Conditions: Diabetic Foot Infections
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 31
Healthy volunteers: no

Brief summary:
Phase I/IIa, five cohort ascending dose with two dosing arms per cohort, study in Type I or II diabetes mellitus subjects with a chronic infected diabetic ulcer defined as having a DUSS score of 0 to 3 and DFI wound score of 1 to 3.

Detailed description:
The study is designed to run the cohorts in series with the completion of the first cohort before initiating the next dosing level. At all study visits the ulcer will be visually examined for any changes and photographed using the Aranz Medical Silhouette™ system that will calculate area and depth of the ulcer.

In Arm 1, eligible subjects will be treated with a single application of Nu-3 or placebo in 4 to 1 ratio to judge the initial safety of Nu-3 over a brief one (1) hour interval and 24-hr interval post application. Bisphosphocin Nu-3 will be applied topically to the chronic infected ulcer, covered with a non-abrasive bandage following the initial observation period. The subject will be released with verbal instructions to leave the bandage on the wound and return for a follow up visit within 24h ± 2h. At the follow up visit, the bandage will be removed, the ulcer visually examined and the subject cleared for the MAD Arm 2 based on the recommendation of the PI and absence of any SAEs.

In Arm 2, eligible subjects which are those who have been approved by the PI after the Visit 2 examination will be instructed in the proper application of bisphosphocin Nu-3. The subjects will be observed applying the first dose in the clinic to ensure compliance. Subjects will then be given a 7 day supply and sent home to continue treatment. Visit 4 or earlier in the case of any adverse events, subjects will return to the clinic for an examination, including visual examination of the ulcer, vital signs, adverse events, photo documentation, collection of a sample for microbiology and concomitant medication use. A final follow up visit will be scheduled +7 days after last dose of study medication (Day 15) for a complete examination as described above.

Arms:
arm 1: Dosage Form: Topical Antimicrobial, Dosage: 1mg/mL, 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL Frequency: QD for day 1, 2x daily for 7 days, Duration: 8 days arm 2: Dosage Form: Diluent, Frequency: QD for day 1, 2x daily for 7 days, Duration: 8 days

Interventions: intervention 1: Bisphosphocin Nu-3 intervention 2: Placebo
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_000.pdf: 
Bisphosphocin NU-3 
Lakewood-Amedex 
Protocol #LAl2014-1 
Page 1 of 44 
CLINICAL INVESTIGATIONAL PROTOCOL 
PROTOCOL NO: LAI2014-1 
Version 3.1 
15 September2016 
A Phase I/Ila, Randomized Double Blind, Placebo-Controlled, Dose Escalating Study to 
Evaluate the Safety and Tolerability of Topically Applied Bisphosphocin™ Nu-3 on 
Infected Diabetic Ulcers of Subjects With Type I or II Diabetes Mellitus 
Lakewood Amedex Inc. 
3030 University Pkwy 
Sarasota, FL 34243 
CONFIDENTIALITY STATEMENT 
This protocol is the confidential property of LAKEWOOD AMEDEX. Any 
unauthorized duplication or dissemination of the contained information is 
prohibited. 
LAl2014-1 _Protocol_ V3. 1_15Sep20163. 1 
Confidential 


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Protocol #LAI2014-1 
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LAI2014-1_Protocol_V3.1_15Sep20163.1 
 
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TITLE PAGE 
 
 
Title: 
A Phase I/IIa, Randomized Double Blind, Placebo-Controlled, 
Dose Escalating Study to Evaluate the Safety and Tolerability of 
Topically Applied Bisphosphocin™ Nu-3 on Infected Diabetic 
Ulcers of Subjects with Type I or II Diabetes Mellitus 
 
Protocol No.: 
LAI2014-1 
 
 
Protocol Version: 
Version 3.1  
 
 
Version Date:  
15 September 2016 
 
 
Investigational Product: 
Bisphosphocin™ Nu-3 
 
 
 
 
Study Director: 
 
 
 
 
Sponsor: 
Lakewood Amedex Inc 
 
3030 University Pkwy 
 
Sarasota, FL 34243 
 
 
Tel.: +1 941 225 2515 
 
Fax: +1 941 225 2511 
 
 
 
 
 
Signature on behalf of the Sponsor: 
 
 
 
Signature: ________________________ Date:_______________ 
 


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INVESTIGATOR PROTOCOL AGREEMENT PAGE 
 
 I, the undersigned, am responsible for the conduct of the trial at this site and agree to the 
following: 
 I understand and will conduct the trial according to the protocol, any approved protocol 
amendments, ICH GCP and all applicable regulatory authority requirements and national 
laws. 
 I have sufficient time to properly conduct and complete the trial within the agreed trial 
period, and I have available an adequate number of qualified staff and adequate facilities 
for the foreseen duration of the trial to conduct the trial properly and safely. 
 I will ensure that all staff at the clinical trial site, who are involved in the trial conduct are 
adequately trained regarding the protocol and their responsibilities.   
 
 
 
 
Signed: 
 
 
 
 
 
______________________________________ 
 
_____________________________ 
Principal Investigator    
 
                        
Date 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood-Amedex 
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LAI2014-1_Protocol_V3.1_15Sep20163.1 
 
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1 
SYNOPSIS 
 
Name of Sponsor: Lakewood Amedex Inc 
Protocol No.: LAI2014-1 
Name of Test Product: Bisphosphocin™ Nu-3 
Clinical Investigation Center(s): Approximately 2 to 4 sites in USA 
Title of Clinical investigation: A Phase I/IIa, Randomized Double Blind, Placebo-Controlled, Dose Escalating 
Study to Evaluate the Safety and Tolerability of Topically Applied Bisphosphocin™ Nu-3 on Infected Diabetic 
Ulcers of Subjects With Type I or II Diabetes Mellitus 
Patient Population:  Male and Female subjects 18-85 years of age suffering from diabetes mellitus and a chronic 
infected diabetic ulcer(s). 
Sample Size: Maximum 50 subjects in total across five study cohorts 
Examination Points:  Day 1, 2, 9, 15 
Study Design 
Multi center, Randomized Double Blind, Placebo-Controlled, Dose Escalating Study 
Clinical investigation Objective(s) and Endpoint: 
 
Objective 
Endpoint 
Variable 
Primary 
To assess the safety 
and tolerability of 
escalating doses of 
topically applied 
bisphosphocin™ Nu-3 
 Treatment emergent 
events related to 
clinical investigation 
product  
 Vital signs,  
 CBC analysis, CMP analysis, 
 Physical examination,   
Secondary 
 To assess the clinical 
and microbiological 
response to 
bisphosphocin™ Nu-3 
 
 To support 
determination of the 
appropriate dose 
range of Nu-3 to be 
employed in future 
clinical studies.  
 Reduction of 
pathogenic bacteria 
 Visual evaluation of 
the ulcer  
 Aerobic and anaerobic culture and 
sensitivity 
 DUSS Score 
Clinical investigation Duration (Per subject) 
Total Clinical investigation duration: (Recruitment period) + screening + treatment and evaluation + follow-up. 
• 
Screening Period: - 7 days to -4 hours 
• 
Treatment: 7 Days 
• 
Follow-up: 24 hours (± 2 hours) after first treatment; 8 days + 1 day) after first treatment 
• 
End of clinical study: 15 days ± 1 day after first treatment 
Randomization 
Randomization will be conducted in blocks of five to a ratio of 4:1 (active:placebo). Qualified subjects will be 
enrolled and randomized in: 8:8:8:8:8:10 ratio to either Nu-3 at one of five possible dose levels (0.1%, 1%, 2%, 5% 
and 10% Nu-3 solution in saline (w/v)), or to placebo, respectively. 


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Inclusion Criteria 
1. Men and women between the ages of 18 and 85.  
2. Voluntary written consent, given before performance of any clinical investigation-related procedure not 
part of standard medical care, and with the understanding that consent may be withdrawn at any time 
without prejudice to future medical care. 
3. Non-hospitalized ambulatory subjects suffering from Diabetes mellitus, Type I or II 
4. Diabetic foot ulcer(s) with a DUSS Score of 0 to 3 
5. Ulcerated area(s) of not more than two (2) ulcers between 0.5 to 6 cm2  
6. Any female of child bearing age must consent to use medically acceptable birth control for the duration of 
the study 
7. Female subjects must meet at least one of the following additional criteria: 
a. 
Surgically sterile with bilateral tubal ligation or hysterectomy. 
b. Post-menopausal for at least one year. 
c. 
If of child-bearing potential, practicing an acceptable method of birth control for the duration of 
the clinical investigation as judged by the Investigator, such as condoms, foams, jellies, 
diaphragm, intrauterine device or abstinence.  
8. Subjects willing to undergo pre-and post-clinical investigation blood collection, physical exams and 
laboratory investigations. 
Exclusion Criteria 
1. A DUSS Score above 3. 
2. DUSS Probing to Bone = “Yes”  
3. An ulcer area(s) greater than 6 cm2 or more than two (2) ulcers 
4. Any subject that has received systemic or topical antibiotics within the last seven (7) days 
5. Any subject on topical antimicrobial treatment for their infected diabetic foot ulcer whose ulcer is 
responding to treatment 
6. Any subject that would be unable to follow the protocol procedures, safely monitor the infection status at 
home, and return for schedule visits 
7. Positive pregnancy test at Screening or Visit 2 
8. Active infection as demonstrated by temperature > 37.5 oC and clinical features of active infection. 
9. Known immunosuppression or taking immunosuppressive agents including systemic steroids.  
10. History of severe co-morbidity with expected patient survival ≤ 6 months.  
11. Pregnancy or lactation 
12. Intake of investigational drugs within 28 days prior to enrollment.  
13. History of concurrent condition that, in the Investigator’s opinion, would jeopardize the safety of the 
subject or compliance with the protocol.  
14. Likely inability to comply with the protocol or cooperate fully with the investigator and site personnel.  
15. Unwillingness or language barrier precluding adequate understanding of the trial procedure or cooperation 
with trial site personnel. 
16. Known or suspected active abuse of alcohol, narcotics or non-prescription drugs. 
17. Other planned surgical procedures within 30 days prior to or 30 days post-index procedure. 
18. Prior enrolment in this clinical trial  


