPATION ........................................................................................... 38 
10.5 
ESSENTIAL DOCUMENTS FOR THE CONDUCT OF A CLINICAL TRIAL .................................................................... 39 
10.6 
ARCHIVING TRIAL DOCUMENTS .............................................................................................................. 39 
11 
ETHICS, LEGAL AND ADMINISTRATIVE ASPECTS ..................................................................................... 40 
11.1 
APPROVAL OF TRIAL DOCUMENTATION .................................................................................................... 40 
11.2 
ETHICAL CONDUCT OF THE TRIAL ............................................................................................................ 40 
11.2.1 
Good Clinical Practice ............................................................................................................. 40 
11.2.2 
Declaration of Helsinki ........................................................................................................... 40 
11.3 
SUBJECT INFORMATION AND CONSENT ..................................................................................................... 40 
11.4 
INSURANCE ....................................................................................................................................... 41 
11.5 
AMENDING THE PROTOCOL ................................................................................................................... 41 
11.6 
DATA PROTECTION .............................................................................................................................. 41 
11.7 
FINANCIAL ASPECT OF THE TRIAL ............................................................................................................. 41 
11.8 
DISCLOSURE OF ALL INFORMATION AND RESULTS ........................................................................................ 41 
11.9 
DECLARATION OF THE END OF TRIAL ........................................................................................................ 42 
11.10 
CLINICAL TRIAL REPORT .................................................................................................................... 42 
11.11 
PUBLICATION PLAN .......................................................................................................................... 42 
11.12 
STUDY SCHEDULE............................................................................................................................ 42 
12 
COROLLARY SUB STUDY OF HIGH RISK POPULATION ............................................................................. 43 
12.1 
INCLUSION AND EXCLUSION COROLLARY SUBSTUDY ...................................................................................... 43 
13 
REFERENCE LIST ..................................................................................................................................... 44 
14 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................................................ 47 

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APPENDIX I ..................................................................................................................................................... 48 
1. 
STUDY DIARY CARD ................................................................................................................................. 48 
2. 
UNIVERSITY OF TEXAS WOUND CLASSIFICATION SYSTEM ................................................................................... 48 
3. 
CARDIFF WOUND IMPACT SCHEDULE ........................................................................................................... 49 
4. 
EQ-5D ................................................................................................................................................ 51 
APPENDIX II .................................................................................................................................................... 53 
WMA DECLARATION OF HELSINKI - ETHICAL PRINCIPLES FOR MEDICAL RESEARCH INVOLVING HUMAN SUBJECTS ................... 53 
 

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1. 
SYNOPSIS 
Title 
A Multi-national, Multi-center, Prospective, Randomized, Double 
Blinded, Placebo-controlled Trial to Evaluate the Efficacy of 
HyperBox Cyclical Topical Wound Oxygen Therapy (TWO2) in the 
Treatment of Chronic Diabetic Foot Ulcers 
Objectives 
To evaluate the efficacy, safety and economic benefits of Cyclical 
Topical Wound Oxygen (TWO2) Therapy in the treatment of chronic 
diabetic foot ulcers 
Device 
HyperBox Cyclical Topical Wound Oxygen Therapy (TWO2) System 
applied 5 times weekly for 90 minutes for up to 12 weeks 
Comparator treatment Identical control device without cycling pressure of oxygen 
Trial design 
Double-blinded, multi-center, prospective, randomized, clinical trial  
Setting 
All subjects will be treated in a home care environment (home or 
equivalent facility)  
Primary endpoint 
Incidence of complete wound closure within 12 weeks  
Secondary endpoints 
 Time to complete wound closure 
 Change in wound size over time 
 Incidence of recurrence 
 Incidence of amputation 
 Incidence of adverse events and adverse device effects  
 Quality of Life – Pain, Mood and Function  
 Economic Analysis (EQ-5D, dressing and related medical history) 
Type of subjects 
 Foot ulcer below or at the malleoli graded as Grade 1 or 2 as 
assessed via the University of Texas Classification  
 Adequate perfusion with ABI > 0.7 AND TcpO2 > 30mmHg OR 
skin perfusion > 30mmHg OR Toe pressure > 30mmHg OR 
Duplex with biphasic waveforms below the knee 
 Ulcer size ≥ 1 cm2 and < 20cm2  after debridement  
 Ulcer duration ≥ 4 weeks but ≤ 1 year 
 Wound size reduction in a 2-week run-in period of ≤ 30% 
Number of centers 
Up to 30 centers in the US, Canada and Europe 
Number of subjects 
Conservative sample size estimation suggested a total sample size of 
220 subjects. The trial will be using a group sequential design (GSD) 
allowing for interim analyses to be performed after one third of 
subjects complete 12 weeks and two thirds of subjects complete 12 
weeks and potentially stopping the trial if stopping criteria are met. 
Length of Study 
It is estimated that subject recruitment of 73 subjects will take about 6 
months. 

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2. 
INTRODUCTION 
2.1. 
Background and Rationale 
Foot disorders, such as ulceration, infection and gangrene are a major source of morbidity 
and a leading cause of hospitalization for persons with diabetes.1 Ulceration, infection, 
gangrene, and amputation are significant complications of the diabetic foot ulcer and are 
estimated to cost billions of dollars each year.2 
 
Foot disorders are also the leading causes of hospitalization in patients with diabetes 
mellitus.2 Diabetic peripheral wounds are a major risk factor for lower extremity amputation.3 
Approximately 40-70% of all lower extremity amputations are performed in patients with 
diabetes, and some 100,000 non-traumatic lower-limb amputations were performed among 
U.S. diabetics in 2008 alone.4 Even superficial diabetic wounds are often difficult to treat and 
show high rates of complications.5 
 
Oxygen (O2) is essential to wound healing. Local tissue hypoxia, caused by disrupted or 
compromised vasculature, is one key factor that limits wound healing.6,7 It is well established 
that O2 is vital in the synthesis of collagen, enhancement of fibroblasts, angiogenesis and 
leukocyte function.8-10 O2 also has key functions in energy metabolism11,12 and in the 
inhibition of microbial growth.13 
 
Clinical use of O2 to promote wound healing began in the 1960’s with the administration of 
systemic full body Hyperbaric Oxygen Therapy (HBO) to treat wounds.13 Today HBO is 
usually administered in single or multi place chambers utilizing pressures of 2,500 mb and 
higher. HBO is reimbursed by the Center for Medicare and Medicaid Services in the US to 
treat certain wounds, including Diabetic Foot Ulcers (DFU’s). A Cochrane review by Kranke 
et al. demonstrated that in people with foot ulcers due to diabetes, HBO significantly reduced 
the risk of major amputation and may improve the chance of healing at one year.14 The 
availability of HBO facilities, contraindications, the need to transfer the patients to the HBO 
facilities and the risks of undesired systemic side effects, limits the widespread use of HBO to 
treat diabetic ulcers on a global basis. 
 
In an effort to address some of these drawbacks, the principle of topical pressurized oxygen 
administration or topical wound oxygen therapy (TWO2) was introduced in the late 60’s.15 The 
approach of topically oxygenating the wound is quite different from HBO. TWO2 does not 
involve pressures anywhere near as high as in HBO. Additionally, TWO2 is portable and can 
be administered in varied care sites, including in the patient’s home. There have been a 
number of studies, including smaller Randomized Control Trials (RCTs) and Case Series 
etc., published demonstrating very positive outcomes with TWO2. 15-22  
 
A previous study with the cycling TWO 2 device in a prospective, controlled clinical trial 
conducted in Canada examined the clinical efficacy of the TWO2 device used in this proposed 
RCT. In outpatients with severe diabetic foot ulcers referred for care to a community wound 
care clinic, 14 out of 17 ulcers (82.4%) in the treatment group and 5 of 11 ulcers (45.5%) in 
the control group healed within 90 days. The median of wound healing in the oxygen group 
was 56 vs. 93 days in the control group (p = 0.04).  
 
The concept of topical oxygen is still relatively unknown to the medical community as a whole 
and a larger well conducted randomized clinical trial is clearly needed to establish topical 
therapy as an important treatment in the management of diabetic foot ulcers. 
 
This trial will evaluate the efficacy and potential economic benefits of TWO2 in the treatment 
of chronic diabetic foot ulcers.  Pre-trial and real-time events captured for each subject will be 

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used to provide data for health economic analyses.  
2.2. 
Medical Device 
The medical device being investigated in this trial is the HyperBox Cyclical Topical Wound 
Oxygen therapy (TWO2) system manufactured by AOTI Ltd., Galway, Ireland.  
 
The TWO2 System operates by applying cyclical oxygen pressure directly to the wound site 
within a sealed and humidified environment. This provides a greater tissue oxygen diffusion 
gradient and increased tissue oxygenation, which enhances antimicrobial actions, stimulates 
angiogenesis and maximizes collagen production. The cyclical nature of the pressure also 
creates a sequential compression effect which helps reduce peripheral edema and stimulates 
wound site perfusion. 
 
The TWO2 system is intended as adjunctive therapy to standard wound care for acute or 
chronic wounds. It can be placed over a wound on the lower leg and when connected to a 
medical grade oxygen supply (Wall supply, Oxygen Concentrator or LOX system) which 
provides oxygen directly to the wound site with a pressure cycling between 5 to 50mbar at a 
frequency of approximately 2 cycles per minute. An integrated humidifier aids in keeping the 
wound moist during treatments.  
 
The devices have US Food and Drug Administration (FDA) 510(k) clearance, Health Canada 
clearance and CE-Mark approval for the following indications: 
 
Treatment of acute and chronic wounds on the lower extremity, such as;  
Diabetic ulcers, Venous stasis ulcers, Post-surgical wounds, Gangrenous lesions, 
Decubitus/pressure ulcers, Amputations/infected stumps, Skin grafts, Burns, Frostbite. 
2.3. 
Risk-Benefit Assessment  
The HyperBox Cyclical Topical Wound Oxygen Therapy System is a non-invasive device that 
has been assessed to provide Non Significant Risk (NSR) per FDA guidelines. The device 
offers the same therapy as the predicate system, which has been used in the marketplace for 
over 20 years with no recorded adverse events. Based on this assessment the potential 
benefit of the therapy is expected to exceed any potential risks envisioned.  
2.4. 
Compliance Statement  
This trial will be conducted in compliance with the approved study protocol and ISO 
14155:2011 (E) Clinical investigation of medical devices for human subjects — 
Good clinical practice as reflected in Good Clinical Practice in accordance with the 
International Conference on Harmonisation (ICH) E6: “Good Clinical Practice: Consolidated 
Guideline,” 62 Federal Register 25,692 (1997) where and as adopted by the FDA, and 
applicable regulatory requirements. Investigators should conduct the trial in compliance with 
these conditions, agreed to with AOTI, Ltd. and approved by Regulatory Authorities and 
IRB/Ethics Committees. Signature of the protocol by the investigator(s) and AOTI, Ltd. 
confirms this agreement. 
 

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3. 
OVERALL STUDY DESIGN AND PLAN 
3.1. 
Overview  
This is a multi-center trial, to be conducted at up to 30 sites in the United States, Canada, 
and Europe. 
 
Inclusion criteria for participation in the trial include the presence of documented diabetes 
(Type I or II) with non-healing, full-thickness, University of Texas Classification of Diabetic 
Foot Ulcers Grade 1 and 2 diabetic foot ulcers of at least 4 weeks duration, but not greater 
than 1 year, measuring 1cm2 or greater and less than or equal to 20 cm2. 
 
Subjects will be asked to sign a written informed consent form, approved by an IRB/Ethics 
Committee, prior to participation in the trial at the screening study visit. Each subject will be 
assigned a Subject Identification Number, which will be used for identification purposes in 
order to ensure subject confidentiality. All subject data held on the study Sponsor’s database 
will be anonymised and identifiable only by Subject Identification Number. 
 
Those who continue to meet all of the eligibility criteria after the 2 week run-in period will be 
enrolled into the trial and randomized to one of two treatment arms: TWO2 therapy or, Control 
therapy. 
 
In this trial a maximum of 220 subjects will be recruited. A Group Sequential Designs (GSD) 
with interims analyses after one third and two third recruited subjects has been chosen that 
will allow the trial to be stopped if TWO2 therapy is obviously better than control therapy. 
 
Subjects will be recruited as outpatients in participating wound care centers and clinics. At 
the screening visit the informed consent document must be signed, the wound will be 
measured and the subject will receive a standardized off-loading device for weight bearing 
wounds. The subject will from then on, be treated with predefined dressings used in the total 
treatment phase of the study. 
 