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Test Product  
 
0.1% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
 
1% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
 
2% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
 
5% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
 
10% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
 
0.9% saline  
Test Kit 
Test Product kits will be labelled and contain a 0.5 oz dropper bottle containing 15 mL of Test Product packaged in 
a cardboard carton with a foam insert for protection.  These kits will be pre-labelled with kit numbers. The contents 
of the kits will not be known to the pharmacist, designee or clinical staff. Upon randomization of the subject, the 
pharmacist or designee will receive a randomization number along with a kit number that is available at that site.  
The kit with the corresponding kit number will be used for dose administration. 
Administration 
Test Product will be administered to the infected diabetic foot ulcer and surrounding intact skin at a dose of 3 drops 
from a dropper bottle per 1 cm2 of ulcer area. This will be explained to test subjects so they know the exact number 
of square  
 
After application of the Test Product is complete, the wound will be left open to air for 5 min and then will be 
dressed as per the following: Owens Gauze will be trimmed to cover the entire dimension of the ulcer and overlaid 
into the ulcer. Gauze dressings (2 x 2 inches) will be placed over the Owens Gauze to a depth of 1/4 inch followed 
by a circumferential wrap of 2 inch Kerlex. The dressing will be secured with paper tape applied to the 
circumferential dressing as to not come in contact with the underlying skin. 
Clinical investigation Procedure  
Subjects found eligible based on the inclusion/exclusion criteria will have the study explained to them and will be 
provided the study specific informed consent form (ICF). The screening evaluations will be performed after the 
subject provides a written informed consent.  For subjects enrolled in the study, activities will consist of a 
Screening/Baseline Visit and three (4) scheduled visits. Screening can take place any time within 7 days to 4 hours 
prior to the start of the study.  Screening will include:  
 
Visit 1 (Day -7 to -4 hours) - Screening/Baseline:  Following signed written informed consent and confirmation of 
eligibility, demographics and medical history, and limited physical examination per protocol, urine pregnancy test 
(if applicable), baseline laboratory tests (blood chemistries, hematology, coagulation profile, and urinalysis), and 
baseline subject disease assessments (as specified in the schedule of events table) will be performed at this visit.     
 
Demography data, medical history, general physical examination of all organ systems, and vital 
signs (blood pressure, heart rate, and temperature): Perform a physical exam and assess vital signs per 
hospital protocol within the specified timeframe for screening.  
 
Vital signs measurements: Weight, temperature, resting blood pressure, heart rate, and respiratory rate  
 
Hematology: WBC, RBC, Hgb, Hct, MCV, MCH, HCHC, RDW, MPV, Platelets, Neutrophils Auto % 
(abs), Immature Granulocyte Auto % (abs), Lymphocytes Auto % (abs), Monocytes Auto % (abs), 
Eosinophils Auto % (abs), Basophils Auto % (abs)  
 
Biochemistry: Sodium, Potassium, Bicarbonate or CO2, Chloride, Total Bilirubin, Creatinine, BUN, 
Calcium, Alkaline Phosphatase, ALT, AST, Total Protein, Albumin, Anion Gap ,eGFR, Globulin, A/G 
ration 
 
For females of child-bearing potential: pregnancy test (urine) to be performed at baseline.  
 
Baseline Subject Disease Assessment: Visual examination of chronic ulcer, score target ulcer using 
DUSS and DFI Wound Scoring System, Swab wound to culture for microbiological examination 
 
Visits 2 (Day 1) SAD Treatment:  Subjects will return after the screening visit for Single Ascending Dose 
Treatment.  Subject disease assessments will be performed as specified in the schedule of events table. 


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 
Vital signs (blood pressure, heart rate, temperature) will be examined within 10 minutes prior to the start 
of treatment 
 
Baseline Subject Disease Assessment: Visual examination of chronic ulcer, score target ulcer using DUSS 
Scoring System, Swab wound to culture for microbiological examination – Not necessary if Visit 2 is the 
same day as Visit 1 
 
Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz Medical Silhouette™ 
System – Not necessary if Visit 2 is the same day as Visit 1 
 
In SAD Treatment, eligible subjects will be treated with a single application of Test Product.  Test Product will be 
administered to the infected diabetic foot ulcer and surrounding intact skin at a dose of 3 drops from a dropper 
bottle per 1 cm2 of ulcer area, left open to air for 5 minutes and then dressed using Owens gauge, Kerlex and tape. 
The ulcer with a non-abrasive bandage following the initial observation period and the subject will be released with 
verbal instructions to leave the bandage on the wound and return for a follow up visit within 24 h ± 2 h. 
 
Visits 3 (Day 2 [24 h ± 2 h.]) MAD Treatment:  Subjects will return after the Single Ascending Dose Treatment. 
Subject disease assessments will be performed as specified in the schedule of events table. 
 
Vital signs (blood pressure, heart rate, temperature) will be examined within 10 minutes prior to the start 
of treatment 
 
Baseline Subject Disease Assessment: Visual examination of chronic ulcer, score target ulcer using DUSS 
Scoring System, Swab wound to culture for microbiological examination 
The Principal Investigator will visually exam the chronic ulcer to determine if a subject is eligible to continue in the 
study. Eligible subjects will be provided with his/her designated test product kit and instructed to apply the product 
twice daily for one (1) week (7 days). Test product application will be demonstrated, and subject instruction sheet 
reviewed, using the assigned test product.  The subjects will be observed applying the first dose in the clinic to 
ensure compliance (administering Test Product to the infected diabetic foot ulcer and surrounding intact skin at a 
dose of 3 drops from a dropper bottle per 1 cm2 of ulcer area, left open to air for 5 minutes and then dressed using 
Owens gauge, Kerlex and tape.). The subject will be scheduled for the next follow-up visit, with a reminder to bring 
the test article kit to the visit.   
 
Visit 4 (Day 9 + 1) Follow-Up to 7 Twice Daily Treatments: 
The study subject will return for a follow-up evaluation to the clinical site on Day 9 ± 1day. At this follow-up visit, 
the subject will undergo the following procedures and evaluations. 
 
Vital signs measurements including weight, temperature, resting blood pressure, heart rate, and respiratory 
rate  
 
Baseline laboratory tests (blood chemistries and hematology) 
 
Visual examination of the ulcer 
 
Scoring of the Ulcer(s) using the DUSS and DFI Wound Scoring System 
 
Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz Medical Silhouette™ 
System 
 
Collection of a sample from the ulcer for microbiological assessment 
 
Subjects will be given a 7 day supply of non-abrasive bandage and written instructions on the proper care and 
hygiene to include keeping the ulcer clean and bandage dry until the Day 15 follow up visit. In addition, subjects 
will be told to call if there is any worsening of the ulcer with regard to pain, infection, or swelling. 
 
Visit 5 (Day 15 ± 1) End of Study (EOS) Visit 
The subject shall attend final end-of-study visit 15 days ± 1 day after receiving Test Product.  At this follow-up 
visit, the subject will undergo the following procedures and evaluations. 
 
Vital signs measurements including weight, temperature, resting blood pressure, heart rate, and respiratory 


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rate  
 
Physical examination  
 
Baseline laboratory tests (blood chemistries and hematology) 
 
Visual examination of the ulcer 
 
Scoring of the Ulcer(s) using the DUSS and DFI Wound Scoring System 
 
Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz Medical Silhouette™ 
System 
 
Collection of a sample from the ulcer for microbiological assessment 
 
At Every Visit: All adverse events (local and systemic), concurrent procedures, and changes in concomitant 
reactions to test product administration include but are not limited to pain, edema, rash, cellulitis, localized 
infectious processes, and any systemic reaction including fever, allergic reaction, and anaphylaxis. 
 
Early Termination Visit:  
If a subject withdraws prior to completing the study, the reason for withdrawal will be documented. If a subject 
withdraws early due to an adverse event, he/she will be followed until resolution/stabilization of the adverse event. 
 
If a subject prematurely withdraws from the study they will be asked to complete the study procedures and 
evaluations performed in the final study visit at the time of withdrawal from the study:  
 
Vital signs measurements including weight, temperature, resting blood pressure, heart rate, and respiratory 
rate  
 
Physical examination  
 
Baseline laboratory tests (blood chemistries and hematology) 
 
Visual examination of the ulcer 
 
Scoring of the Ulcer(s) using the DUSS System 
Statistical Analysis 
Summary tables (descriptive statistics and/or frequency tables) will be provided for all baseline variables, efficacy 
variables, and safety variables.  Continuous variables will be described by descriptive statistics (n, mean, standard 
deviation, minimum, maximum, median, 95% confidence limits for the mean).   
 
Frequency tables will be used to report adverse event frequency by system organ class (SOC), preferred term (PT), 
severity, and relationship to test article.  All subjects receiving the test article will be included in the safety 
analyses.   
 
Safety data including laboratory evaluations and vital signs assessments are summarized by dose group and time 
point of collection. Descriptive statistics are calculated for quantitative safety data, and frequency counts are 
compiled for classification of qualitative safety data. In addition, a mean change from baseline table is provided for 
vital signs and a shift table describing out of normal range shifts is provided for clinical laboratory results.  
Changes in physical examinations are described in the text of the final report.  


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Justification of Sample Size 
This is a pilot dose-ranging study.  No sample size calculations were conducted. 
 
Analysis Populations 
All statistical processing will be performed using SAS® unless otherwise stated.  All subjects enrolled in the study 
that were dispensed and applied test article at least once will be included in the analysis of safety and efficacy and 
will be considered the intent-to-treat (ITT) population.  Last-observation-carried-forward (LOCF) will be used to 
impute missing values for efficacy variables.   
 
A subject will be included in the per-protocol (PP) efficacy analysis population if all of the following criteria are 
met: 
 
Subject meets the inclusion/exclusion criteria; 
 
Subject has not taken or applied any interfering concomitant medications  
 
Subject has completed Visit 4/Day 15 ( 2 days) and has missed no more than one Follow-Up Visit;  
 
Subject has been compliant with the dosing regimen (i.e., subject must take at least 80% of the expected 
doses up to and including the final visit). 
 