A diary card will be given to every subject.  Subjects will be instructed on keeping the simple 
diary card to record the use of the TWO2 or control device, dressing changes and to collect 
data for health economic analysis. A study ID card will also function as an alert to other 
providers of participation in a clinical study (example Appendix I).  
 
After a run-in period of 2 weeks the subject will be randomized into the study if the wound-
size reduction is less than 30% of the initial measurement and all other inclusion and 
exclusion criteria are met. 
 
All subjects will receive 90 minutes of either TWO2 therapy or control therapy at their home or 
nursing care facility 5 times a week for the treatment phase of up to 12 weeks. To secure 
double blinding no TWO2 treatment will be done at the study centers. Delivery, installation 
and training on use of the TWO2 device or control device may differ according to 
geographical location and shall be performed by trained accredited home health service 
providers utilizing nurses or other registered healthcare providers, or by trained 
home/durable medical equipment (DME) providers and an expert on the TWO2 device or 
control device.  The subject, or in some cases caregiver or healthcare professional (such as 
a home health nurse or aide), applying the TWO2 therapy or control therapy will be 
adequately trained and assessed for competency on the use of the device, which will be 
reviewed monthly.  Dressing changes at home (or equivalent) may be performed either by 
the subject, caregiver or healthcare professional such as a home health nurse or aide.  Both 
the TWO2 therapy and control therapy groups will visit the study center once per week for on 

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treatment study visits. Dressings will be pre-specified advanced moist wound therapy 
(AMWT) dressings, which will be changed at a minimum of once weekly according to the 
PI/Sub Investigator’s (SubI) clinical judgment. Subjects will receive standardized off-loading 
for a weight bearing wounds or an open heel boot for posterior heel wounds.  A Charcot 
Restraint Orthotic Walker boot (CROW boot) may be used where there is a need to protect a 
Charcot Foot deformity. Debridement will be performed as needed according to the PI/SubI 
clinical judgment.  
 
The treatment phase of the study will be 12 weeks. At every on treatment study visit at the 
clinic, foot ulcers are photographed. The initial (baseline) measurement after debridement will 
serve as the standard. Digital images will be sent to a blinded central assessor to be 
assessed and measured. 
 
Once wounds are initially considered closed, that visit will serve as the first of two 
confirmatory visit.  All subjects will continue treatment for an additional 2 weeks, to confirm 
wound closure.23 During this period, subjects will continue to receive TWO2 therapy or 
Control therapy, use and change dressings, and use the off-loading device, where it is 
required, per the protocol.  For subjects whose wounds close at week 11 or later the 
treatment phase will continue to the second confirmatory visit.   
 
 
All subjects whose wounds are confirmed closed or who finished the treatment phase of 12 
weeks – whichever is sooner - will enter into the post-treatment follow-up phase of 12 and 38 
weeks.  
 
Subjects who are withdrawn by the PI/SubI from the study treatment phase will be asked to 
return for the final week 12 on treatment visit and follow-up phase at 12 and 38 weeks.   
 
The maximum duration for participation in the trial is 54 weeks. During the follow-up phase, 
subjects will receive standard care according to the clinician’s recommendation and will be 
asked not to participate in another wound care trial in this period.  
 
The start of the trial is scheduled for July 2014.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3.2. 
Flowchart of Subject Flow 
     
   
TWO2 
N= 110 
90 minutes treatment / 5 times weekly 
Control 
N= 110 
90 minutes treatment / 5 times weekly 
Patients considered for participation (Screening) 
 
Follow up  
For 38 week (post allocated treatment) 
Perform allocated treatment for a total of 12 weeks or until wound 
closure, whichever is first 
Run in period of 2 weeks with off-loading  
 
Patients fulfilling inclusion criteria 
 
Randomization 
N=220 
End 
of 
Study 
no 
Wound size 
reduced < 
30% after 2 
Follow up  
For 12 week (post allocated treatment) 

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3.3. 
Endpoints  
3.3.1. 
Primary Study Endpoints  
The primary endpoint is the: 
 
Incidence of complete wound closure within 12 weeks.  
 
Closure is defined as 100% skin re-epithelialization without dressing requirements which is 
confirmed by 2 consecutive study visits 2 weeks apart.  
3.3.2. 
Secondary Study Endpoints  
1. Time to complete wound closure 
2. Change in wound size over time 
3. Incidence of reoccurrence 
4. Incidence of amputation 
5. Incidence of adverse events 
6. Quality of Life – Pain, Mood and Function 
7. Health Economic Analysis  
3.3.3. 
Safety Endpoints  
Incidence, duration and severity of adverse events and device effects will be documented in 
every case. This includes: 
 
Incidence of severe infections needing hospitalization 
 
Malfunctioning of the device 
 
Worsening of the wound 
 
Death 

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3.3.4. 
Justification for Trial Design 
 
The choice of using the “Incidence of complete wound closure” as the primary endpoint is in 
accordance with the recommendation of the FDA document “Guidance for Industry, Chronic 
Cutaneous Ulcer and Burn Wounds, Developing Products for Treatment”.23 
 
The choice of the parallel group design is the most appropriate for a wound healing study.  
 
The treatment duration of 12 to 16 weeks is a standard in randomized clinical trials in wound 
care.24-29 Two recent studies with the TWO2 system in venous and diabetic ulcers showed 
that the majority of ulcers treated with topical oxygen show wound closure after 12 weeks.21,22 
 
A double blind trial is the gold standard in trial design.  
 
Although there are a number of published reports reporting positive results on topical oxygen 
treatment and diabetic foot ulcers there is no comparative trial so far. In order to overcome 
the uncertainty surrounding the expected benefit, the trial will be undertaken using group 
sequential design (GSD) allowing for interim analysis to be performed at intervals during the 
trial recruitment. 
 
For the purposes of standardizing to reduce treatment variability the choice of dressings for 
this trial will be specifically limited.   
 
All “active” dressings will be excluded from the trial. These include: antimicrobial dressings 
containing silver, iodine or Polyhexamethylene Biguanide (PHMB), dressings containing 
enzymes, and growth factors (Regranex® or dermal skin substitutes like Apligraf® or 
Dermagraft®). 
 
Adjunctive treatments such as Hyperbaric oxygen therapy (HBO), Vacuum assisted closure 
(VAC), Heat therapy, Laser Therapy, Mechanical constant tension, Larval/maggot therapy 
(Biodebridement), Artificial Skin and Electrical stimulation for Tissue Repair (ESTR) are not 
allowed in this study. 
 
No local antimicrobial treatments are allowed in the study. Systemic antibiotic treatment of 
infection will be prescribed according to local standards. 
 
Histological studies in pigs treated with topical oxygen showed signs of improved 
angiogenesis and tissue oxygenation. Immunohistochemical analyses revealed a stronger 
presence of Vascular Endothelial Growth Factor (VEGF) in the tissue from oxygen treated 
wounds in pigs compared to the non-treated controls.17 As no studies have investigated 
these effects in humans, punch biopsies from 20 randomized subjects may be planned at 
selected centers and taken at baseline and after 4 weeks. 
 
Recent RCTs with patients with diabetic foot ulcers from Blume et al., 2008 investigating 
Negative Pressure Wound Therapy versus advanced Moist Wound Therapy and the paper 
from Löndahl, 2010 where Hyperbaric Oxygen Therapy was evaluated as an adjunctive 
therapy did not exclude infected ulcers. As oxygen is critical to fight infection in chronic 
wounds33-35 and the TWO2 device has been shown to eliminate infections and multi-resistant 
staphylococcus aureus (MRSA) 21,22,36, in this trial wound infections will not be excluded. 
However, any subject with wound infection must be under treatment at the time of 
randomization. 

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4. 
SELECTION OF TRIAL POPULATION 
Healing chronic ulcers will have a positive impact on patient well-being and quality of life. In 
this study we aim to examine the efficacy of Topical Wound Oxygen (TWO2) therapy with 
AMWT in the healing of diabetic foot ulcers and compare the outcome to that of AMWT and 
Control therapy in a double blind study. 
 
In the event that a subject being considered for study enrollment has multiple diabetic foot 
ulcers, the ulcer to be considered as the index ulcer will be the largest one, which still meets 
all other inclusion criteria (Index ulcer).  
4.1. 
Inclusion Criteria  
A subject must meet all of the following criteria to be eligible for enrollment in the trial.   
 
Ulcer characteristics 
• 
Subject has a documented diagnosis of Diabetes mellitus Type 1 or 2  
• 
Foot ulcer at or below ankle with duration of more than 4 weeks but no longer than 1 
year 
• 
If the index ulcer is a post amputation wound date of surgery must be > 30 days 
• 
2 week run in period with less than 30% wound size reduction 
• 
University of Texas Grade 1A, 1B, 1C, 1D, 2A, 2B, 2C, or 2D (Appendix I) 
• 
Ulcer is ≥1cm2 and ≤ 20cm2 after debridement at start of run-in period  
• 
If more than one ulcer is present on the foot, only the largest is considered in the 
study (Index ulcer) 
• 
Index ulcer must be ≥ 1cm away from any other ulcers present on the foot 
 
Vascular status 
• 
Adequate perfusion with ABI > 0.7 And  
TcpO2 > 30mmHg OR skin perfusion > 30mmHg OR Toe pressure > 30mmHg OR 
Duplex with biphasic waveforms below the knee 
• 
No planned revascularization procedure or vascular surgery within the last/next 30 
days  
 
Device and study related 
• 
Males and Females 18-89 years 
• 
Subject and/or caregiver are willing and able to comply with all specified care and visit 
requirements  
• 
Subject has a reasonable expectation of completing the study; according to the 
Investigator’s clinical judgment 
 

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4.2. 
Exclusion Criteria: 
Ulcer characteristics 
• 
Evidence of gangrene on any part of affected limb 
• 
Documented evidence of osteomyelitis on any part of affected limb  
• 
Index ulcer has exposed bone 
• 
Index ulcer exhibits signs of severe clinical infection that requires hospitalization or 
immediate surgical intervention 
• 
Active Charcot foot on the study limb 
 
Device and study related 
• 
Subject participated in another investigational device, drug or biological trial within last 
30 days 
• 
Uncontrolled diabetes: HbA1c > 12 % 
• 
Renal dialysis or serum creatinine > 2.5 mg/dl 
• 
Known immune insufficiency 
• 
Chronic steroid use or immunosuppressive agents within the last three (3) months or 
is anticipated to require them during the course of the study 
• 
Active treatment for malignancy (not specific to study limb) 
• 
Patient has a Deep Vein Thrombosis within the last 30 days 
• 
Subject has received growth factor therapy (e.g., autologous platelet-rich plasma gel, 
becaplermin, bilayered cell therapy, dermal substitute, extracellular matrix) within the 
screening period 
• 
Subject may not be pregnant at the time of treatment 
4.3. 
Removal of Subjects from Therapy or Assessment  
Subjects may discontinue study treatment and withdraw from the study at any time. The 
reasons for withdrawal may include the subject withdrawing their consent to participate in the 
study, or Investigator may decide to withdraw the subject from the study because of Adverse 
Events and Device Effects (ADE) or subject non-compliance. 
 
Where subjects are removed from study treatment (TWO2 therapy or Control therapy) for 
other than non-compliance and continue to provide consent to participate, may continue with 
study visits to asses for primary and secondary endpoints.  This may include: 
 
12 week final treatment visit  
 
12 week follow up 
 
38 week follow up 
 
If treatment is terminated and no follow up visits are anticipated, the final evaluation will be 
performed as completely as possible. In addition, any comments (spontaneous or elicited) or 
complaints made by the subject and the reason for termination will be recorded in the CRF 
and the subject’s medical records.  
 

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5. 
RANDOMIZATION 
The Sponsor will act as the central randomization center.  After successful assessment of 
inclusion/ exclusion criteria at the 2 week run in visit, the subject will be randomized to one of 
two treatment arms: TWO2 group or, Control group. Study sites will notify the Sponsor when 
a subject has met the criteria for randomization.   In accordance with local requirements, the 
device service provider will be notified by the study site , who will provide the contact details 
of the subject for device delivery, set-up and training. 
 
Randomization will then be centrally performed by the Sponsor by utilizing randomized 
permuted blocks of number using the ralloc function in Strata 11.0 Subjects will be 
randomized to the next available treatment group. The local Device Service Provider will be 
notified of the assigned device, by the Randomisation Centre, to be delivered directly to the 
successfully randomized subject. The Central Reading Centre, Investigators and Trial 
monitors will not be aware of the randomization group assigned.   
 
5.1. 
Identification numbers 
Each subject who has signed a written IRB/Ethics Committee approved Informed Consent 
Form will receive a unique subject ID number. The number will have 6 digits. The first two 
numbers will be the country code, the next two digits will define the center and the last two 
digits will be consecutive screened subjects in this center.  
 
After all baseline medical data is recorded and the wound is documented at the 2 week 
baseline/randomization visit, the subject’s number will become the subject identification 
number (Subject ID) for the remainder for the study. The investigator will not know to which 
treatment arm the subject is allocated throughout the trial.  
5.2. 
Allocation concealment 
Randomization will be coordinated by a central randomization center. Neither investigator nor 
subject will know their randomization allocation or can influence their allocation (=allocation 
concealment). 
 

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6. 
 
TRIAL TREATMENT MODALITIES 
6.1. 
Trial Treatment 
The TWO2 group will receive topical oxygen as an adjunctive treatment to standard of care 
treatment. The TWO2 treatment consists of the Topical Wound Oxygen therapy system 
(AOTI Ltd.) as well as an oxygen concentrator. Oxygen will be delivered to the device at a 
flow rate of at least 10 liters/ minute.  
 
After connecting the TWO2 Single patient-use leg chamber to the controller and concentrator, 
the controller initiates 4 phases of the treatment: 
 
Phase 1:  
The outer leg chamber is inflated. The limb with the ulcer can then be placed into the 
chamber.  
 
Phase 2:  
The integrated cuff is inflated to seal the inside of the chamber against the limb. 
 
Phase 3:  
The therapy phase: Cyclical pressurized oxygen is delivered to the limb inside the chamber.  
 
Phase 4:  
The cuff and outer leg chamber is deflated and removed from subject’s limb. 
 
 
This setup delivers cyclical pressurized oxygen to the wound site within a sealed and 
humidified environment. 
  
The affected limb will be placed in the chamber for 90 minutes daily, 5 days a week, for a 
period of up to 12 weeks (plus an additional 2 visits, 2 weeks apart to confirm wound closure 
for index ulcers closing at week 9 or later) or until full closure of the wound, whichever is 
sooner.  
 
All treatments can be done with or without removing the dressings. 
 
 
The picture below shows the TWO2 Single patient-use leg chamber and the controller. 
 
 
 
 

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6.2. 
Control Treatment 
The control treatment will look and act exactly like the TWO2 treatment. The only difference 
is that the controller has technical modifications.  
 
In phase 3, the control therapy will not supply oxygen or pressure to the affected limb during 
the treatment but mimics the therapy on the display with pressure values that go up and 
down. 
 
All other phases of device operation are identical. 
6.3. 
Standard Care Treatment 
Subjects in both groups will receive the same standard care regime that includes sharps 
debridement of all callus/necrotic tissue, AMWT and off-loading, where it is required. In a 
multi-center trial the value of study site consistency in standard care regimens within a trial 
cannot be over-emphasized because of the profound effects these procedures have on 
clinical outcome for chronic wounds.23 Consistency in standard care regimens is important for 
minimizing variability and allowing assessment of treatment effect. Every attempt will be 
made to minimize deviations from the procedures described in the protocol and subject 
compliance recorded in CRFs. 
 
In order to minimize variability in standard of care between sites: 
 
 
All centers will perform a standardized training before the trial 
 
All centers will be monitored closely by a study site monitor and the study director 
6.3.1. 
Debridement 
Adequate debridement is key for wound healing in diabetic ulcers. All wounds will receive 
baseline sharps debridement of all callus/necrotic tissue and additional debridement as 
needed throughout the trial.  Debridement will be performed where it is determined to be 
clinically necessary by the PI/SubI.  
 
Any surgical debridement has to be documented on CRFs, reported as an adverse event and 
will be included in analysis of product safety and efficacy.  
 
Standardized debridement will be part of the Investigators training. To ensure the same 
standard of debridement/wound preparation in all centers, photographs of all subjects will be 
evaluated by a central assessor. 
 
Enzymatic debridement products, like other concomitant topical products, can confound 
results in topical wound-treatment product trials and therefore must be prohibited.  

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6.3.2. 
Off-Loading 
Excessive pressure on the sole of the foot is one of the key factors in the pathogenesis of 
neuropathic plantar ulcers. Consequently pressure relief is of fundamental importance in the 
treatment of the diabetic foot ulcer30 and has been intensively proven in well designed 
randomized clinical trials. 24,26,31,32  
 
Therefore all subjects in the present study will be provided with an off-loading device (for 
weight bearing wounds) from Salvatelli srl, Italy (Piaggesi study): 
 
 
 
Optima Diab 
Optima CLHeel 
 
During the weekly wound assessment, subjects will be educated about the importance of the 
off-loading (where it is required). 
 
 
 
 
 

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6.3.3. 
Dressing Changes and Wound Dressing 
Dressing changes will be performed a minimum of once a week according to the 
Investigators recommendation and consist of the following procedures: 
 
1. Dressing removal 
2. Cleansing with saline  
3. Application of new dressing 
 
At each dressing change the wound will be cleansed with saline.  
 
Antiseptic substances like Octenisept®, Prontosan®, or similar products CANNOT be used in 
this trial. 
 
All wound dressings will be from Convatec/Kendall. The wound dressings will be limited to 
the following: 
 
Primary Dressing 
Kendall Foam Dressing,  
 
Wound filling dry wounds 
Curafil™* Gel Wound Dressing 3 Ounce Tube  
 
Secondary Dressing 
Non-sterile Kerlix Rolls  
 
Surgical Tape 
Generic surgical tape one roll 
 
Saline 
Generic sterile saline 0.9% 
 
The wound dressings selected by the Investigator for each subject, will be used in 
accordance with the manufacturer’s guidelines and institutional protocols and healthcare 
professional’s judgment.  
 
The number of dressing changes will be documented in the CRFs. 
 
Dressing changes can be performed by the subject, caregiver or healthcare professional 
(such as a home health nurse or aide). 
 
 
 
 
 
 

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6.3.4. 
Monitoring and treatment of wound infection  
Infection will be assessed clinically via symptoms and signs including purulent drainage, 
erythema, warmth, exudation, odor, pain, fever, and leukocytosis as well as wound size and 
time to wound healing. In patients with diabetes, fever, pain, and leukocytosis may be 
absent.37-41 
 
Mild and moderate infections can be included into the study. No local antimicrobial 
treatments are allowed in the study. Systemic antibiotic treatment of infection will be 
prescribed according to local standards (refer to section 3.3.4.). 
 
An infection that needs hospitalization is regarded as a severe adverse event or device effect 
and the subject will be suspended from the study for up to 4 weeks, up to 2 times, and may 
rejoin the study on resolution and compliance with other inclusion criteria.     
 
6.3.5. 
Education 
Subjects will be educated during each study visit about the importance of maintaining normal 
blood glucose levels and encouraged on continued appropriate diabetes management 
including diet and medications to achieve or maintain a stable glycemic state.  Subjects will 
also be educated on how to care for their feet. 
 
Routine laboratory testing of blood glucose or HbA1c is included in this trial and will be drawn 
at the screening visit and at the end of the treatment period. 
 
Comorbidities are documented but no special treatment of comorbidities is part of this trial. 
6.4. 
Treatment modalities not allowed in the study 
Other adjunctive or alternative treatments, such as; Hyperbaric oxygen therapy (HBO), 
Vacuum assisted closure (VAC), Heat therapy, Laser therapy, Mechanical constant tension, 
Larval/maggot therapy (Biodebridement), Electrical stimulation (ESTR), Artificial skin 
(Dermagraft) are not allowed during this study. 

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7. 
 
ASSESSED VARIABLES 
7.1. 
Efficacy Assessment 
7.1.1. 
Primary Efficacy Assessment 
 
The primary endpoint is the “Incidence of complete wound closure within 12 weeks”. 
 
Only one ulcer (the index ulcer) will be assessed per subject. In subjects with multiple ulcers, 
this will be the largest ulcer present fulfilling all other inclusion criteria.  
 
Closure is defined as skin re-epithelialization without drainage or dressing requirements 
which is confirmed at 2 consecutive trial visits 2 weeks apart.23 
 
Wound closure checks will be performed weekly. The closure of the wound is a clinical 
judgment by the investigator. A digital picture reviewed by the central assessor will also 
provide evidence of wound closure.  
 
Ulcers will be measured by the central assessor.   A deidentified digital photograph of the 
ulcer will be taken with and transmitted by the study sites using a wifi/wireless enabled tablet 
device. The ulcer’s surface area, circumference, maximum and minimum diameters will be 
recorded at baseline assessment of the subject.  
7.1.2. 
Secondary Efficacy Assessments 
 
Time to complete wound closure 
The time (in days) from first treatment at baseline of the ulcer until it closes completely will be 
recorded.  
 
Change in wound size over time 
Ulcer size over time will be assessed from the digital photographs of the ulcer taken at each 
visit.  
 
Incidence of recurrence  
Recurrence is identified as an interruption in the epithelium following complete healing of the 
index ulcer. If the subject does have a recurrence, this will be documented and the re-opened 
ulcer will be measured and recorded  
 
Incidence of Amputation  
All amputations will be recorded. The investigator will judge whether the amputation was 
done because of the index ulcer or for different reasons. 
 
Incidence of adverse events and device effects  
 
Quality of Life   
Pain - A Visual Analogue Scale will be used to determine pain levels. 
Mood and Function - The Cardiff Wound Impact Schedule (CWIS) assesses levels 
of mood and function and is the tool used to determine Quality of Life (Appendix I).  
(Subjects in countries where the CWIS has not been validated will be debriefed on its 
use at the end of the study).    
 
Economic Analysis 
Economic assessments will be determined from the Euroqol-5D (EQ-5D), subject diary and 

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medical records documenting dressing changes, time, location and healthcare professional 
assistance and management (Appendix I).  
 
Cost effectiveness will be determined by examining the difference in the number of 
healed ulcers in relation to the difference in cost in each randomized group 
 
Incremental cost per additional ulcer healed= Cost of treatment (TWO) - Cost treatment (Control)  
                             
 
 
   Healed ulcers (TWO) - Healed ulcers (Control) 
Cost Utility will be determined by examining the difference in Quality Adjusted Life 
Years (QALYs) associated with the different treatment regimens and their associated 
costs.  Health utilities will be derived from the EQ-5D tool and used to calculate QALYs 
in each group.  This will be related to overall treatment costs using the following formula 
Incremental cost per QALY = Cost of treatment (TWO)-cost of treatment (control) 
                                                QALY (TWO) – QALY(control) 
 
See 7.2 Safety Endpoints 
7.2. Safety Endpoints 
This study is being conducted in North America and Europe. The study will align assessment, 
documentation and reporting of safety outcomes with the best available guidance that is 
derived from ICH GCP, ISO 14155:2011(E) and 21 CFR Part 812.   
 
Each subject will be carefully monitored throughout the trial and asked for an account of 
adverse events and device effects at each study visit.  An assessment must be made of the 
seriousness, intensity and relationship of each occurrence to the administration of TWO2 
therapy or control therapy.   
 
The subject population for this study is known to have a high incidence of morbidity and 
mortality.  Known co-morbidities related to diabetes include cardiovascular disease, 
cerebrovascular disease, renal failure and deterioration of foot ulcers that may require 
hospitalization. The study will capture data on adverse events and device effects and serious 
adverse events and device effects related to the study’s secondary outcome measures and 
will also record concomitant medication related to these occurrences. Serious adverse 
events that are anticipated in this population do not require expedited reporting, but they will 
be recorded as they may affect the status of subjects in the study. Detailed documentation of 
pharmaceutical interventions are not required for the safety evaluation of the device, but will 
be recorded where in the opinion of the PI/SubI they may directly or indirectly relate to the 
index ulcer.  
  
7.2.1. 
Definition of Adverse Event 
An adverse event (AE) is defined as: 
An AE is: Any untoward medical occurrence, unintended disease or injury, or untoward 
clinical signs (including abnormal laboratory findings) in subjects, users or other persons, 
whether or not related to the investigational medical device 
NOTE 1 This definition includes events related to the investigational medical device or the 
comparator. 
NOTE 2 This definition includes events related to the procedures involved. 
NOTE 3 For users or other persons, this definition is restricted to events related to 
investigational medical devices. 
ISO 14155:2011E (3.2) 

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7.2.2. 
Definition of Adverse Device Effects 
An adverse device effect (ADE) is defined as: 
An adverse event related to the use of an investigational medical device. 
NOTE 1 This definition includes any effect resulting from insufficiencies or inadequacies in 
the instructions for use or the deployment of the device. 
NOTE 2 This definition includes any effect that is a result of user error (ISO 14155:2011(E) 
(3.1)). 
7.2.3. 
Definition of Unanticipated Adverse Device Effects 
An unanticipated adverse device effect (UADE) is defined as: 
Unanticipated adverse device effect is any serious adverse effect on health or safety, any 
life-threatening problem or death caused by, or associated with a device, if that effect, 
problem, or death was not previously identified in nature, severity, or degree of incidence in 
the application; or any other unanticipated serious problem associated with a device that 
relates to the rights, safety, or welfare of subjects. (CFR 21 Part 812.3(s)) 
7.2.4. 
Serious Adverse Device Effect 
A serious adverse device effect (SADE) is defined as: 
Adverse device effect that has resulted in any of the consequences characteristic of a 
serious adverse event (ISO 14155:2011(E) (3.36)).  
7.2.5. 
Definition of Serious Adverse Events 
A serious adverse event (SAE) is defined as: 
Any untoward medical occurrence which in any way:  
 
 
requires inpatient hospitalization or prolongation of existing 
hospitalization 
 
is life-threatening 
 
results in death or results in persistent or significant 
disability/incapacity  
 
is a congenital anomaly/birth defect 
 
or is considered serious by the investigator* 
* Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance. They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above.   
 