Interim Analysis 
A safety review will be conducted after each cohort to confirm proceeding to the next dose level.  
Safety Assessments 
Adverse events: Treatment-emergent adverse events will be coded according to the Medical Dictionary for 
Regulatory Activities (MedDRA). The incidence of treatment-emergent adverse events will be summarized by 
body system and preferred term. 
 


Bisphosphocin NU-3 
Lakewood-Amedex 
2 
SCHEDULE OF EVENTS 
VISIT/CONTACT 
DAY 
Eligibility Screening, Inclusion, Exclusion 
Informed Consent 
Demographics & Medical Histo1y 
Physical Exam 
Vital Si=s 
Urine Pregnancy Test 1 (WOCBP) 
Laboratorv Tests - Safetv Labs 
Visual Examination of Chronic Ulcer 
Photo Documentation and Calculation of target ulce1· area and 
depth - ARANZ Silhouetten 1 Medical System 
Score Target Ulcer using DUSS System 
Ratimz of the infected ulcer usine: DFI Wound Sco1ine: svstem 
Swab wound to culture for Microbiological Evaluation 
Investigation Product Single Treatment 
Subject Instrnction Sheet Reviewed & Distributed 
Subject Dia1y Distributed 
Subiect Diarv Collected 
Dispense Test Article 
Test Alt icle Accountability 
Concomitant Medication 
Adverse Events 
Screening/ 
Baseline 
Visit 1 
-7 Day 
to -4 hour 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Protocol #LAl2014-1 
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Treatment 
Visit 2 
Visit 3 
Day 1 
Day 2 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
EOS 
Visit 4 
Visit 5 
Day 9 + 1 
Day 15±1 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
<-----------------------------------------------------------> 
<-----------------------------------------------------------> 
Early 
Termination 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
1 Women of childbearing potential (WOCBP) include any female who has experienced menarche and who has not tmdergone successful surgical sterilization (hysterectomy, bilateral tubal 
ligation, or bilateral oophorectomy) or is not postmenopausal [defined as amenoffhea >12 consecutive months]. Even women who are using oral, implanted, or injectable contraceptive 
hormones, an intrauterine device (IUD), baffier methods (diaphragm, condoms, spermicidal) to prevent pregnancy, practicing abstinence, or where partner is sterile (e.g., vasectomy 
pe1fo1med at least six months prior to the subject's initiation of treatment) should be considered to be of childbearing potential. 
LAl2014-1_Protocol_ V3.1_15Sep20163.1 
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3 
TABLE OF CONTENTS 
 
1 
SYNOPSIS ........................................................................................................................................... 4 
2 
SCHEDULE OF EVENTS ............................................................................................................... 10 
3 
TABLE OF CONTENTS ................................................................................................................. 11 
4 
ABBREVIATIONS AND DEFINITIONS ...................................................................................... 14 
5 
BACKGROUND ............................................................................................................................... 16 
6 
RATIONALE .................................................................................................................................... 16 
7 
OBJECTIVE ..................................................................................................................................... 17 
7.1 PRIMARY OBJECTIVE ...................................................................................... 17 
7.2 SECONDARY OBJECTIVES ................................................................................ 17 
8 
STUDY DESIGN .............................................................................................................................. 17 
8.1 OVERALL STUDY DESIGN ............................................................................... 17 
8.2 NUMBER OF CENTERS ..................................................................................... 17 
8.3 COHORTS ...................................................................................................... 18 
8.4 PLACEBO CONTROL ....................................................................................... 18 
8.5 NUMBER OF SUBJECTS .................................................................................... 18 
8.6 STUDY VISITS ............................................................................................... 18 
8.7 STUDY DURATION ......................................................................................... 18 
8.8 DOSE SELECTION AND RATIONALE ................................................................... 18 
8.9 FIRST DOSE PLAN .......................................................................................... 18 
8.10 SAFETY REVIEW ............................................................................................ 19 
8.10.1 
Safety Review Committee ................................................................................... 19 
8.10.2 
Safety Review Procedure .................................................................................... 19 
8.10.3 
Cohort 1 Day 1a Experience .............................................................................. 20 
8.10.4 
Unblinding for Safety Review ............................................................................ 20 
8.10.5 
Decision Criteria................................................................................................. 20 
8.11 SUBSEQUENT COHORT DOSE PLAN ................................................................... 20 
9 
SELECTION AND WITHDRAWAL OF SUBJECTS ................................................................. 21 
9.1 INCLUSION AND EXCLUSION OF SUBJECTS ......................................................... 21 
9.1.1 Inclusion Criteria ..................................................................................................... 21 
9.1.2 Exclusion Criteria .................................................................................................... 21 
9.2 WITHDRAWAL OF SUBJECTS ............................................................................ 22 
10 
INVESTIGATIONAL PRODUCTS ............................................................................................... 23 
10.1 STUDY DRUG AND PLACEBO ........................................................................... 23 


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10.2 DOSE SCHEDULE............................................................................................ 23 
10.3 DRUG SHIPPING, PACKAGING AND LABELING .................................................... 24 
10.4 STUDY DRUG HANDLING AND STORAGE ........................................................... 24 
10.4.1 
Handling and Storage ........................................................................................ 24 
10.4.2 
Study Drug Accountability ................................................................................. 24 
10.5 RANDOMIZATION ........................................................................................... 25 
10.6 BLINDING AND UNBLINDING ........................................................................... 25 
10.7 STUDY DRUG PREPARATION ............................................................................ 25 
10.8 STUDY DRUG ADMINISTRATION....................................................................... 25 
10.8.1 
Study Drug Administration Procedure .............................................................. 25 
10.8.2 
Disposition of Dose Administration Materials .................................................. 26 
11 
CONCOMITANT MEDICATIONS AND EXPOSURES ............................................................ 26 
11.1 PRIOR MEDICATIONS ...................................................................................... 26 
11.2 CONCOMITANT MEDICATIONS ......................................................................... 26 
12 
TREATMENT OF SUBJECTS AND CONDUCT OF STUDY ................................................... 26 
12.1 INFORMED CONSENT ...................................................................................... 26 
12.2 VISIT 1 (DAY -7 TO -4 HOURS) - SCREENING ...................................................... 27 
12.3 VISIT 2 (DAY 1) SAD TREATMENT ................................................................... 28 
12.4 VISIT 3 (DAY 2 [24 H ± 2 H.]) MAD TREATMENT ............................................... 28 
12.5 VISIT 4 (DAY 9 ± 1) FOLLOW-UP TO 7 TWICE DAILY TREATMENTS ...................... 29 
12.6 VISIT 5 (DAY 15 ± 2) END OF STUDY (EOS) VISIT .............................................. 29 
12.7 AT EVERY VISIT ............................................................................................ 30 
12.8 EARLY TERMINATION VISIT ............................................................................ 30 
12.9 ADVERSE EVENT MONITORING ........................................................................ 30 
13 
ASSESSMENT OF SAFETY ........................................................................................................... 30 
13.1 PHYSICAL EXAMINATION ................................................................................ 30 
13.2 VITAL SIGNS ................................................................................................. 30 
13.3 CLINICAL CHEMISTRY ..................................................................................... 31 
13.4 ADVERSE EVENTS .......................................................................................... 31 
14 
ASSESSMENT OF EFFICACY ...................................................................................................... 35 
14.1 PRIMARY EFFICACY ENDPOINT ASSESSMENT ..................................................... 35 
14.2 SECONDARY EFFICACY ENDPOINT ASSESSMENT ................................................ 35 
15 
PHARMACOKINETIC ASSESSMENT ........................................................................................ 35 
16 
STATISTICAL CONSIDERATIONS ............................................................................................ 35 
16.1 JUSTIFICATION OF SAMPLE SIZE AND POWER ANALYSIS ...................................... 36 


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16.2 ANALYSIS POPULATIONS ................................................................................ 36 
16.3 INTERIM ANALYSIS ........................................................................................ 36 
16.4 ANALYSIS OF BASELINE DATA......................................................................... 36 
16.5 ANALYSIS OF PRIMARY OUTCOME MEASURE .................................................... 37 
17 
REGULATORY AND ADMINISTRATIVE ISSUES .................................................................. 37 
17.1 SUBJECT INFORMATION AND INFORMED CONSENT ............................................. 37 
17.2 PRE-STUDY DOCUMENTATION ......................................................................... 37 
17.3 INDEPENDENT/ INSTITUTIONAL ETHICS COMMITTEE APPROVAL ........................... 38 
17.4 ETHICAL CONDUCT OF THE STUDY ................................................................... 38 
18 
DATA RECORDING AND COLLECTION.................................................................................. 39 
18.1 SOURCE DOCUMENTS ..................................................................................... 39 
18.2 CASE REPORT FORMS (CRFS) ......................................................................... 39 
19 
MONITORING, AUDIT, RETENTION AND CONFIDENTIALITY OF STUDY 
RECORDS ................................................................................................................................................. 39 
20 
STUDY TERMINATION ................................................................................................................ 40 
21 
CONFIDENTIALITY AND PUBLICATION POLICY ............................................................... 40 
22 
REFERENCES ................................................................................................................................. 41 
 
 
 


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4 
ABBREVIATIONS AND DEFINITIONS 
Abbreviation 
Meaning 
 C  
degrees Celsius 
 F 
degrees Fahrenheit 
AE 
Adverse Event/Adverse Experience 
ALT 
Alanine Aminotransferase 
API 
Active Pharmaceutical Ingredient 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Unit 
CLSI 
Clinical and Laboratory Standards Institute 
CMP 
Comprehensive Metabolic Panel 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTCAE 
Common Terminology Criteria for Adverse Events v4.0 
DFI 
Diabetic Foot Infection 
DSMB 
Data and Safety Monitoring Board 
DUSS 
Diabetic Ulcer Severity Score 
ELSD 
Evaporative Light Scatter Detection 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
hCG 
Human Chorionic Gonadotropin 
HCV 
Hepatitis C Virus 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HPLC 
High Performance Liquid Chromatography 
IB 
Investigator’s Brochure 
ICH 
International Committee on Harmonization 
ICMJE 
International Committee of Medical Journal Editors 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IR 
Infrared Spectroscopy 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
LAI 
Lakewood-Amedex, Inc 
LCMS 
Liquid Chromatography Mass Spectrometry 
LLN 
Lower Limit of Normal 