NOTE: The term ‘life-threatening,’ in the definition of ‘serious,’ refers to an event in which the 
subject was at risk of death at the time of the event; it does not refer to an event which 
hypothetically might have caused death had it been more severe.  
7.2.6. 
Relationship of Events to Study Treatment 
The causal relationship of each AE to the investigational product will be assessed by the 
study Investigator using the following definitions: 
 
Definite 
Clear cut temporal association, and no other possible cause. 
Probable Good reasons and sufficient documentation to assume a causal 
relationship in the sense of plausible, conceivable, likely but not 
necessarily highly probable. 
Possible 
Sufficient information to accept the possibility of a causal relationship 
in the sense of not impossible and not unlikely, although the 
connection is uncertain or doubtful, e.g. due to missing data or 

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insufficient evidence. 
None 
The AE is completely independent of the trial product administration 
and/or evidence exists that the event is definitely related to another 
etiology. 
 
7.2.7. 
Severity of Events and Effects 
Classifications of the AE severity include the following: 
 
Mild 
Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 
Symptoms do not require therapy or a medical evaluation; signs 
and symptoms are transient. 
Moderate 
Events introduce a low level of inconvenience or concern to the 
participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate 
experiences may cause some interference with functioning. 
Severe 
interrupt the participant’s normal daily activities and generally 
require systemic drug therapy or other treatment; they are 
usually incapacitating.  
7.2.8. 
Anticipated Adverse Device Effects 
There are no known ADEs reported with the use of the TWO2 device.  The following AEs are 
possible secondary outcomes and must be reported in the CRFs:  
 Mild to Moderate Adverse Events 
Mild and moderate adverse events can include, but are not necessarily limited to, the 
following clinical signs and symptoms: 
 
 
 
Pain 
 
Dermatitis  
 
Cellulitis  
 
New or satellite areas of breakdown 
 
Erythema 
 
Severe Adverse Events 
Severe adverse device effects can include, but are not necessarily limited to, the following 
clinical signs and symptoms: 
 
Infection requiring hospitalization 
7.2.9. 
Reporting Serious Adverse Effects 
Any adverse device effects (ADEs) will be recorded and if an considered serious 
unanticipated adverse device effect (SUADEs) will be reported to the IRB/Ethics Committee 
within 10 working days (CFR 21 Part 812.150(a)(1)) or sooner if required by Regulatory 
Authorities and institutional guidelines and in accordance with the principles of ICH GCP.   
Any study related occurrence that could adversely affect the safety of subjects or the conduct 
of the trial should be reported to AOTI Ltd. and if locally required to the local IRB/Ethics 
Committee by telephone and fax within 24 hours of being noted. An expedited reporting form 
should be used.  
 
The information reported must at least include the following: 
Name, address and telephone number of the reporting investigator. 
 
Title of the trial. 

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 
Study code. 
 
Subject identification number, sex and year of birth. 
 
Description of the AE, measures taken and outcome. 
 
Preliminary classification of causal relationship by the investigator. 
Within 48 hours of sending the initial information the investigator must send a completed 
copy of the form to AOTI Ltd by fax or email (where local policy allows).  
 
AOTI Ltd Reports must be addressed to: 
Study Director Contact Details for Adverse Event Reporting: 
 
Name: Mike Griffiths DProf, DMS, CRT, FCMI 
 Fax: +1 760-859-3911 (US sites only) 
Email: TWO2DFUstudy@aotinc.net  
 
 Telephone: +1 760 672 1920  
 
7.2.10. 
Follow-up of Adverse Device Effects 
The investigator will initiate appropriate therapeutic measures and investigations for adverse 
effects(s) and concurrent illness(es) that occur during the study and must treat with 
established standards of care. The investigator must continue to follow all serious adverse 
effects and those non-serious effects possibly related to a study treatment until they are 
resolved or until the investigator considers them to be stable. This follow-up may extend 
beyond the end of the trial. 
7.2.11. 
Reporting Safety Information 
AOTI Ltd. will promptly notify the Investigator and the Regulatory Authorities of findings that 
could adversely affect the safety of subjects, impact on the conduct of the trial, or alter the 
IRB/Ethics Committee approval/favorable opinion of the trial. The Investigator must promptly 
inform the relevant IRB/Ethics Committee of all occurrences deemed to involve increased 
risks to subjects (if required).  
 

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8. 
TRIAL CONDUCT 
8.1.1. 
Overview of Screening Treatment and Follow-Up Requirements 
 
Procedure / 
Examination 
Screening 
& Start 
Run-In 
Baseline * 
(2 weeks 
later) 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
12 wks 
Follow 
up 
38 wks 
Follow 
up 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In-& Exclusion criteria 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse Effects and 
Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical History/ 
Preexisting Conditions 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Record Concomitant 
Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Compliance 
assessment (treatment, 
dressings) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Height and Weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Wound History  
(Duration; course of 
healing last 12 months 
and previous treatment 
modalities) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dressing removal / 
wound cleansing and 
re-dressing 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Wound assessment 
and measurement 
(location, size)** 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Pain assessment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Neuropathic 
assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Infection Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
ABI and Secondary 
Vascular Assessment 
*** 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dig. Photographs ( post 
debridement) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Debridement if needed 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Cardiff Wound Impact 
Schedule ****  
X 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
Lab tests  
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Punch biopsies ***** 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Education on diabetic 
foot care/off-
loading***** 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Economic assessment 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
Device competency 
assessment 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
Note:  *Baseline visit window is -1/+2 days. All follow-up visit windows are +/-2 days. 12 and 38 Week follow-up +/- 2 weeks 
** Measurement recorded from central assessor report plus manual measurement at baseline 
*** Secondary vascular testing only needed if not within 4 months of screening date  
**** Cardiff Wound Impact Schedule (debriefing of subjects week 38 Follow up where not previously validated) 
***** Optional (none for sub-study)  
***** Off-loading with study boot or CROW boot where required 
****** Health Economic Assessment (EQ-5D, dressing history up to 8 weeks and significant related medical 
events)   

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8.1.2. 
Wound Closure Assessment 
 
Procedure / Examination 
Wound Closure 
Visit # 1 
Wound Closure 
Visit # 2 
Verify Informed Consent  
X 
X 
Adverse Events  
X 
X 
Record Concomitant Medications 
X 
X 
Compliance assessment (treatment, dressings) 
X 
X 
Confirm Wound Closure Assessment 
X 
X 
Dressing removal / wound cleansing and re-dressing 
X 
 
Pain assessment 
X 
X 
Dig. Photographs  (post Debridement if needed) 
X 
X 
Debridement if needed 
X 
X 
Cardiff Wound Impact Schedule (If at week 12 on treatment or last visit before follow up) 
X 
X 
Education on diabetic foot care/off-loading Study dressings 
X 
 
Education on diabetic foot care/off-loading Standard of care dressings per PI/Sub I 
 
X 
Economic assessment (if week 4, 8 or 12 on treatment or last visit before follow up) 
X 
X 
Note: 
* The wound closure visits may occur within the 12 week follow up period  
* *Visit window is +/- 2 days. 
8.2. 
Assessment by Time Point 
8.2.1. 
Screening Assessment 
Potential subjects should sign a written IRB/Ethics Committee approved informed consent. 
Inclusion criteria and exclusion criteria are checked. The index ulcer is chosen as the largest 
ulcer (in surface area) on the foot. The wound has to be measured and assessed. The 
vascular assessment is performed, and blood taken for the lab tests. Previous treatment 
modalities for the index ulcer attempted are documented. 
 
If the subject meets all criteria for inclusion into the study the 2 week run-in period is initiated.  
 
The following assessments will be done: 
1) 
Obtain informed consent 
2) 
Complete subject contact information and demographics 
3) 
Assign subject a screening ID number 
4) 
Verify subject eligibility by checking against inclusion/exclusion criteria 
5) 
Quality of Life Assessment using CWIS 
6) 
Medical history 
7) 
Concomitant medication 
8) 
Height and Weight 
9) 
Tobacco use 
10) 
Identify index ulcer 
11) 
Wound History 
12) 
Wound assessment: Location, size and appearance  
13) 
Pain assessment of wound using the Visual Numerical Rating Scale 
14) 
Neuropathic assessment  
15) 
Infection assessment 
16) 
Digital Photograph (post debridement) 
17) 
Debridement if needed 
18) 
Dress wound with study dressings 
19) 
Vascular assessment (ABI and  toe pressure or tissue pressure or TcPO2 or 
duplex (below knee)) 
20) 
Record lab data or blood sample taken to obtain values for: 
 
Electrolytes  
 
Creatinine  

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 
HbA1c, blood glucose 
 
WBC 
 
Albumin and pre-albumin (where routinely available) 
 
CBC  
 
C-reactive protein  
21) 
Provide and educate on off-loading boot/CROW boot, study dressings and diary 
card and ID card 
22) 
Educate on diabetes management and foot care 
23) 
Urine pregnancy for women of childbearing potential  
The subject is supplied with the off-loading boot/CROW boot and treated with the dressings 
defined for this study for 2 weeks until the baseline assessment takes place. 
8.2.2. 
Baseline Procedures (after 2 week run-in period) 
Prospective subjects must meet all inclusion criteria to be eligible to participate in the study. 
Missing data for the final verification of all inclusion and exclusion criteria will be performed 
including HbA1c which has to be available at the baseline visit. 
 
The following data will be collected  at the baseline visit prior to randomization treatment with 
the TWO2 therapy or Control therapy:  
1) 
Verify Informed Consent 
2) 
Record adverse events 
3) 
Record changes in concomitant medication 
4) 
Verify subject eligibility by checking against inclusion/exclusion criteria 
5) 
Quality of Life Assessment using CWIS 
6) 
Collect diary card/Compliance assessment (treatment/dressings) 
7) 
Remove dressings/wound cleansing 
8) 
Pain Assessment using the Visual Numerical Rating Scale 
9) 
Digital photograph (post debridement) 
10) 
Debridement if needed 
11) 
Wound assessment: Location, size  and appearance  
12) 
Infection assessment 
13) 
Dress wound with study dressings 
14) 
Record lab data if not previously recorded 
15) 
Education on diabetes management and foot care and off-loading boot/CROW 
boot 
16) 
Dispense study dressings and diary card 
17) 
Health Economic Assessment (including EQ-5D) 
18) 
Punch biopsies  
19) 
Device competency assessment (Completed in subjects home or equivalent after 
baseline clinic visit) 
8.2.3. 
Randomization Procedures 
Subjects will be randomized to receive either TWO2 therapy as an adjunctive therapy or a 
control therapy. 
8.2.4. 
Weekly procedures during treatment period 
All subjects (TWO2 therapy and control therapy) will have a weekly assessment in the clinic 
until the index ulcer is closed or 12 weeks have passed – whichever is sooner. 
 
Each Assessment Visit will include the following procedures:  
 

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1) 
Verify Informed Consent 
2) 
Record adverse events/device effects 
3) 
Record changes in concomitant medication  
4) 
Quality of Life assessment using CWIS (week 12 only) 
5) 
Collect diary card/Compliance assessment (treatment/dressings) 
6) 
Remove dressings/wound cleansing 
7) 
Pain Assessment using the Visual Numerical Rating Scale 
8) 
Digital photograph (post debridement) 
9) 
Debridement if needed 
10) 
Wound assessment: Location, size, depth and appearance  
11) 
Infection assessment 
12) 
Blood sample taken to obtain values for (visit 12 only): 
 
Electrolytes  
 
Creatinine  
 
HbA1c, blood glucose 
 
WBC 
 
Albumin and pre-albumin (where available) 
 
CBC 
 
C-reactive protein 
13) 
EQ-5D) (week 4, 8 and 12 only) 
14) 
Education on diabetes management and foot care and off-loading boot/CROW 
boot 
15) 
Dispense study dressings and diary card 
16) 
Device competency assessment (completed in subject’s home or equivalent. 
Week 4 and 8 only) 
8.2.5 
Punch biopsies 
Punch biopsies may be taken at baseline and 4 weeks for up to 20 subjects enrolled in the 
study at selected centers. These subjects will have specifically agreed to this procedure on 
their Informed Consent Form.  
 