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MAD 
Multiple Ascending Dose 
MBC 
Minimum Bactericidal Concentration 
MIC 
Minimum Inhibitory Concentration 
MOA 
Mechanism of Action 
MRSA 
Methicillin Resistant Staphylococcus aureus 
MS 
Mass Spectroscopy 
NDM-1 
New Delhi Metallo-Beta Lactamase-1 
NMR 
Nuclear Magnetic Resonance 
PBS 
Phosphate Buffered Saline 
PI 
Principal Investigator 
RBCs 
Red Blood Cells 
SAD 
Single Ascending Dose 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
TS 
Trypic Soy 
TSBD 
Trypic Soy Broth with 1% Dextrose 
ULN 
Upper Limit of Normal 
US 
United States 
WBC 
White Blood Cell 
 


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BACKGROUND 
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Lakewood-Amedex Inc. has developed a novel class of synthetic broad spectmm 
antimicrobials, tenned bisphosphocins. Bisphosphocins™ function through a novel directly 
bactericidal mechanism of action which is not mediated through a single receptor/target 
protein. 
The bisphosphocin ™ mechanism of action appears specific for microbial 
membranes with minimal effect on mammalian cell membranes. In addition to possessing 
activity against all the ESKAPE2 bacteria strains, including those resistant to antibiotics, 
bisphosphocins™ are effective against stationary, slow-growing, and bacteria encased in 
biofilm. 
Bisphosphocins™ represent a new class of antimicrobials and a potentially 
important new class of therapeutics needed to ti·eat the increasing number of antibiotic 
resistant bacterial and fungal infections. 
Nu-3 is a thymidine analogue with butylphosphate blocking groups at both the 3' and 5' 
positions 
.... . 
Nu-3 exhibits effective in vitro spectrum of 
activity at killing 70 different sti·ains of bacteria, including all Catego1y A pathogens and in 
vivo infections caused by Francisella tularensis, Helicobacter pylori, and Pseudomonas 
aeruginosa. 
Diabetic foot infections are one of the most common complications for people who suffer 
from diabetes and a frequent cause of hospitalizationError! Reference source not found .. 
ccording to the American Diabetes Association approximately 8.3% of the US population 
suffers from diabetes and in 2006 there were 65,700 non-ti·aumatic amputations perfo1med on 
diabetics. In addition, almost half of diabetic patients that have an amputation will develop 
an ulcer on the remaining limb within 18 months after surge1y. The ti·eatinent of a diabetic 
foot infection is complicated as it requires both antimicrobial therapy to cure the infection 
and proper wound care management to heal the ulcer. Curing the infection is pa1ticularly 
challenging since the infection tends to be polymicrobial in nature requiring a broad specti11m 
antibiotic, the ulcer is prone to re-infection, and the pathogenic bacteria are increasingly 
becoming resistant to most front line therapies. In addition, recent studies have estimated that 
approximately 60-80% of chronic infections involve biofilm fonnation which makes the 
bacteria more resistant to ti·aditional antibioticsError! Reference source not found.. proper 
anagement of an infected diabetic foot ulcer requires an antimicrobial therapy to cure or clear 
the infection to allow proper wound management or therapy to heal the ulcer and prevent 
relapse. 
6 
RATIONALE 
Nu-3 is a thymidine-based therapeutic compound indicated for the reduction of pathogenic 
bacteria associated with chronic diabetic foot ulcers. The animal data from both rodent and 
swine models of wound injmy and its anti-fungal as well as anti-bacterial activity suggest that 
ti·eatment with Nu-3 may reduce and potentially eliminate pathogenic bacteria in patients 
2 In recent years, the Infectious Diseases Society of America has highlighted a faction of antibiotic-
resistant bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter 
baumannii, Pseudomonas aeruginosa and Enterobacter spp.) -
acronymically dubbed 'the ESKAPE 
pathogens' - capable of 'escaping' the biocidal action of antibiotics and mutually representing new 
paradigms in pathogenesis, transmission and resistance. 
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with chronic diabetic foot ulcers.  The topical route of administration will be the appropriate 
method of delivery for humans.  The low order of toxicity in rodents suggests that Nu-3 has a 
very low risk of toxicity to humans at the proposed doses. The doses selected for evaluation 
are within the FDA guidelines of the recommended safety margin of one tenth of the 
NOAEL.   
 
Prior to determining the efficacy of Nu-3 in patients, safety of Nu-3 related to skin irritation  
was established in male and female healthy volunteers in a Human Repeat Insult Patch Test. 
Briefly, 20 microliters of positive control (0.1% sodium lauryl sulfate), test product (2% Nu-3 
in 0.85% saline), or negative control (0.9% saline) was randomly applied using occlusive 
patches to either abraded or non-abraded (intact) skin for 20 consecutive days on at least 30 
subjects. A total of 35 subjects completed the study and were scored on all six sites.  There 
was a difference among products for both abraded and intact skin sites. The conclusion from 
the study with regards to irritation was as follows: 
 
Abraded Skin: Positive Control > 2% Nu-3 > Negative Saline control 
 
Intact Skin: Positive Control > 2% Nu-3 = Negative Saline Control 
 
7 
OBJECTIVE 
The objective of this study is to evaluate Nu-3 for safety, tolerability, and microbiological 
response to bacteria in patients with chronic diabetic foot ulcer, but excluding any ulcer 
caused by an underlying DUSS Probing to Bone = “Yes”. 
This study will specifically examine the ability of different doses of Nu-3 to eliminate 
pathogenic bacteria in patients with chronic diabetic foot ulcers. Dose and regimen 
information will be obtained for future clinical studies of Nu-3. 
 
7.1 
Primary Objective 
The primary objective of this study is  
 To assess the safety and tolerability of a twice daily, 7-day repeat topical doses of Nu-
3 in adult male and female patients with chronically infected diabetic ulcer. 
 
7.2 
Secondary Objectives  
The secondary objectives of the study are: 
 
To assess the clinical and microbiological response to bisphosphocin™ Nu-3. 
 
To support determination of the appropriate dose range of Nu-3 to be employed in 
future clinical studies. 
 
8 
STUDY DESIGN 
8.1 
Overall Study Design 
This study is a Phase 1/2a prospective, multi-center, blinded, randomized (with respect to 
placebo), dose escalation, twice daily for 7 consecutive days study of Nu-3 solution 
administered topically in patients with chronic diabetic foot ulcer.  
 
8.2 
Number of Centers 
This study will be conducted at two to four investigative site. 


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8.3 
Cohorts 
The dose of Nu-3 administered will be escalated in five planned sequential cohorts, with 
10 subjects in each cohort, to be administered Placebo (0.9% saline) or Nu-3 doses of 0.1% 
solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) (Cohort 1), 1% solution of 
Bisphosphocin™ Nu-3 in 0.9% saline (w/v) (Cohort 2), 2% solution of Bisphosphocin™ Nu-
3 in 0.9% saline (w/v) (Cohort 3), 5% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) 
(Cohort 4), 10% solution of Bisphosphocin™ Nu-3 in 0.9% saline (w/v) (Cohort 5) at twice 
daily for 7 consecutive days.  
 
Additional cohorts may be enrolled, if needed, as provided by protocol. 
 
8.4 
Placebo Control 
In each Cohort, 8 subjects will be randomized to receive Nu-3, and 2 subjects will be 
randomized to receive Placebo. 
 
8.5 
Number of Subjects 
Approximately 50 subjects will be enrolled, of which approximately 40 will be exposed to 
Nu-3. Numbers may vary due to replacement of subjects, addition of cohorts as permitted by 
protocol, or stopping of study. 
 
8.6 
Study Visits  
Subjects will have 5 nominally scheduled study visits:  Screening (Day -7 to -4 hours), Single 
Dose Visit (Day 1), Safety Review and Multiple Dose Visit (Day 2), Safety Review Visit 
(Day 9 ±1 day) and Final Visit (Day 15 ± 2 days).  Additional visits for screening procedures 
and/or follow-up (e.g., in follow-up of Adverse Events) may be necessary.  
 
8.7 
Study Duration 
From consent and screening through final visit, a subject’s duration of participation may vary 
between approximately 2 and 3 weeks. No individual subject will be randomized more than 
once in this study. 
 
8.8 
Dose Selection and Rationale 
While mechanisms of the demonstrated therapeutic effect of Nu-3 in the animal models, and 
of the proposed therapeutic effect in humans, are unknown, structural characteristics of Nu-3 
have not suggested homology with any genetically encoded human biological molecule. 
There is no known suggestion of ligand-receptor specificity, and in particular no suggestion 
of immune system effect at the proposed doses. Preclinical studies have not suggested a 
species specificity for any pharmacodynamic effect.  
 
Accordingly, Nu-3 is not regarded as a higher-risk agent, and a conventional method for 
determination of maximum safe starting dose is considered appropriate for this first-in-human 
study. 
 
8.9 
First Dose Plan 
The dosing schedule for all cohorts is intended to minimize potential risk to subjects’ safety. 
It is known that the types, severities, time courses and pharmacokinetic correlates of adverse 
effects observed in preclinical studies are not necessarily predictive of experience in human 
subjects, at any dose. This plan further recognizes that major acute toxicities of 
pharmaceuticals in human subjects may on rare occasions not be predicted by preclinical 


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experience, warranting additional caution in administering first doses to humans. As such 
caution has been advised in particular regarding biopharmaceuticals, it is noted that Nu-3 is 
not categorized as a biopharmaceutical. A traditional risk-reduction strategy has been to 
apply large safety factors (divisors of human equivalent doses corresponding to NOAELs in 
appropriate animal toxicology models) in determining the Maximum Safe Starting Dose for 
first-in-human study. A further risk-reduction strategy, adopted here, is to separate first and 
second exposures more widely in time than has been typical study procedures.  
 