The biopsies will be taken utilizing a standard 4 mm dermal punch biopsy kit and will be 
taken following routine debridement of the wounds to avoid collection of dead necrotic tissue. 
Prior to biopsy collection, the wound bed is prepared with local antiseptics. As per subject 
request, local anesthesia is administered. This is not standard because most subjects in this 
study will be insensate in the area of the wound. 
 
In addition it is important to have some tissue samples without local anesthetic to determine 
whether the use of anesthetic agents impacts the molecular studies planned with the biopsy.  
Biopsies will be taken from the wound edge and from the center of the wound. 
 
Samples will be collected, packed and transported per the procedure outlined by the testing 
laboratory instructions. The samples will be transported from clinic to the testing laboratory in 
properly secured biohazard-safe containers under appropriate temperature conditions. 
 
The samples will be analyzed histologically for signs of infection, collagen and neo-
angiogenesis.  Additional immune-histochemical analyses for such values as VEG-F and 
matrix metalloproteinases (MMPs) are also envisioned. 
 
8.2.6 
Premature Withdrawal of a Subject from the Trial 
Subjects are free to withdraw from the trial at any time without penalty or prejudice to their 
further treatment. Subjects may stop the trial treatment for any reason. 

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All subjects should be invited to do the End of Trial Visit to complete a final assessment, 
including wound status, complete quality of life questionnaire, etc. 
The End of Trial Visit should occur on or as close as possible to treatment week 12. Though 
encouraged to do so in all cases, some subjects may still decline to attend this visit, in which 
case this will be recorded in the CRF (along with any reasons if given).  Subjects who are 
withdrawn from the treatment will be asked if they consent to be followed up after withdrawal 
to determine whether their ulcer heals within the 12 weeks from randomization. 
 
In addition, a follow-up after 12 weeks and/or 38 weeks will be undertaken wherever possible 
in those that were withdrawn to confirm whether they healed according to intention-to-treat 
(ITT). 
8.2.7 
Procedures for confirmation of wound closure 
Subjects with index ulcers that are observed to close will be followed for 4 weeks to confirm 
wound closure. During this period, subjects will continue to receive treatment and dressing 
changes and wear the standard off-loading device. If wound closure occurs within the 11th 
week, treatment will continue for an additional 2 weeks to confirm closure (up to a maximum 
of 14 weeks of treatment).  
 
The wound closure confirmation assessment will be done at confirmation visits 2 weeks after 
closure. 
 
1) 
Verify Informed Consent 
2) 
Record adverse events/device effects 
3) 
Record changes in concomitant medication 
4) 
Quality of Life Assessment using CWIS (If week 12 on treatment or last visit 
before follow up) 
5) 
Collect diary card/Compliance assessment (treatment/dressings) 
6) 
Confirm wound closure for past week 
7) 
Pain Assessment using the Visual Numerical Rating Scale 
8) 
Wound History: time of wound closure 
9) 
Digital Photograph of the closed index ulcer 
10) 
Education on diabetes management and foot care/off-loading boot/CROW boot 
11) 
Dispense study dressings (confirmation visit 1 only) /dressings (if indicated 
confirmation visit 2) and diary card (confirmation visit 1 only) 
12) 
EQ-5D (if week 4, 8 or 12 on treatment or last visit before follow up) 
13) 
Blood sample taken to obtain values for (if at week 12 from randomization or at 
last closure visit if before): 
 
Electrolytes  
 
Creatinine  
 
HbA1c, blood glucose 
 
WBC 
 
Albumin and pre-albumin (where available) 
 
CBC 
 
C-reactive protein 
 
Subjects whose index ulcers are confirmed as closed will enter into the follow-up phase and 
will be terminated from further treatment with TWO2 therapy or Control therapy. Subjects will 
be encouraged to continue to wear the standard off-loading boot/CROW boot during the 
follow-up phase. 

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8.2.8 
12 Week Follow up 
All subjects will be reviewed and followed up 12 weeks after “wound closure” or the 12-week 
“end of treatment phase”, whichever is sooner +/- 2 weeks.  Data for 12 week follow up 
analysis will be collected per the subject’s preference by telephone or in the clinic if attending 
a routine visit for wound care. Index ulcers not yet healed, will continue to be treated with 
best available therapy as determined by the treating medical provider.  
The following assessments will be performed on all subjects: 
 
1) 
Verify Informed Consent 
2) 
Record adverse events  
3) 
Record changes in concomitant medication 
4) 
Quality of Life Assessment using CWIS 
5) 
Pain Assessment using the Visual Numerical Rating Scale (where possible, if 
applicable) 
6)  
Digital Photograph of the open index ulcer 
7) 
Health Economic Assessment (including EQ-5D) 
8) 
Education on diabetes management (and foot care/off-loading when indicated) 
 
8.2.9 
38 Week Follow up 
The 38 week follow up will take place 26 weeks +/- 2 weeks from the time of the last study 
visit (12 week follow up).  Data for 38 week follow up analysis will be collected per the 
subject’s preference by telephone or in the clinic if attending a routine visit for wound care.  
Index ulcers not yet healed, will continue with best available therapy as determined by the 
treating medical provider.  
 
1) 
Verify Informed Consent 
2) 
Record adverse events 
3) 
Record changes in concomitant medication 
4) 
Quality of Life Assessment using CWIS 
5) 
Pain Assessment using the Visual Numerical Rating Scale (where possible, if 
applicable) 
6)  
Digital Photograph of the open index ulcer (where possible) 
7) 
Health Economic Assessment (including EQ-5D) 
8) 
Education on diabetes management (and foot care/off-loading when indicated) 
9) 
Debriefing of subjects where CWIS not previously validated 
8.2.10 
Definition of the End of Trial 
For the purposes of notifying the IRB/Ethics Committee and the Competent Authorities, the 
end of the trial is defined as completion of the final 38 week follow-up on the final subject 
remaining in the trial.  
8.2.11 
Premature Termination of the Trial 
If AOTI Ltd. or the principal investigator discovers conditions arising during the trial which 
indicate that the trial should be halted, it must be terminated after appropriate consultation 
between AOTI Ltd. and the principal investigator. Conditions that may warrant termination 
include, but are not limited to: 
 
1. The discovery of an unexpected, significant or unacceptable risk to 
the subjects enrolled in the trial. 
2. Failure of the investigator to enter subjects into the study at an 

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acceptable rate. 
3. Insufficient adherence to the protocol. 
4. If study data at interim analysis indicates that treatment with TWO2 
therapy is of no clear benefit to diabetic patients with diabetic foot 
ulcers. 
 
If the trial is prematurely terminated or suspended for any reason, the investigator and 
subjects will be informed promptly. Appropriate therapy and follow up for subjects will be 
assured. 
 
The investigator must notify the IRB/Ethics Committee of any premature 
termination of the trial, within 15 days, using the “Declaration of End of Clinical 
Trial” form available from the EudraCT/and other relevant websites. 
 
AOTI Ltd. will notify the Regulatory Authorities within 15 days using the same 
process. 
8.2.12 
Subjects Lost-to-follow-up 
Every effort should be made for all subjects enrolled in this study to complete all study visits 
and assessments including the follow-up phase. A minimum of three (3) documented 
telephone calls will be made to any subject who does not return for scheduled treatment or 
follow up visits. Should the telephone calls be unsuccessful in contacting the subject, a 
certified letter will be sent. Only after a minimum of four (4) attempts to get the subject to 
return to the clinic, will the subject be considered "lost-to-follow-up".  
 
If the subject has to stay in the hospital for another medical condition or because of the 
diabetic ulcer, all reasonable efforts will be undertaken to continue the trial treatment on 
discharge, if the hospitalization is no longer than 4 weeks. 

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9 
STATISTICAL ANALYSIS 
9.1 
Sample Size and Power Analysis 
All sample size estimations in this section of the protocol were undertaken using the sampsi 
function in Stata 11 (Statcorp LP, Texas, USA).  Comparisons of groups was made assuming 
no continuity correction.  
 
Little data are available on similar studies using topical oxygen therapy within randomized 
clinical trials making sample size estimation problematic. Large trials of dermal substitutes 
and negative pressure have been undertaken that can be used to indicate the potential 
healing in the control group. The three large trials have control healing of between 18% and 
28.9% around 12 weeks after entering the trial, with a mean of 28%.25,27,29 
 
The expected improvement in healing using the active device is more problematic as there 
have been few comparative studies published. Aburto and Frye undertook a randomized 
study of 20 venous ulcers and 20 diabetic foot ulcer patients. 42 Topical oxygen demonstrated 
higher healing in the DFU patients after 90 days of 90% versus 40% in the control group. 
Blackman et al randomized 28 patients with DFUs and obtained a similar response (82.4% 
versus 45.5%).22 The potential difference between treatment groups was therefore 50% and 
36.9% respectively. Using these studies a total sample size of 28 and 52 would be expected 
based on 80% power at the 5% level of significance. Neither of these studies was blinded. It 
is therefore likely that the effect is likely to be substantially smaller than that experienced in 
these two studies.  
 
Because of the small size of these trials and lack of blinding it has been decided to offer a 
more cautious approach to the calculation of sample size based on a smaller expected 
benefit of oxygen therapy. By combining the results of the two randomized studies already 
undertaken the control group achieved a healing rate of 9/21 (42.8%).  In the actively treated 
group healing occurred in 23/27 (85.2%).  Assuming an expected control rate of 43% and a 
difference of half that experienced in these trials of 21% (to 64%) would require a sample 
size of 88 patients in each group (total=176), with a power of 80% and a level of significance 
set at p<0.05.  
In order to overcome the uncertainty surrounding the expected benefit, the trial will be 
undertaken using group sequential design (GSD) allowing for interim analysis to be 
performed at intervals during the trial recruitment. The interim analysis will take place after 
one third of subjects have been recruited, after two thirds and then a final analysis at full 
recruitment. The Pocock method of interim analysis will be used, which requires an equal 
level of significance (p<0.022) over the three analyses.43 If the difference in primary outcome 
produces a probability less than this in any of the analyses then the null hypothesis is 
rejected and the alternative hypothesis of a difference in healing has been shown.  In order to 
achieve this level of significance a total of 110 patients need to be recruited to each arm of 
the trial (total=220).   
 
Since all patients will achieve a 12 week end point (healed versus unhealed) no uprating of 
this sample size will be made to take into consideration patients lost to follow up (see section 
9.2).   
 

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9.2 
General Statistical Methods 
Analysis Populations 
Two analysis populations will be evaluated: Intent To Treat (ITT) and Per Protocol 
(PP). The ITT population will be considered the primary population of interest and 
will include all subjects who were randomized to study treatment. The PP population will 
include all subjects who were randomized to study treatment and had no significant protocol 
violations that might affect the outcome of treatment.  
 
All participants will be followed up to the primary end point (12 weeks) irrespective of whether 
they were on active treatment. Participants that withdraw from treatment continue to be 
followed up to the 12 week end-point. Participants that die, undergo amputation or who are 
lost to follow up will be considered as treatment failures. 
 
Continuous demographic variables (age, ulcer duration, etc.) will be summarized for the 
study population as descriptive statistics (number, mean, median, SD, minimum and 
maximum value), and by randomized groups. No formal inferential statistical testing will take 
place of baseline values in relation to randomized groups. 
 
Categorical demographic variables, such as gender, will be summarized as a proportion of 
the intention-to-treat population.  
 
All analyses will be undertaken using the STATA 11 statistical package. 
9.3 
Primary outcome analysis  
The primary endpoint is the “Incidence of complete wound closure within 12 weeks”. 
 
The null hypothesis to be tested is that there is no difference between treatment groups in 
terms of the rate of wound healing after 12 weeks of study treatment. The alternative 
hypothesis is that such a difference exists.  
 
Uncorrected Chi squared analyses will be used to determine the difference between 
treatment groups in terms of wound closure rate within 12 weeks, at the interim analysis. 
95% confidence intervals will be estimated using the normal approximation interval. 
 
Logistic regression analysis will be used to determine the effects of confounding on the 
primary outcome at the trial end. In particular, gender, age, ulcer duration, size and diabetic 
factors will be considered in the model. A stepwise procedure will be used to eliminate non-
significant covariates from the model.  The effect of different centers’ outcomes will be 
entered as a random effect into the model. 
9.4 
Secondary outcome analyses  
9.4.1 
Time to complete wound closure  
The analysis will use the Cox proportional hazards model to determine time to event 
(healing). Subjects who withdraw from treatment will be entered into the analysis and 
censored at the time they withdraw from the trial or are lost to follow up. The analysis will 
consider covariates that might influence the outcome in the trial. Factors considered 
potentially important in predicting the outcome of treatment will be tested in a Cox model. 
Stepwise elimination of non significant covariates will be undertaken and only those 
covariates that remain statistically significant will be retained in the model. Tests of 
proportionality will be undertaken using graphical (Nelson Aalen) and quantitative methods 
(Schoenfeld test). The data will be presented graphically using Kaplan Meier graphs. The 

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results of the multivariable analysis will be presented as Hazard ratios together with 95% 
confidence intervals and p-values generated from the model. 
 
9.4.2 
Change in wound size 
An important secondary outcome measure is partial wound healing, which is a function of 
wound area and perimeter change. Care must be taken to choose an appropriate measure 
for this outcome, as measures such as reduction in area or percentage reduction in area 
usually lead to biased comparisons. It is proposed that the trial uses the linear healing 
measure of Gowland-Hopkins & Jamieson44 and described in Gilman45. This measure is 
defined as: 
 
d = ΔA/pavg 
  
 
where ΔA is the change in wound area between two successive visits and pavg is the average 
of the wound perimeters measured at these two visits. ‘d’ will be calculated weekly after 
randomization to treatment.  
 
Comparisons of the two treatment groups will be carried out in terms of d, at each of these 
assessments. An unpaired t test will be carried out at each assessment, to test the 
hypothesis that the treatment groups do not differ in terms of d, against the alternative 
hypothesis that the treatment groups differ in terms of d.  A 95% confidence interval for the 
difference between treatment groups in terms of ‘d’ will be calculated, based on the t test 
results.  
9.4.3 
Wound recurrence rate. 
The wound recurrence rates will be analyzed with a time to event strategy as in 9.4.1. 
9.4.4 
Incidence of amputation 
Fisher’s Exact Test will be used. An approximate 95% confidence interval for the difference 
between treatment groups in terms of amputation rate will be estimated using the normal 
approximation interval.  
9.4.5 
Safety and Tolerability 
Safety and tolerability data will be presented in summary form and in detailed data listings. 
No significance testing will be applied to these data.  
9.5 
Missing and Spurious Data  
No procedures are planned for the imputation of missing data. Subjects with missing data will 
be omitted from the relevant analyses.  
 
If main outcomes are missing every attempt should be made to discover why and rectify 
wherever possible. If it is still not possible to discover the outcomes then the reason for this 
must be recorded e.g. death, refusal, incapacity. 
 
For the health economic analysis it is standard procedure to impute missing data where 
necessary in order that an appropriate estimate can be given of the total costs of care. 
 
 

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9.6 
Independent Data Monitoring Committee 
As suggested by international guidelines on data monitoring committees, an independent 
Data Monitoring Committee (DMC) will be formed to decide whether the predefined criteria 
for early stopping of the trial are met.46,47 The DMC will meet at the interim analysis to be 
performed after 73 and 146 subjects and at the end of study.  In the event of new 
unanticipated and related adverse effects being reported, the DMC will convene for an 
unscheduled meeting and will determine at that time the need for additional review and or 
action.  
 
The Data Monitoring Committee will consist of three to five independent experts outside of 
the study group. These members: 
 
will not be involved in the conduct of the clinical trial  
 
will not have any financial interest in the outcome of the study 
 
will have no planned authorship in publications on study results  
 
will not serve in parallel on the DMC of a clinical trial with diabetic patients with a 
different sponsor 
 

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10 
 
DATA HANDLING 
10.1 
Quality Assurance and Quality Control of Data 
The Investigator will permit trial-related monitoring, audits, IRB/Ethics Committee review, and 
regulatory inspection(s), provide direct access to source data and documents by suitably 
qualified and authorized persons from AOTI, Ltd. (and their representatives) and/or the 
bodies stated above. 
 
The Investigator will obtain the prior written consent of the subject for such direct access. 
AOTI Ltd. will conduct a site visit to verify the qualifications of each Investigator, inspect the 
site facilities, and inform the Investigator and all trial staff of their responsibilities and the 
procedures for ensuring adequate and correct documentation. 
 
The Investigator is responsible for ensuring the accuracy and credibility of the data by 
ensuring that a process is in place for the review of completed CRFs prior to any inspection 
by the study monitor. 
10.2 
Case Report Forms and Source Documents 
All study data will be recorded in a Case Report Form. Source data will not be recorded 
directly onto the CRF with the exception of the EQ-5D and CWIS.   Data from source 
documents such as  paper medical records or original electronic reports shall be signed and 
dated by a member of the study team and must be available in the subject’s file.  
All Case Report Forms (CRFs) are to be completed by site personnel delegated 
responsibility by the Investigator and reviewed and signed by the Investigator. All data 
recorded in the CRFs must be consistent with the subject’s source documents. Entries in the 
CRF for each subject will be checked against the source documents at site by the study 
monitor. Missing or un-interpretable data will be discussed with the Investigator for resolution. 
 
The database used for the compilation and storage of CRFs is the property of AOTI Ltd. and 
will be retained by AOTI Ltd. at the end of the trial. A copy of CRF data will be retained by 
each site in the provided Investigator Site File. 
10.3 
Direct Access to Source Data 
The Investigator will permit trial-related monitoring, audits, IRB/Ethics Committee review, and 
regulatory inspection(s), providing direct access to source data and documents by suitably 
qualified and authorized persons from AOTI Ltd. (and their representatives) and/or the bodies 
stated above. 
 
The Investigator will obtain the prior written consent of the subject for such direct access. 
During the trial a study monitor representing AOTI Ltd. will regularly visit the trial site to 
review protocol compliance, verify entries in the CRFs with source data in the subject’s 
medical records and ensure the trial is being conducted in accordance with the regulatory 
and ICH GCP requirements. 
 
The Investigator assures the Sponsor of the necessary support at all times. 
10.4 
Documentation of Subject Participation 
For all subjects who give written informed consent regardless of whether they receive TWO2 
therapy or control therapy, the Investigator must record subject identification data in the 
"Subject Identification List" (full name, initials, date of birth, subject identification code). The 

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Subject Identification List must allow for the definite identification of any subject that takes 
part in the trial. The Investigator must keep the list of subject identification codes until 
informed in writing by the Sponsor that the list can be destroyed. 
 
A statement acknowledging the participation of a subject in this clinical trial must be 
documented in the subject's medical file/notes and, where the trial physician is not the 
primary care physician, it is recommended that the subject’s primary care physician is 
informed of the subject’s participation in this clinical trial, provided the subject gave his/her 
consent. 
10.5 
Essential Documents for the conduct of a Clinical Trial 
At the beginning of the trial, an Investigator Site File will be established at the trial site. The 
Investigator is responsible for maintaining the trial documents and must take measures to 
prevent accidental or premature destruction of these documents. 
 
The essential documents to be included in the Investigator Site File are listed in ICH E6 
Section 8. 
10.6 
Archiving Trial Documents 
All trial documents belonging to the Investigator Site File will be archived at site (or off-site at 
a suitably secure location meeting ICH GCP requirements if specified and agreed in writing 
by the Sponsor) for a period of 15 years from the date of overall closure of the trial. This date 
will be communicated to all sites by the Sponsor at the end of the trial. A written record of the 
location of archived documents is to be forwarded to the Sponsor by each centre at the end 
of the trial (e.g. in the study closure report). 
 
All trial documents belonging to the trial master file will be archived by the Sponsor at a 
suitably secure location meeting ICH GCP requirements, for a period of 15 years from the 
date of overall closure of the trial.  
 
 
 

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11 
ETHICS, LEGAL AND ADMINISTRATIVE ASPECTS 
11.1 
Approval of Trial Documentation 
All trial related documentation must be approved by AOTI Ltd. and Principal Investigator, in 
writing, before it is released. Prior to initiation of the trial, the protocol, informed consent form, 
subject information sheet and any other relevant trial documentation will be submitted to the 
responsible IRB/Ethics Committee and Regulatory Authorities. Written approval of the trial 
must be obtained and forwarded to AOTI Ltd. before the trial center can be initiated or trial 
materials released to the investigator.  
 
Furthermore, any necessary extension or renewal of the IRB/Ethics Committee approval 
must be obtained and forwarded to the sponsor. In particular, change(s) to any aspect of the 
trial, such as modification(s) of the protocol, the written informed consent form, the written 
information provided to subjects, and/or other procedures must be approved, in writing, by 
the IRB/Ethics Committee. 
 
The Investigator will report promptly to the IRB/Ethics Committee, any new information that 
may adversely affect the safety of subjects or the conduct of the trial. Similarly, the 
Investigator will submit written summaries of the trial status to the IRB/Ethics Committee 
annually, or more frequently, if requested by the IRB/Ethics Committee. Upon completion of 
the trial, the Investigator will provide the IRB/Ethics Committee with a brief report of the 
outcome of the trial, if required. 
11.2 
Ethical Conduct of the Trial  
11.2.1 
Good Clinical Practice 
The procedures detailed in this protocol are designed to ensure that the Sponsor and 
Investigator abide by the principles of the Good Clinical Practice guidelines of the 
International Conference on Harmonization (ICH). 
11.2.2 
Declaration of Helsinki 
The trial will adhere to the guidelines of the World Medical Association Declaration of Helsinki 
in its revised edition (Fortaleza, Brazil, October 2013), as well as the demands of national 
drug/medical device and data protection laws and other applicable regulatory requirements. 
A copy of the Declaration of Helsinki is supplied in the Appendices (Appendix II). 
11.3 
Subject Information and Consent 
The Investigator is responsible for ensuring that no patient is subjected to any trial-related 
examination or activity before that patient has given informed consent. Written consent must 
be given by the patient/their legally authorized representative after the receipt of detailed 
information. The verbal explanation will cover all the elements specified in the written 
information provided for the patient. 
 
The Investigator will inform the patient of the aims, methods, anticipated benefits and 
potential hazards of the trial including any discomfort that it may entail. The patient must be 
given every opportunity to clarify any points he/she does not understand and if necessary, 
ask for more information. At the end of the interview the patient may be given time to reflect if 
this is required, or if the patient requests more time. Patients will be required to sign and date 
the informed consent form. After completion, informed consent forms will be kept and 
archived by the Investigator in the Investigator's trial master file.  
 

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It should be emphasized that the subject is at liberty to withdraw their consent to participate 
at any time, without penalty or loss of benefits to which the subject is otherwise entitled. 
Patients who refuse to give, or who withdraw written informed consent will not be included or 
continued in the trial. 
11.4 
Insurance 
The company carries insurance to cover the use of the products for the devices for the 
indications defined in this trial. 
11.5 
Amending the Protocol 
No substantial amendment or deviation should be implemented without agreement from the 
sponsor and prior review and documented approval/ favorable opinion from the IRB/Ethics 
Committee and Regulatory Authorities except where necessary to eliminate an immediate 
hazard(s) to trial subjects. In such case, as soon as possible, the implemented deviation or 
change, the reasons for it, and if appropriate, the proposed protocol amendment(s) must be 
submitted to AOTI Ltd. for approval and then submitted to the IRB/Ethics Committee and 
Regulatory Authorities for approval. 
 
When an amendment is thought to be non-substantial, it must be confirmed by AOTI Ltd. and 
the Investigator in advance, in writing, and the reason for the non-substantial amendment 
must be documented by both the Investigator and the Sponsor. 
11.6 
Data Protection 
To protect the subject's identity, a unique subject identification code (subject ID) will be 
assigned by the Investigator to each trial subject and used in lieu of the subject's name when 
the Investigator reports AEs and/or other trial related data. Thus, the subject identification 
code, and not the subject’s name or hospital number, will appear on all documents.  
 
Personal information will be treated as confidential, but may need to be reviewed by monitors 
from AOTI Ltd., auditors and authorized representatives of the IRB/Ethics Committee and 
Regulatory Authority. The subject’s consent to direct access to the subject’s original medical 
records for data verification purposes has to be obtained prior to that patient's participation in 
the trial. 
 