Preclinical experience suggests that possible adverse effects after a single dose topical 
exposure to NU-3 would be unlikely. Accordingly, the dose plan is as follows: 
 Qualified patients for all Cohorts are screened from Day -7 to -4 hours. Patients 
understand (disclosed in informed consent) that their qualification for enrollment is 
not finalized until the time of enrollment. They understand that their order of 
enrollment is randomly determined.  
 On Day -7 to -4 hours, baseline procedures are performed (demographics and medical 
history, and limited physical examination per protocol, urine pregnancy test (if 
applicable), baseline laboratory tests (blood chemistries, hematology, coagulation 
profile, and urinalysis), and baseline subject disease assessments).  
 On Day 1, the patients is randomized and dosed to either Nu-3 or Placebo. 
 On the next following day, Day 2, if the Investigator observes no dose-limiting 
toxicity (Section 8.10.6) in the patient dosed on Day 1, the Investigator will distribute 
the investigational product kit and instructed to apply the product twice daily for one 
(1) week (7 days). 
 If the Investigator observes a suggestion of dose-limiting toxicity, the procedure 
specified in Section 8.10.3 is followed. 
 
8.10 
Safety Review 
8.10.1 
Safety Review Committee  
Membership of the Safety Review Committee includes the Sponsor’s designated Medical 
Monitor, the Medical Director of the CRO, Medical Director of Lakewood Amedex, and 
other member(s) as deemed appropriate by Sponsor.  
 
8.10.2 
Safety Review Procedure  
Decisions regarding dose escalation and/or modification (including option of next cohort 
repeating at same dose, or doses intermediate to or reduced from those planned per protocol) 
are based upon review by the Safety Review Committee of safety data to the date of the 
Safety Review Visit (including data at least through the Day 8 Safety Review Visit) for the 
current cohort and safety data of the preceding cohorts to the date of the Safety Review Visit.  
 Safety Review meeting may be conducted by teleconference.  
 Decisions of the Safety Review Committee must be unanimous among the members 
participating in the review and are recorded in meeting minutes.  
 Draft minutes of Safety Review Committee meetings are reviewed by all members 
who attended. Proposed changes are reviewed by all members who attended. Final 
draft of minutes is approved by consensus.  
 Individual members’ approvals of final draft minutes are documented to study 
regulatory file.  
 Final draft of minutes is placed in study regulatory file and provided to IRB. 


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8.10.3 
Cohort 1 Day 1a Experience  
If a Subject dosed on Day 1 of Cohort 1 experiences any therapeutic adverse event around the 
wound area: 
 Further dosing of Cohort 1 is suspended. 
 The Safety Review Committee meets to consider available safety data. 
 The Safety Review Committee decides and documents rationale for further course of 
action, which may include resumption of study conduct. 
8.10.4 
Unblinding for Safety Review  
If the Safety Review Committee decides that unblinding of dose randomization is necessary 
for decision regarding continued dosing and/or dose escalation, the unblinding is done in a 
manner that minimizes the extent of unblinding and of its potential effects on assessments of 
adverse events. This action is documented in the minutes of the Safety Review Committee 
meeting. 
8.10.5 
Decision Criteria 
8.10.5.1 
Stopping of Dose Administration and/or Escalation 
Further study drug dose administration or escalation will be stopped: 
1. by Sponsor, for any reason at any time; 
2. by Principal Investigator or Safety Review Committee, if subjects’ safety is 
considered to be at unacceptable risk; 
3. by Safety Review Committee, according to these criteria: 
 If 2 or more of the Nu-3-treated subjects in a cohort experience a DLT, 
dose escalation is stopped; 
 If 1 of the Nu-3-treated subjects experiences a DLT, the cohort is 
expanded to include 4 additional subjects (Nu-3:Placebo ratio of 3:1); 
and 
 If 2 or more of the Nu-3-treated subjects in the expanded cohort (of 
8+4=12 subjects) experience a DLT, dose escalation is stopped;  
 If only 1 of the Nu-3-treated subjects in the expanded cohort 
experiences a DLT (i.e., no additional subjects experience a DLT 
following cohort expansion), stopping of dose escalation is not 
required. 
8.10.5.2 
Definition of Dose-Limiting Toxicity (DLT)  
Dose-Limiting Toxicity in an individual subject is defined as any Grade 2 or higher Adverse 
Event (AE) toxicity according to National Cancer Institute Common Terminology Criteria for 
Adverse Events, Version 3.0 (CTCAE – See Appendix 16.1) that is considered by the 
Investigator to be at least possibly related to study drug.  
 
8.10.5.3 
Definition of Maximum Tolerated Dose (MTD) 
If dose escalation is stopped according to criteria of 8.10.4 and the Safety Review Committee 
does not decide to administer a dose lower than the level at which dose escalation was 
stopped and higher than the next lowest tested level, the next lowest tested level will be 
considered the maximum tolerated dose (MTD).   
 
8.11 
Subsequent Cohort Dose Plan 
Anticipated Dose Plan – Upon completion of Safety Review of experience to date, if the 
Safety Review Committee determines that there is no reason to do otherwise, the dose plan 
for the subsequent cohort is: 


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 Randomization and dosing proceed no sooner than 7 days after the last Day 1 dosing 
of the preceding cohort.  
 Dose of study drug administered to subjects in the next cohort is the next higher dose 
anticipated by protocol (e.g., 1% Nu-3 solution dose cohort after 0.1% Nu-3 solution 
dose cohort). 
 Ten subjects are randomized such that eight dose with Nu-3 and two dose with 
Placebo. 
 A single subject is dosed at a given scheduled time. 
 Dosing of successive subjects are separated in time by an interval that may vary 
between cohorts but must be at least 20 minutes. Safety Review Committee may 
specify this interval. 
 
9 
SELECTION AND WITHDRAWAL OF SUBJECTS 
Subjects with diabetic chronic foot ulcer and with a DUSS Score of 0 to 3, ulcerated area(s) 
of not more than two (2) ulcers between 0.5 to 6 cm2, and no underlying DUSS Probing to 
Bone = “Yes” will be enrolled.  This study will enrol 10 evaluable subjects per group in 5 
groups for a total of 50 evaluable subjects. An evaluable subject is one who has received 
investigational product and has returned for the 15 ± 2 Day follow-up visit. Up to 
approximately 55 patients will be enrolled in order to have a population minimum of 50 
evaluable subjects at the end of the trial. 
 
9.1 
Inclusion and Exclusion of Subjects 
To be included in this study, subjects must meet the following inclusion/exclusion criteria: 
 
9.1.1 
Inclusion Criteria 
1. Men and women between the ages of 18 and 85.  
2. Voluntary written consent, given before performance of any clinical investigation-
related procedure not part of standard medical care, and with the understanding that 
consent may be withdrawn at any time without prejudice to future medical care. 
3. Non-hospitalized ambulatory subjects suffering from Diabetes mellitus, Type I or II 
4. Diabetic foot ulcer(s) with a DUSS Score of 0 to 3 
5. Ulcerated area(s) of not more than two (2) ulcers between 0.5 to 6 cm2  
6. Any female of child bearing age must consent to use medically acceptable birth 
control for the duration of the study 
7. Female subjects must meet at least one of the following additional criteria: 
d. Surgically sterile with bilateral tubal ligation or hysterectomy. 
e. Post-menopausal for at least one year. 
f. If of child-bearing potential, practicing an acceptable method of birth control 
for the duration of the clinical investigation as judged by the Investigator, such 
as condoms, foams, jellies, diaphragm, intrauterine device or abstinence.  
8. Subjects willing to undergo pre-and post-clinical investigation blood collection, 
physical exams and laboratory investigations.. 
9.1.2 
Exclusion Criteria 
1. A DUSS Score above 3. 
2. DUSS Probing to Bone = “Yes”  
3. An ulcer area(s) greater than 6 cm2 or more than two (2) ulcers 


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4. Any subject that has received systemic or topical antibiotics within the last seven (7) 
days 
5. Any subject on topical antimicrobial treatment for their infected diabetic foot ulcer 
whose ulcer is responding to treatment 
6. Any subject that would be unable to follow the protocol procedures, safely monitor 
the infection status at home, and return for schedule visits 
7. Positive pregnancy test at Screening or Visit 2 
8. Active infection as demonstrated by temperature > 37.5 oC and clinical features of 
active infection. 
9. Known immunosuppression or taking immunosuppressive agents including systemic 
steroids.  
10. History of severe co-morbidity with expected patient survival ≤ 6 months.  
11. Pregnancy or lactation 
12. Intake of investigational drugs within 28 days prior to enrollment.  
13. History of concurrent condition that, in the Investigator’s opinion, would jeopardize 
the safety of the subject or compliance with the protocol.  
14. Likely inability to comply with the protocol or cooperate fully with the investigator 
and site personnel.  
15. Unwillingness or language barrier precluding adequate understanding of the trial 
procedure or cooperation with trial site personnel. 
16. Known or suspected active abuse of alcohol, narcotics or non-prescription drugs. 
17. Other planned surgical procedures within 30 days prior to or 30 days post-index 
procedure. 
18. Prior enrolment in this clinical trial 
 
9.2 
Withdrawal of Subjects 
A subject must be withdrawn from the study prior to completion for any of the following 
reasons: 
 Whenever the subject decides that it is in his/her best interest to withdraw 
 Whenever the investigator decides that it is in the subject’s best interest to be 
withdrawn 
 An adverse event or illness which may, in the investigator’s opinion, significantly 
affect the subject or study 
 Non-compliance 
 Sponsor administrative reasons. 
 
Withdrawal of consent for follow-up should be accompanied by documentation of the reason 
for withdrawal. Withdrawal of consent for treatment should be distinguished from withdrawal 
of consent for follow-up visits and from withdrawal of consent for non-patient contact 
follow-up, e.g., medical records checks. Subjects requesting withdrawal should be informed 
that withdrawal of consent for follow-up will jeopardize the public health value of the study. 
 
Subjects who withdraw should be explicitly asked about the contribution of possible adverse 
events to their decision to withdraw consent, and any adverse event information elicited 
should be documented. 
 


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Preferably the subject should withdraw consent in writing and, if the subject or the subject’s 
representative refuses or is physically unavailable, the site should document and sign the 
reason for the subject’s failure to withdraw consent in writing. The informed consent for the 
study should note that although a subject is free to leave the study and stop taking study 
medication, the investigators hope the patient will remain for follow up status evaluations. 
 
Data from subjects who withdraw from the study will still be used in the statistical analysis. 
A decision to replace a withdrawn subject will be made on a case-by-case basis after 
discussions between the investigator, Medical Monitor, and Sponsor.  Generally speaking, a 
subject who does not meet the definition of “evaluable” (i.e., received investigational product 
and has returned for the Day 2 follow-up visit) may be replaced. However, all subjects, 
whether withdrawn or not, will be included in ITT population for safety evaluations of Nu-3. 
 
10 
Investigational Products 
 
10.1 
Study Drug and Placebo 
 
 
Nu-3 
Placebo 
Manufacturer 
Xcelience LLC  
5415 West Laurel Street 
Tampa, Florida 33607 USA 
Route 
Topical 
Formulation 
0.1% Nu-3 in 0.9% saline (w/v) 
1% Nu-3 in 0.9% saline (w/v) 
2% Nu-3 in 0.9% saline (w/v) 
5% Nu-3 in 0.9% saline (w/v) 
10% Nu-3 in 0.9% saline (w/v) 
0.9% sterile saline for injection 
 
All study drug is provided in a 0.5 oz. oval low density ethylene dropper bottle.  The bottle 
opening is plugged with a 13 mm extended controlled dropper tip and sealed with a 15/415 
extend tip closure. 
 
 
10.2 
Dose Schedule 
The anticipated dosing plan is that each subject will receive a single topical administration of 
Nu-3 or Placebo on Day 1 followed by twice daily for 7 day administration of Nu-3 or 
Placebo on Day 2 as outlined in the following table. Actual randomization assignments and 
conduct of study may vary from this, as provided in 8.10 Safety Review.  
 
Cohort 
Nu-3 in 0.9% saline (w/v) 
Total Dosed  
Nu-3 + Placebo 
Dose 1 
0.1% 
Dose 2 
1% 
Dose 3 
2% 
Dose 4 
5% 
Dose 5 
10% 
1 
8 
-- 
-- 
-- 
-- 
10 
2 
-- 
8 
-- 
-- 
-- 
10 
3 
-- 
-- 
8 
-- 
-- 
10 
4 
-- 
-- 
-- 
8 
-- 
10 
5 
-- 
-- 
-- 
 
8 
10 
 
50 
 
 


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10.3 
Drug Shipping, Packaging and Labeling 
Study drug is shipped to the investigative site from
 in standard packing which 
consists of an outer shipping container (20 x 17 x 12.5), an inner container (12 x 12 x 6) and 
two foam inserts each holding a quantity of 25 vials. Ice is packed on the bottom and around 
all three sides of the foam containers. Contents of the label(s) will meet all applicable 
regulatory requirements. 
 
Study drug is packaged in a 0.5 oz. oval low density ethylene dropper bottle plugged with a 
13 mm extended controlled dropper tip and sealed with a 15/415 extend tip closure 
 
The following draft label will be affixed to all containers of Nu-3 drug product, indicating the 
appropriate storage condition. 
 
DRAFT LABEL 
 
GC: X; LID: X; CARTON 
Protocol #: 
LAI2014-1 
Product: 
Bisphosphocin Nu-3 topical antibiotic or placebo 
Bottle Number: 
#### 
Lot Number*: 
14K### 
Directions for Use: 
As directed, apply twice daily to the affected area 
Storage: 
Room Temperature^ 
Caution: 
New Drug-Limited by federal law to Investigational Use 
Keep out of reach of children 
Manufacturer: 
Lakewood Amedex Inc., Sarasota, FL (941)225-2515 
*Lot number represents the group of kits being assembled for each cohort. 
^Room temperature is defined according to the USP as the temperature that is prevailing in a work area. 
 
10.4 
Study Drug Handling and Storage 
10.4.1 
Handling and Storage 
Upon arrival of study drug shipping container at the investigative site, it is immediately 
opened by pharmacy personnel in a secure location, and contents are verified to match the 
shipping documentation. 
 
Study drug is stored at the investigative site in a secure location routinely accessible only to 
Pharmaceutical Services personnel, under these environmental conditions: at ambient 
temperature. 
 
10.4.2 
Study Drug Accountability 
The investigative site maintains records of study drug accountability and reconciliation 
throughout the course of the study. Records show the amount(s) of study drug received from 
Sponsor, administered to subjects, wasted/lost (with description of circumstances), and 
returned to Sponsor and/or Sponsor’s designee. Only subjects enrolled and randomized in this 
study may receive study drug.  


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10.5 Randomization 
Subjects are assigned to dosing with Nu-3 or Placebo according to the randomization 
schedule prepared prior to study start and provided by Sponsor/CRO.  In the event that an 
expanded or unscheduled cohort is added, Sponsor/CRO will provide the randomization 
schedule for that cohort prior to admission of the cohort to the investigative site. The 
randomization scheme was predetermined and ordered in blocks of five (5). Five study kits 
are provided to each investigational site utilized to maintain the randomization scheme. 
 
10.6 
Blinding and Unblinding 
This is a double-blind study. The randomization schedule for each cohort is provided by 
Sponsor/CRO as a sequential list in a sealed envelope. At the investigative site, procedural 
precautions are taken to avoid unblinding due to visual inspection of Nu-3 and/or Placebo 
prepared for dosing. 
 
Unblinding occurs only in the event of an Adverse Event, or pregnancy of a subject’s partner, 
and only if knowledge of the administered study drug is judged likely to affect the medical 
treatment decisions related to the subject or pregnant partner, or if required for decision-
making by the Safety Review Committee. In the event of unblinding, the Investigator notifies 
the Sponsor within 24 hours of breaking the blind and provides formal, written 
documentation to the Sponsor within 5 working days. This documentation includes the 
identity of the individual(s) who have become aware of randomization assignment(s), the 
date on which the blind was broken, and a description of the event motivating the unblinding. 
The Investigator also records in relevant source documents that blind was broken, reason for 
unblinding, and the date/time of that occurrence.  
 
The Safety Review Committee may request unblinding to assist in Safety Review and 
decisions regarding further conduct of study. To the extent possible while meeting the need 
for appropriate Safety Review, the Investigator and other investigative site personnel are not 
unblinded. The existence of unresolved Adverse Events at the time of unblinding is given 
consideration. If an Investigator is unblinded, that Investigator is not subsequently involved 
in the management or assessment of AEs for Subjects whose dose assignments have been 
unblinded.  
 
10.7 
Study Drug Preparation 
Study drug is kept under supervision of pharmacy personnel until delivered to the area 
designated for study drug administration.  
 
10.8 
Study Drug Administration 
 
10.8.1 
Study Drug Administration Procedure 
Licensed Clinical Operations personnel at the investigative site administer each dose of Study 
Drug to study.  Test Product will be administered to the infected diabetic foot ulcer and 
surrounding intact skin at a dose of 3 drops from a dropper bottle per 1 cm2 of ulcer area.  
 
After application of the Test Product is complete, the wound will be left open to air for 5 min 
and then will be dressed as per the following: Owens Gauze will be trimmed to cover the 
entire dimension of the ulcer and overlaid into the ulcer. Gauze dressings (2 x 2 inches) will 
be placed over the Owens Gauze to a depth of 1/4 inch followed by a circumferential wrap of 


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2 inch Kerlex. The dressing will be secured with paper tape applied to the circumferential 
dressing as to not come in contact with the underlying skin. 
 
10.8.2 
Disposition of Dose Administration Materials 
Following study drug administration, materials possibly containing remaining visible study 
drug or Placebo solution are placed in a closed container and returned to the Pharmacy for 
storage at ambient temperature until shipped to the Sponsor. 
 
11 Concomitant Medications and Exposures 
 
11.1 
Prior Medications  
All medications and herbal supplements used by a patient within 14 days prior to screening 
are recorded in the medical history.  Dose, frequency, and purpose of use are recorded, as 
best the volunteer can recall.  
 
11.2 
Concomitant Medications 
The Investigator notifies the Medical Monitor regarding all medications taken by a subject 
during study participation, for treatment of Adverse Events or pre-existing conditions.  
 
12 TREATMENT OF SUBJECTS AND CONDUCT OF STUDY 
Subjects can be screened up to seven days to 4 hours before treatment. Subjects who have 
been found eligible for the study based on the inclusion and exclusion criteria will have the 
study explained in detail. If the subject decides to participate in the study, the subject will be 
given a study specific ICF to sign. Only after the subject undergoes the informed consent 
process and provides a written informed consent, the screening evaluations shall be 
performed. Subjects can be consented at any time once identified by the site study staff as 
potentially eligible. 
If a female of child-bearing potential is found to be pregnant, or is planning to become 
pregnant within one week of administering the study medication, then she is not eligible for 
the study. 
Male or female patients of at least 18 years of age are candidates for this study. If the patient 
may meet entry criteria, the patient will be informed about the study. The investigator should 
also consider the patient’s risk of injury prior to screening. Even after the inclusion and 
exclusion criteria are reviewed, if the investigator considers the patient a risk for injury, the 
patient should not be enrolled in the study. 
 
12.1 
Informed Consent 
All patients are to give informed consent in accordance with the origins of the Declaration of 
Helsinki and applicable regional regulatory requirements (e.g. 21 CFR Part 50).  
The patient will sign the Informed Consent Form before she/he enters the study, i.e., before 
screening bloods, screening assessments or any other study-related activity. The patient will 
be given sufficient time to consider the study’s implications before deciding whether to 
participate. The format and content of the ICF must be agreed upon by Lakewood Amedex 
and the Institutional Review Board. 
Should there be any amendments to the final protocol; the patient must agree to sign the 
amended ICF indicating that they re-consent to participate in the trial. 
 