Documents which identify the subject (e.g. signed consent forms) must not be submitted to 
the sponsor at the end of the trial, but must be maintained in confidence by the Investigator. 
11.7 
Financial Aspect of the Trial 
The Sponsor, AOTI, shall provide payment for this study in accordance with the following 
schedule: 
 
1) 
The Sponsor will pay all fees for Institutional Review Board / Ethics Committee 
approval fees 
2) 
The Sponsor will reimburse directly all study related costs for TWO2 medical 
devices, dressings, and Salvatelli off-loading boot 
11.8 
Disclosure of all Information and Results 
In signing the final protocol, the Investigator agrees to keep all information and results 
concerning the trial confidential for as long as the data remains unpublished. The 
confidentiality obligation applies to all personnel involved at the investigational site.  

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Publication of trial results requires prior approval from AOTI Ltd. 
11.9 
Declaration of the End of Trial 
The Investigator is responsible for notifying the IRB/Ethics Committee of the end of the trial, 
within 90 days of its completion. AOTI Ltd. is responsible for notifying the competent authority 
of the end of the trial, within 90 days of its completion. 
 
If the trial ends prematurely the IRB/Ethics Committee and the competent authority must be 
informed within 15 days. 
11.10 
Clinical Trial Report 
AOTI Ltd. is responsible for the writing of the clinical trial report. The format of the report will 
be in accordance with the ICH GCP guidelines (E3).  
11.11 
Publication plan 
It is planned to publish the main paper of this trial in a premium journal like The Lancet, New 
England Medical Journal or Diabetes Care. 
 
Secondary analyses will be published depending on subject and clinical relevance.  
11.12 
Study Schedule 
 
Study Start Date: July 2014 
Estimated First Interims Analyses: January 2017 
Estimated Second Interims Analyses:  May 2017 
If needed Final Analyses: December 2017 
Reporting/Submission date: 6 month after data analyses 
 
 

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12 
COROLLARY SUB STUDY OF HIGH RISK POPULATION 
In many cases TWO2 has been shown to be particularly effective in severe wounds of 
multimorbid patients. Diabetic patients frequently suffer from ischemia, renal insufficiency or 
more severe wounds. A significant fraction of these patients use corticosteroids for other 
chronic diseases. 
 
Due to the low chance of healing, a high mortality, and other reasons such as reimbursement 
issues these patients are normally excluded from clinical trials. However, as these patients 
represent an increasing percentage of all patients with diabetic ulcers we shall also include a 
sub study with high risk patients. In this sub study 24 patients will be randomized to either the 
active or the control group.  
 
All procedures and outcomes will be the same as in the main arm of the study except for two 
differences: 
 
No biopsies will be taken 
 
Different inclusion and exclusion criteria 
 
12.1 
Inclusion and exclusion corollary substudy 
All inclusion and exclusion criteria remain the same as in the main study population except 
the ones stated below: 
 
INCLUSION CRITERIA 
 
ALL University of Texas Grades allowed  
(Grade 1 and 2 in the main study) 
 
ABI can be as low as 0.5  
 
 
(0.7 in the main study) 
 
May have osteomyelitis  
(excluded in the main study) 
 
May have exposed bone 
(excluded in the main study) 
 
Subject may require renal dialyses 
(excluded in the main study) 
 
Subject may use chronic steroids  
(excluded in the main study) 
 
EXCLUSION CRITERIA 
 (See previous exclusions that are now allowed in this substudy)  
 
Gangrene is still excluded 
 
 
 

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13 
REFERENCE LIST  
1. Frykberg, R. Diabetic Foot Ulcers: Pathogenesis and management. Am Fam Physicians. 
2002;66: 1655-1562. 
2. Frykberg, RG, Zgonis T, Armstrong D, et al. Diabetic foot disorders: A clinical practice 
guideline (2006 revision). J Foot Ankle Surg.2006; 45: 1-66. 
3. Younes NA, Albsoul AM, Awad H. Diabetic heel ulcers: a major risk factor for lower extremity 
amputation. Ostomy Wound Management. 2004; 50(6): 50-60. 
4. Driver VA and de Leon JD. Health Economic Implications for Wound Care and Limb 
Preservation. Journal of Managed Care Medicine. 2008; 11: 13-19. 
5. Ulbrecht JS, Cavanagh PR and Caputo GM. Foot problems in diabetes: an overview. Clin 
Infect Dis. 2004; 39 (suppl 2): 73–82. 
6. Gordillo GM and Sen CK. Revisiting the essential role of oxygen in wound healing. Am. J. 
Surg. 2003; 186(3): 259-263. 
7. Khanna S and Wallace WA. Wound healing: oxygen and emerging therapeutics. Antiox. 
Redox. Signal. 2002; 4: 961-963. 
8. Prockop DJ, Kivirikko KI and Guzman NA. The biosynthesis of collagen and its disorders (part 
1). N Engl J Med. 1979; 301: 13-23. 
9. Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen and its 
disorders (part 2). N. Engl. J. Med. 1979; 301: 77-85. 
10. Hopf HW; Hunt TK, West JM, et al. Wound tissue oxygen tension predicts the risk of wound 
infection in surgical patients. Arch J Surg. 1997, 132: 997-1004. 
11. Patel V, Chivukula IV, Roy S, et al. Oxygen: from the benefits of inducing VEGF expression to 
managing the risk of hyperbaric stress. Antioxid Redox Signal. 2005; 7: 1377-1387. 
12. Kairuz, E, et al. Hyperbaric oxygen stimulates epidermal reconstruction in human skin 
equivalents. Wound Repair Regen. 15, 2007, pp. 266-274. 
13. Hunt TK, Ellison EC and Sen CK. Oxygen: at the foundation of wound healing – introduction. 
World J Surg. 2004; 28 291-293. 
14. Kranke P, Bennett M, Roeckl-Wiedmann I, Debus S. Hyperbaric oxygen therapy for chronic 
wounds. Cochrane Database of Systematic Reviews. 2004, (2): CD004123. CD004123. 
15. Fisher, BH. Topical Hyperbaric Oxygen Treatment of Pressure Sores and Skin Ulcers. Lancet. 
1969;2(7617): 405-409. 
16. Davis, SC, Cazzaniga, AL and Ricotti, C. Topical oxygen emulsion: A novel wound therapy. 
Arch Dermatol. 2007; 143: 1252-1256. 
17. Fries, RB, Wallace, WA and Roy, S. Dermal excisional wound healing in pigs following 
treatment with topically applied pure oxygen. Mutat Res. 2005; 579: 172-181. 
18. Kalliainen LK, Gordillo GM, Schlanger R, Sen CK. Topical Oxygen as an Adjunct to Wound 
Healing: A Clinical Case Series. Pathophysiology. 2003; 9: 81-87. 
19. Heng MC, Harker J, Csathy G, Marshall C, Brazier J, Sumampong S, Paterno Gomez E. 
Angiogensis in Necrotic Ulcers Treated with Hyperbaric Oxygen. Ostomy Wound 
Management. 2000, 46, pp. 18-32. 
20. Heng, MCY. Enhanced Healing and Cost-Effectiveness of Low Pressure Oxygen Therapy in 
Healing Necrotic Wounds: A Feasability Study of Technology Transfer. Ostomy Wound 
Management 2000; 46 (3): 52-62. 
21. Tawfick W and Sultan S. Does Topical Wound Oxygen (TWO2) Offer an Improved Outcome 
Over Conventional Compression Dressings (CCD) in the Management of Refractory Venous 
Ulcers (RVU)? A Parallel Observational Comparative Study. Eur J Vasc Endovasc Surg. 2009 
May 21.  

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22. Blackman, E et al. Topical Wound Oxygen Therapy in the Treatment of Severe Diabetic Foot 
Ulcers: A Prospective Controlled Study. Ostomy Wound Manage. 2010 Jun;56(6):24-31. 
23. US Department of Health and Human Services, FDA: Guidance for Industry, Chronic 
Cutaneous Ulcer and Burn Wounds, Developing Products for Treatment. FDA Website March 
2011; Available from URL: 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm071324.pdf 
24. Faglia E, et al. Effectiveness of removable walker cast versus nonremovable fiberglass off-
bearing cast in the healing of diabetic plantar foot ulcer: a randomized controlled trial. 
Diabetes Care. 2010 Jul;33(7):1419-23. Epub 2010 Mar 31. 
25. Blume PA, et al. Comparison of negative pressure wound therapy using vacuum-assisted 
closure with advanced moist wound therapy in the treatment of diabetic foot ulcers: a 
multicenter randomized controlled trial. Diabetes Care. 2008 Apr;31(4):631-6. Epub 2007 Dec 
27. 
26. Armstrong et al. Evaluation of Removable and Irremovable Cast Walkers in the Healing of 
Diabetic Foot Wounds. DIABETES CARE, VOLUME 28, NUMBER 3, MARCH 2005 
27. Marston WA, et al. The Efficacy and Safety of Dermagraft in Improving the Healing of Chronic 
Diabetic Foot Ulcers. Diabetes Care. 2003 Jun;26(6):1701-5. 
28. Carravagi C, et al. HYAFF 11–Based Autologous Dermal and Epidermal Grafts in the 
Treatment of Noninfected Diabetic Plantar and Dorsal Foot Ulcers. Diabetes Care. 2003 
Oct;26(10):2853-9.  
29. Veves A, et al. Graftskin, a Human Skin Equivalent, Is Effective in the Management of 
Noninfected Neuropathic Diabetic Foot Ulcers. Diabetes Care. 2001 Feb;24(2):290-5. 
30. Frykberg RG, et al. Role of neuropathy and high foot pressures in diabetic foot ulceration. 
Diabetes Care. 1998 Oct;21(10):1714-9. 
31. Carravagi C, et al. Effectiveness and safety of a nonremovable fiberglass off-bearing cast 
versus a therapeutic shoe in the treatment of neuropathic foot ulcers: a randomized study. 
Diabetes Care. 2000 Dec;23(12):1746-51.  
32. Katz IA, et al. A randomized trial of two irremovable off-loading devices in the management of 
plantar neuropathic diabetic foot ulcers. Diabetes Care. 2005 Mar;28(3):555-9. 
33. Hunt TK, Ellison EC, Sen CK. Oxygen: at the foundation of wound healing – introduction. 
World J Surg. 2004;28:291–293. 
34. Jonsson K, Jensen JA, Goodson WH III, et al. Tissue oxygenation, anemia, and perfusion in 
relation to wound healing in surgical patients. Ann Surgery. 1991;214:605–613. 
35. 29. Sen CK. Wound healing essentials: let there be oxygen. Wound Rep Reg. 2009;17:1–18. 
36. Fisher BH. Topical hyperbaric oxygen treatment of pressure sores and skin ulcers. Lancet. 
1969;2(7617):405–409. 
37. Lipsky BA, Pecoraro RE, Wheat LJ The diabetic foot: soft tissue and bone infection. Infect. 
Dis. Clin. North Am. 1990; 4:409–432 
38. Frykberg RG, Veves A. Diabetic foot infections. Diabetes/Metab. Rev. 1996; 12:255–270 
1996. 
39. Armstrong DG, Perales TA, Murff RT, Edelson GW, Welchon JG. Value of white blood cell 
count with differential in the acute diabetic foot infection. J. Am. Podiatr. Med. Assoc. 1996; 
86:224–227 
40. Caballero E, Frykberg RG. Diabetic foot infections. J. Foot. Ankle Surg. 1998; 37:248–255 
41. Armstrong DG, Lavery LA, Sariaya M, Ashry H. Leukocytosis is a poor indicator of acute 
osteomyelitis of the foot in diabetes mellitus. J. Foot Ankle Surg. 1996; 35:280–283 
42. Aburto I, Frye C; A Randomized Controlled Trial to Evaluate Different Treatment Regimes with 
Topical Wound Oxygen (TWO2) on Chronic Wounds. Oral presentation at the International 
Symposium on Diabetic Foot, Noordwijkerhout, May 13th, 2011 

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43. Pocock SJ. “”Group sequential methods in the design and analysis of clinical trials.” 
Biometrika 1977; 64: 191-9  
44. Gowland-Hopkins, NF & Jamieson, CW. Br J Surg 1983; 70:532-34. 
45. Gilman,T. Int J Lower Extremity Wounds 2004; 3:125-132. 
46. European Medicines Agency. Pre-authorisation Evaluation of Medicines for Human Use. 
Committee for medicinal products for human use (CHMP). Guideline on data monitoring 
committees. 2006. Doc. Ref. EMEA/CHMP/EWP/5872/03 Corr.  
47. U.S. Department of Health and Human Services. Food and Drug Administration. Guidance for 
Clinical Trial Sponsors. Establishment and Operation of Clinical Trial Data Monitoring 
Committees. 2006. OMB Control No. 0910-0581. Expiration Date: 6/30/2012. 