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12.2 
Visit 1 (Day -7 to -4 hours) - Screening 
After granting of informed consent the patient will undergo screening. Screening includes the 
following: 
 Demography data, medical history, general physical examination of all organ 
systems, and vital signs (blood pressure, heart rate, and temperature): Perform a 
physical exam and assess vital signs per hospital protocol within the specified 
timeframe for screening.  
 Vital signs measurements: Weight, temperature, resting blood pressure, heart rate, 
and respiratory rate  
 Hematology: WBC, RBC, Hgb, Hct, MCV, MCH, HCHC, RDW, MPV, Platelets, 
Neutrophils Auto % (abs), Immature Granulocyte Auto % (abs), Lymphocytes Auto 
% (abs), Monocytes Auto % (abs), Eosinophils Auto % (abs), Basophils Auto % (abs)  
 Biochemistry: Sodium, Potassium, Bicarbonate or CO2, Chloride, Total Bilirubin, 
Creatinine, BUN, Calcium, Alkaline Phosphatase, ALT, AST, Total Protein, 
Albumin, Anion Gap ,eGFR, Globulin, A/G ration 
 For females of child-bearing potential: pregnancy test (urine or serum -HCG) to 
be performed at baseline.  
 Baseline Patient Disease Assessment: Visual examination of chronic ulcer, score 
target ulcer using DUSS Scoring System, Swab wound to culture for microbiological 
examination 
Diabetic Ulcer Wound Scoring System (DUSS) 
Parameter 
Score 
Palpable Pedal Pulses 
Presence = 0 
Absence = 1 
Probing to Bone 
No = 0 
Yes = 1 
Location of Ulcer 
Toe = 0 
Foot = 1 
Number of Ulcerations 
Single = 0 
Multiple = 1 
Score Range  
0 
4 
 
Diabetic Foot Ulcer Wound Infection Score 
 
Parameter 
Wound Infection Score 
0 
1 
2 
3 
Purulent discharge 
Absent 
 
 
Present 
Nonpurulant drainage (serious, 
sanguinous) 
Absent 
Mild 
 
 
Erythema 
None 
Mild pink, 
barely 
perceptible 
Moderate pale 
red, defined 
edges 
Severe red to 
dark red 
Induration 
None 
Mild 
Moderate 
Severe 
Tenderness (sign) 
None 
Mild 
Moderate 
Severe 
Pain (symptoms) 
None 
Mild 
Moderate 
Severe 
Local Warmth (relative to uninfected 
contralateral foot) 
Same 
Mildly 
increased 
Moderately 
increased 
Severely 
increased 


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Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ as described in Appendix A 
 
A past medical/surgical history will be recorded for any conditions or treatments that are, in 
the opinion of the investigator, relevant to the current diagnosis.  The previous condition will 
be recorded as a diagnosis rather than a previous symptom. The time of the previous 
diagnosis relative to the current admission will be provided (i.e., “6 months ago”) to the best 
of the patient’s knowledge. If the diagnosis was more than two years ago, then it can be 
recorded as “more than two years ago”. 
 
Prior medications will be recorded. Prior medications include medications that the patient is 
currently taken or has taken within the past fourteen days. 
 
Physical examinations are performed at screening by the investigator or designee.  The 
screening examinations will include general appearance, height, weight, skin, neck, eyes, 
ears, nose, throat, heart, lungs, abdomen, cervical and axillary lymph nodes, extremities, and 
nervous system. Height is measured in stocking or bare feet, and recorded in centimeters 
(cm). Weight is recorded in kilograms (kg). 
 
Results of these investigations/assessments and baseline measurements obtained from the 
local laboratory will be reviewed by the Investigator prior to the start of the treatment. 
Patients who do not meet study entry criteria will be promptly notified and excluded from 
further participation in the study. Patients who continue to meet entry criteria will be enrolled 
in the study and assigned a patient number by the database. The patient can be randomized at 
any time following the screening.  
 
12.3 Visit 2 (Day 1) SAD Treatment 
Patients will return after the screening visit for Single Ascending Dose Treatment.  Patient 
disease assessments will be performed as specified in the schedule of events table. 
 Vital signs (blood pressure, heart rate, temperature) will be examined within 
10 minutes prior to the start of treatment 
 Baseline Patient Disease Assessment: Visual examination of chronic ulcer, score 
target ulcer using DUSS Scoring System, Swab wound to culture for microbiological 
examination – Not applicable if Visit 2 is the same day as Screening Visit 
 Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ system  - Not applicable if Visit 2 is the same day as Screening 
Visit 
 
In SAD Treatment, eligible patients will be treated with a single application of Test Product.  
Test Product will be administered to the infected diabetic foot ulcer and surrounding intact 
skin at a dose of 1 to 3 drops from a dropper bottle per 1 cm2 of ulcer area, left open to air for 
5 minutes and then dressed using Owens gauge, Kerlex and tape. The ulcer with a non-
abrasive bandage following the initial observation period and the patient will be released with 
verbal instructions to leave the bandage on the wound and return for a follow up visit within 
24 h ± 2 h. 
 
12.4 
Visit 3 (Day 2 [24 h ± 2 h.]) MAD Treatment 
Patients will return after the Single Ascending Dose Treatment. Patient disease assessments 
will be performed as specified in the schedule of events table. 


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 Vital signs (blood pressure, heart rate, temperature) will be examined within 10 
minutes prior to the start of treatment 
 Baseline Patient Disease Assessment: Visual examination of chronic ulcer, score 
target ulcer using DUSS Scoring System, Swab wound to culture for microbiological 
examination 
 Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ System 
The Principal Investigator will visually exam the chronic ulcer to determine if a patient is 
eligible to continue in the study. Eligible patients will be provided with his/her designated 
test product kit and instructed to apply the product twice daily for one (1) week (7 days). Test 
product application will be demonstrated, and patient instruction sheet reviewed, using the 
assigned test product.  The patients will be observed applying the first dose in the clinic to 
ensure compliance (administering Test Product to the infected diabetic foot ulcer and 
surrounding intact skin at a dose of 1 to 3 drops from a dropper bottle per 1 cm2 of ulcer area, 
left open to air for 5 minutes and then dressed using Owens gauge, Kerlex and tape.). The 
patient will be scheduled for the next follow-up visit, with a reminder to bring the test article 
kit to the visit.   
 
12.5 
Visit 4 (Day 9 + 1) Follow-Up to 7 Twice Daily Treatments 
The study patient will return for a follow-up evaluation to the clinical site on Day 9 + 1. At 
this follow-up visit, the patient will undergo the following procedures and evaluations. 
 Vital signs measurements including weight, temperature, resting blood pressure, heart 
rate, and respiratory rate  
 Baseline laboratory tests (blood chemistries and hematology) 
 Visual examination of the ulcer 
 Scoring of the Ulcer(s) using the DUSS and DFI Wound n System 
 Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ System 
 Collection of a sample from the ulcer for microbiological assessment 
 Collection of Subject Home Treatment Log 
 Collection of Investigational Product (IP) 
 
Patients will be given a 7 day supply of non-abrasive bandage and written instructions on the 
proper care and hygiene to include keeping the ulcer clean and bandage dry until the Day 15 
follow up visit. In addition, patients will be told to call if there is any worsening of the ulcer 
with regard to pain, infection, or swelling. 
 
12.6 
Visit 5 (Day 15 ± 1) End of Study (EOS) Visit 
The patient shall attend final end-of-study visit 15 ± 1 days after receiving Test Product.  At 
this follow-up visit, the patient will undergo the following procedures and evaluations. 
 Vital signs measurements including weight, temperature, resting blood pressure, heart 
rate, and respiratory rate  
 Physcial examination  
 Baseline laboratory tests (blood chemistries and hematology) 
 Visual examination of the ulcer 
 Scoring of the Ulcer(s) using the DUSS and DFI Wound Scoring System 
 Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ System 


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 Collection of a sample from the ulcer for microbiological assessment 
 
12.7 
At Every Visit 
 
All adverse events (local and systemic), concurrent procedures, and changes in concomitant 
medications during the study will be recorded on the source documents and case report forms 
(CRFs).  Local reactions to test product administration include but are not limited to pain, 
edema, rash, cellulitis, localized infectious processes, and any systemic reaction including 
fever, allergic reaction, and anaphylaxis. 
 
12.8 
Early Termination Visit 
 
If a subject withdraws prior to completing the study, the reason for withdrawal will be 
documented. If a subject withdraws early due to an adverse event, he/she will be followed 
until resolution/stabilization of the adverse event. 
 
If a subject prematurely withdraws from the study they will be asked to complete the study 
procedures and evaluations performed in the final study visit at the time of withdrawal from 
the study:  
 Vital signs measurements including weight, temperature, resting blood pressure, heart 
rate, and respiratory rate  
 Physcial examination  
 Baseline laboratory tests (blood chemistries and hematology) 
 Visual examination of the ulcer 
 Scoring of the Ulcer(s) using the DUSS System 
 
12.9 
Adverse Event Monitoring 
Subjects will be monitored for any adverse events throughout the screening and until the end-
of-study visit on Day 15. Adverse events will be specifically assessed by observation at Day 
1 following administration of the first dose of investigational product, at Day 2 following the 
second dose administration, at Day 9 visit and at Day 15 visit. Abnormal and clinically 
significant measurements obtained from the local lab will be used to note adverse events. 
 
Vital signs (blood pressure and heart rate) can be monitored per hospital/site protocol 
following administration of the first dose of investigational product, and at Day 2 following 
the second dose administration. Deviations from normal will be recorded as adverse events.  
 
13 ASSESSMENT OF SAFETY 
13.1 Physical Examination 
Physical examinations are performed at screening and at the Day 15 End of Study Visit.  The 
examinations will include general appearance, skin, neck, eyes, ears, nose, throat, heart, 
lungs, abdomen, cervical and axillary lymph nodes, extremities, and nervous system. Height 
is measured in stocking or bare feet, and recorded in inches, at screening. Weight is recorded 
in pounds at screening and Day 15 visit. 
 
13.2 
Vital Signs 
Vital Signs, including one or more of systolic and diastolic blood pressure, pulse, and 
temperature, are recorded at screening, at Day 1 visit, at Day 2 visit, at Day 9 visit, and at the 
Day 15 Final Visit. See Schedule of Events for detail. 


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Consistent BP measurement in one arm is preferred to facilitate comparisons of serial BP 
measurements.  However, optimal performance of other procedures (e.g., blood sampling) 
may require changing BP measurement from one arm to the other.  
 