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14 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
AE  
Adverse Event 
ADE 
Adverse device effect 
ABI 
Ankle Brachial Index 
AMWT 
Advanced Moist Wound Therapy 
AOTI  
Advanced Oxygen Therapy Ltd. 
CE  
The initials "CE" do not stand for specific words but are a declaration by 
a manufacturer that a product meets the requirements of applicable 
European Directives. 
CRF  
Case Report Form 
CWIS 
Cardiff Wound Impact Schedule 
EQ-5D 
Euroqol-5D 
GSD 
Group Sequential Design 
ICF 
Informed Consent Form 
ICH GCP 
International Conference on Harmonization Good Clinical Practice 
IEC  
Independent Ethics Committee 
IRB 
Institutional Review Board 
ITT 
Intention-to-treat 
mbar  
Millibars 
NSR 
Non Significant Risk 
PO2  
Pressure of Oxygen 
PP  
Per Protocol 
SADE 
Serious Adverse Device Effect 
SAE  
Serious Adverse Event 
SAP  
Statistical Analysis Plan 
TWO2  
Topical Wound Oxygen 
UADE  
Unanticipated Adverse Device Effect 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX I 
1. Study Diary Card (Example) 
 
2. University of Texas Wound Classification System 
 
 
 
I 
II 
III 
A 
Superficial Index Ulcer not 
involving tendon, capsule or 
Index Ulcer penetrating to 
tendon or capsule 
Index Ulcer penetrating to 
bone or joint 
B 
Infected 
Infected 
Infected 
C 
Ischemic 
Ischemic 
Ischemic 
D 
Infected and ischemic 
Infected and ischemic 
Infected and ischemic 
 
 

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3. Cardiff Wound Impact Schedule 
 
 

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4. EQ-5D 

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APPENDIX II 
WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving 
Human Subjects 
 
Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964 
and amended by the: 
29th WMA General Assembly, Tokyo, Japan, October 1975 
35th WMA General Assembly, Venice, Italy, October 1983 
41st WMA General Assembly, Hong Kong, September 1989 
48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 
52nd WMA General Assembly, Edinburgh, Scotland, October 2000  
53rd WMA General Assembly, Washington DC, USA, October 2002 (Note of Clarification 
added) 
55th WMA General Assembly, Tokyo, Japan, October 2004 (Note of Clarification added) 
59th WMA General Assembly, Seoul, Republic of Korea, October 2008 
64th WMA General Assembly, Fortaleza, Brazil, October 2013 
 
Preamble 
1.         The World Medical Association (WMA) has developed the Declaration of Helsinki as 
a statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data. 
            The Declaration is intended to be read as a whole and each of its constituent 
paragraphs should be applied with consideration of all other relevant paragraphs. 
2.         Consistent with the mandate of the WMA, the Declaration is addressed primarily to 
physicians. The WMA encourages others who are involved in medical research involving 
human subjects to adopt these principles.  
General Principles 
3.         The Declaration of Geneva of the WMA binds the physician with the words, “The 
health of my patient will be my first consideration,” and the International Code of Medical 
Ethics declares that, “A physician shall act in the patient's best interest when providing 
medical care.” 
4.         It is the duty of the physician to promote and safeguard the health, well-being and 
rights of patients, including those who are involved in medical research. The physician's 
knowledge and conscience are dedicated to the fulfilment of this duty. 
5.         Medical progress is based on research that ultimately must include studies involving 
human subjects. 

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6.         The primary purpose of medical research involving human subjects is to understand 
the causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best proven 
interventions must be evaluated continually through research for their safety, effectiveness, 
efficiency, accessibility and quality. 
7.         Medical research is subject to ethical standards that promote and ensure respect for all 
human subjects and protect their health and rights. 
8.         While the primary purpose of medical research is to generate new knowledge, this 
goal can never take precedence over the rights and interests of individual research subjects. 
9.         It is the duty of physicians who are involved in medical research to protect the life, 
health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects. The responsibility for the protection of research subjects 
must always rest with the physician or other health care professionals and never with the 
research subjects, even though they have given consent. 
10.       Physicians must consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable international 
norms and standards. No national or international ethical, legal or regulatory requirement 
should reduce or eliminate any of the protections for research subjects set forth in this 
Declaration. 
11.       Medical research should be conducted in a manner that minimises possible harm to the 
environment. 
12.       Medical research involving human subjects must be conducted only by individuals 
with the appropriate ethics and scientific education, training and qualifications. Research on 
patients or healthy volunteers requires the supervision of a competent and appropriately 
qualified physician or other health care professional. 
13.       Groups that are underrepresented in medical research should be provided appropriate 
access to participation in research. 
14.       Physicians who combine medical research with medical care should involve their 
patients in research only to the extent that this is justified by its potential preventive, 
diagnostic or therapeutic value and if the physician has good reason to believe that 
participation in the research study will not adversely affect the health of the patients who 
serve as research subjects. 
15.       Appropriate compensation and treatment for subjects who are harmed as a result of 
participating in research must be ensured. 
Risks, Burdens and Benefits  
16.       In medical practice and in medical research, most interventions involve risks and 
burdens. 

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           Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the risks and burdens to the research subjects. 
17.       All medical research involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and groups involved in the 
research in comparison with foreseeable benefits to them and to other individuals or groups 
affected by the condition under investigation. 
           Measures to minimise the risks must be implemented. The risks must be continuously 
monitored, assessed and documented by the researcher.  
18.       Physicians may not be involved in a research study involving human subjects unless 
they are confident that the risks have been adequately assessed and can be satisfactorily 
managed. 
           When the risks are found to outweigh the potential benefits or when there is conclusive 
proof of definitive outcomes, physicians must assess whether to continue, modify or 
immediately stop the study.            
Vulnerable Groups and Individuals 
19.       Some groups and individuals are particularly vulnerable and may have an increased 
likelihood of being wronged or of incurring additional harm. 
           All vulnerable groups and individuals should receive specifically considered 
protection. 
20.       Medical research with a vulnerable group is only justified if the research is responsive 
to the health needs or priorities of this group and the research cannot be carried out in a non-
vulnerable group. In addition, this group should stand to benefit from the knowledge, 
practices or interventions that result from the research. 
Scientific Requirements and Research Protocols  
21.       Medical research involving human subjects must conform to generally accepted 
scientific principles, be based on a thorough knowledge of the scientific literature, other 
relevant sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected. 
22.       The design and performance of each research study involving human subjects must be 
clearly described and justified in a research protocol. 
           The protocol should contain a statement of the ethical considerations involved and 
should indicate how the principles in this Declaration have been addressed. The protocol 
should include information regarding funding, sponsors, institutional affiliations, potential 
conflicts of interest, incentives for subjects and information regarding provisions for treating 
and/or compensating subjects who are harmed as a consequence of participation in the 
research study. 

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           In clinical trials, the protocol must also describe appropriate arrangements for post-trial 
provisions. 
Research Ethics Committees 
23.       The research protocol must be submitted for consideration, comment, guidance and 
approval to the concerned research ethics committee before the study begins. This committee 
must be transparent in its functioning, must be independent of the researcher, the sponsor and 
any other undue influence and must be duly qualified. It must take into consideration the laws 
and regulations of the country or countries in which the research is to be performed as well as 
applicable international norms and standards but these must not be allowed to reduce or 
eliminate any of the protections for research subjects set forth in this Declaration.  
           The committee must have the right to monitor ongoing studies. The researcher must 
provide monitoring information to the committee, especially information about any serious 
adverse events. No amendment to the protocol may be made without consideration and 
approval by the committee. After the end of the study, the researchers must submit a final 
report to the committee containing a summary of the study’s findings and conclusions.  
Privacy and Confidentiality  
24.       Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information. 
Informed Consent  
25.       Participation by individuals capable of giving informed consent as subjects in medical 
research must be voluntary. Although it may be appropriate to consult family members or 
community leaders, no individual capable of giving informed consent may be enrolled in a 
research study unless he or she freely agrees. 
26.       In medical research involving human subjects capable of giving informed consent, 
each potential subject must be adequately informed of the aims, methods, sources of funding, 
any possible conflicts of interest, institutional affiliations of the researcher, the anticipated 
benefits and potential risks of the study and the discomfort it may entail, post-study provisions 
and any other relevant aspects of the study. The potential subject must be informed of the 
right to refuse to participate in the study or to withdraw consent to participate at any time 
without reprisal. Special attention should be given to the specific information needs of 
individual potential subjects as well as to the methods used to deliver the information. 
           After ensuring that the potential subject has understood the information, the physician 
or another appropriately qualified individual must then seek the potential subject’s freely-
given informed consent, preferably in writing. If the consent cannot be expressed in writing, 
the non-written consent must be formally documented and witnessed.       
           All medical research subjects should be given the option of being informed about the 
general outcome and results of the study. 
27.       When seeking informed consent for participation in a research study the physician 
must be particularly cautious if the potential subject is in a dependent relationship with the 

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physician or may consent under duress. In such situations the informed consent must be 
sought by an appropriately qualified individual who is completely independent of this 
relationship. 
28.       For a potential research subject who is incapable of giving informed consent, the 
physician must seek informed consent from the legally authorised representative. These 
individuals must not be included in a research study that has no likelihood of benefit for them 
unless it is intended to promote the health of the group represented by the potential subject, 
the research cannot instead be performed with persons capable of providing informed consent, 
and the research entails only minimal risk and minimal burden. 
29.       When a potential research subject who is deemed incapable of giving informed 
consent is able to give assent to decisions about participation in research, the physician must 
seek that assent in addition to the consent of the legally authorised representative. The 
potential subject’s dissent should be respected. 
30.       Research involving subjects who are physically or mentally incapable of giving 
consent, for example, unconscious patients, may be done only if the physical or mental 
condition that prevents giving informed consent is a necessary characteristic of the 
research  group. In such circumstances the physician must seek informed consent from the 
legally authorised representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the specific 
reasons for involving subjects with a condition that renders them unable to give informed 
consent have been stated in the research protocol and the study has been approved by a 
research ethics committee. Consent to remain in the research must be obtained as soon as 
possible from the subject or a legally authorised representative. 
31.       The physician must fully inform the patient which aspects of their care are related to 
the research. The refusal of a patient to participate in a study or the patient’s decision to 
withdraw from the study must never adversely affect the patient-physician relationship. 
32.       For medical research using identifiable human material or data, such as research on 
material or data contained in biobanks or similar repositories, physicians must seek informed 
consent for its collection, storage and/or reuse. There may be exceptional situations where 
consent would be impossible or impracticable to obtain for such research. In such situations 
the research may be done only after consideration and approval of a research ethics 
committee. 
Use of Placebo 
33.       The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best proven intervention(s), except in the following circumstances: 
           Where no proven intervention exists, the use of placebo, or no intervention, is 
acceptable; or 
           Where for compelling and scientifically sound methodological reasons the use of any 
intervention less effective than the best proven one, the use of placebo, or no intervention is 
necessary to determine the efficacy or safety of an intervention  

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           and the patients who receive any intervention less effective than the best proven one, 
placebo, or no intervention will not be subject to additional risks of serious or irreversible 
harm as a result of not receiving the best proven intervention.  
           Extreme care must be taken to avoid abuse of this option. 
 
Post-Trial Provisions 
34.       In advance of a clinical trial, sponsors, researchers and host country governments 
should make provisions for post-trial access for all participants who still need an intervention 
identified as beneficial in the trial. This information must also be disclosed to participants 
during the informed consent process. 
Research Registration and Publication and Dissemination of Results 
35.       Every research study involving human subjects must be registered in a publicly 
accessible database before recruitment of the first subject. 
36.       Researchers, authors, sponsors, editors and publishers all have ethical obligations with 
regard to the publication and dissemination of the results of research. Researchers have a duty 
to make publicly available the results of their research on human subjects and are accountable 
for the completeness and accuracy of their reports. All parties should adhere to accepted 
guidelines for ethical reporting. Negative and inconclusive as well as positive results must be 
published or otherwise made publicly available. Sources of funding, institutional affiliations 
and conflicts of interest must be declared in the publication. Reports of research not in 
accordance with the principles of this Declaration should not be accepted for publication. 
Unproven Interventions in Clinical Practice 
37.       In the treatment of an individual patient, where proven interventions do not exist or 
other known interventions have been ineffective, the physician, after seeking expert advice, 
with informed consent from the patient or a legally authorised representative, may use an 
unproven intervention if in the physician's judgement it offers hope of saving life, re-
establishing health or alleviating suffering. This intervention should subsequently be made the 
object of research, designed to evaluate its safety and efficacy. In all cases, new information 
must be recorded and, where appropriate, made publicly available. 
Copyright, World Medical Association. All Rights Reserved. Reproduced with permission.