The investigator or designee will perform a thorough physical examination at the start of the 
study and brief physical examination at 15 days post dose administration. Any changes in the 
overall health and well-being of the subject during the study will be reported as an adverse 
event. If the subject is unable to return for the 15 day visit, then the physical exam is not 
mandatory.  In this case, the subject’s well-being may be assessed by phone. 
An investigator will be in the premises during the investigational product administration and 
on call until the end of the study. 
13.3 Clinical chemistry 
A CMP (biochemistry and hematology) will be performed by the local lab at screening, and 
then repeated at Day 9 and Day 15 after the first dosing. Changes from baseline will be 
assessed as a safety parameter. If a change in a clinical chemistry parameter is observed and 
considered to be clinically significant by the investigator, the observation will be written up 
as an adverse event. 
 
13.4 Adverse events 
Subjects will be monitored for any adverse events throughout the screening and until the end-
of-study visit on day 15. Adverse events will be specifically assessed by observation at Day 1 
following administration of the first dose of investigational product, at Day 2 following the 
second dose administration, at Day 9 visit and at Day 15 visit.  
 
The recording of adverse events is an important aspect of study documentation. It is of utmost 
importance that all staff members at the site involved in the study are familiar with the 
content of this section in order to document all adverse events according to the detailed 
guidelines set out below. During the study where there is safety evaluation, the principal 
investigator or site staff will be responsible for detecting, documenting and reporting AEs and 
SAEs. 
 
An adverse event (AE) is any untoward medical occurrence associated with the use of a drug 
in humans, whether or not considered drug related.  Events that are expected as a result of a 
procedure (other than drug administration of investigational product) that the subject 
undergoes should not be listed as an AE unless the event is more severe than expected or 
deemed significant by the Principal Investigator. 
 
Timely and complete reporting of all AEs assists the medical staff and sponsor in identifying 
any untoward medical occurrence, thereby allowing:  
1. protection of the safety of study subjects;  
2. a greater understanding of the overall safety profile of the test article;  
3. recognition of dose-related test article toxicity; 
4. appropriate modification of study protocols;  
5. improvements in study design or procedures; and  
6. adherence to worldwide regulatory requirements.   
 
Test article is defined as a pharmaceutical form of an active ingredient (or “primary 
operational component” for devices) or vehicle/placebo being tested or used as a reference in 


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the study, whether blinded or unblinded. AEs may be either spontaneously reported or 
elicited during questioning and examination of a subject. All AEs must be completely 
recorded on the within the electronic source document system. If known, the Investigator 
should report the diagnosis of the underlying illness or disorder, rather than its individual 
symptoms. Subjects experiencing AEs that cause interruption or discontinuation of test 
article, or those experiencing AEs that are present at the end of their participation in the study 
will receive follow-up as appropriate. If possible, report the outcome of any AE that caused 
permanent discontinuation or that was present at the end of the study particularly if the AE 
was considered by the investigator to be definitely, probably, or possibly related to test 
article. 
 
The investigator will instruct the subject to report any adverse events that may occur during 
the study. At each visit, the investigator will ask the subject, in non-directive fashion, about 
any change in the subject’s overall condition since the previous visit. 
 
The severity of each adverse event, as judged by the Investigator, will be graded according to 
Common Terminology Criteria for Adverse Events v4.02 (CTCAE). For AEs not listed in the 
CTCAE table, the severity of adverse changes in physical signs or symptoms will be 
classified as follows (consistent with CTCAE): 
 
Grade 1 Mild - asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated. 
Grade 2 Moderate - minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental activities of daily living. 
Grade 3 Severe - medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
activities of daily living. 
Grade 4 Life-threatening - urgent intervention indicated. 
Grade 5 Death - Death related to AE. 
 
The investigator must determine the relationship of the AE to the test article according to the 
following categories: 
 
Definite - An event that follows a reasonable temporal sequence from administration of 
the test article; that follows a known or expected response pattern to the test article; and 
that is confirmed by improvement on stopping or reducing the dosage, and reappearance 
of the event on repeated exposure (re-challenge). 
Probable - An event that follows a reasonable temporal sequence from administration of 
the test article; that follows a known or expected response pattern to the test article; and 
that is confirmed by improvement on stopping or reducing the dosage of the test article; 
and that is unlikely to have been caused by concurrent/underlying illness or other drugs, 
procedures, or other causes. 
Possible - An event that follows a reasonable temporal sequence from administration of 
the test article; that follows a known or expected response pattern to the test article; but 
may have been caused by concurrent/underlying illness, other drug, procedure, or other 
causes. 
Unlikely - An event that does not follow a reasonable temporal sequence from 
administration of the test article; that does not follow a known or expected response 
pattern to the test article, or most likely was caused by concurrent/underlying illness, 
other drug, procedure, or other causes, because of their known effects.  


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Not Related - An event almost certainly caused by concurrent/underlying illness, other 
drug, procedure, or other causes. 
 
An adverse reaction is any AE caused by a drug. Adverse reactions are a subset of all 
suspected adverse reactions for which there is reason to conclude that the drug caused the 
event. For the purposes of prescription drug labelling, the term adverse reaction means an 
undesirable effect, reasonably associated with the use of a drug that may occur as part of its 
pharmacological action or may be unpredictable in its occurrence.  
 
A suspected adverse reaction is any adverse event for which there is a reasonable possibility 
that the drug caused the event.  
 
For the purposes of safety reporting, “reasonable possibility” means there is evidence to 
suggest a causal relationship between the drug and the adverse event. A suspected adverse 
reaction implies a lesser degree of certainty about causality than adverse reaction, which 
means any adverse event caused by a drug.   
 
An event that is a serious adverse event (SAE) must be recorded on the AE eCRF and 
requires expeditious handling to comply with regulatory requirements. 
 
An adverse event or suspected adverse reaction is considered “serious” if, in the opinion of 
either the investigator or sponsor, it results in any of the following outcomes: 
 Death. 
 Life-threatening event. 
 Inpatient hospitalization or prolongation of existing hospitalization.   
 Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
 Congenital anomaly/birth defect. 
 Is an important medical event - defined as a medical event(s) that may not result in 
death, be life-threatening, or require hospitalization but, based upon appropriate 
medical judgment, may jeopardize the patient/subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in the definition above. 
Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do 
not result in inpatient hospitalization, or the development of drug dependency or drug 
abuse.  
 
Events NOT considered to be SAEs are: 
 Hospitalizations for the treatment, which was elective or pre-planned, of a pre-
existing condition that did not worsen. A hospitalization planned after the index 
admission, such as a staged PCI procedure, or a planned prolonged hospitalization, 
such as coronary artery bypass surgery related to the index admission, will not be 
considered an SAE.   
 Treatment on an emergency, outpatient basis, for an event not fulfilling any of the 
definitions of “serious” given above and not resulting in hospital admission. 
 


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The Medical Monitor, Lead Principal Investigator, or Sponsor may be consulted with any 
questions regarding the definition of “planned”. The Medical Monitor may be consulted 
regarding other questions about SAEs. 
 
Adverse events classified as “serious” require expeditious handling and reporting to the 
Medical Monitor, Safety Officer or designee to comply with regulatory requirements. All 
SAEs, whether related or unrelated to test article, must be immediately reported by 
telephone to the Medical Monitor, Safety Officer or, in the event that neither is 
available, to the Project Manager listed on the first page of the protocol. All SAEs 
should be entered in the eCRF as soon as possible. These include those SAEs listed in the 
protocol or Investigator Brochure and must include an assessment of whether there is a 
reasonable possibility that the drug caused the event.   
 
All SAE are required to be captured on the study supplied SAE report form. Please contact 
the Safety Officer at 
 
 
 
 
If only limited information is initially available, follow-up reports are required. In the event 
of death, if an autopsy is performed, a copy of the report should be sent to the Sponsor or 
designee, if available. 
 
As required, the Sponsor or designee will notify participating investigators of all suspected 
adverse reactions that are serious and unexpected. This notification will be in the form of an 
IND safety report of potential serious risks as soon as possible but no later than 15 calendar 
days after the Sponsor determines that the information is “reportable” according to the 
criteria listed in Section 312.32(c)(1)(i) to (iv) of the CFRs. These are: 
 Serious and unexpected suspected adverse reactions, 
 Findings from other studies including epidemiological studies, pooled analyses or 
other clinical studies that suggest a significant risk in humans exposed to the test 
articles, 
 Findings from animal or in vitro tests that suggest a significant risk to humans 
exposed to the test articles, or reports of significant organ toxicity at or near the 
expected human exposure, and 
 Clinically important increases in the rate of occurrence of serious suspected adverse 
reactions. 
 
Upon receiving such notices, the investigator must review and retain the notice with the 
Investigator Brochure and immediately submit a copy of this information to the responsible 
IRB according to local regulations. The investigator and IRB will determine if the ICF 
requires revision. The investigator should also comply with the IRB procedures for reporting 
any other safety information. Where required, submission of safety updates by the 
investigator to Health Authorities should be handled according to local regulations. 
 
All AEs must be recorded on the AE CRF. AEs should be followed to resolution or 
stabilization (if possible), and reported as SAEs if they become serious. AEs will be collected 
starting when the subject signs the ICF and ending with the end-of-study visit on Day 15. For 
subjects who withdraw from the study, the collection of AEs will end 15 days after the first 
dose administration. If any adverse event is present when a subject completes the study or if a 


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subject is withdrawn from the study and the adverse event has still not resolved at this time, 
additional follow-up will be performed, as appropriate. The investigator will continue to 
follow-up all serious adverse events or other adverse events that are considered to be related 
to the investigational products, until it is resolved or assessed to be stable by the Investigator. 
This follow-up may be extended beyond the end of the study period.  An adverse event can 
reach one of the following outcomes: 
 Completely recovered from the AE 
 Recovered with sequelae 
 Ongoing 
 Ongoing at the time of death 
 Death 
 Unknown 
 
14 ASSESSMENT OF EFFICACY 
14.1 Primary Efficacy Endpoint Assessment 
The primary efficacy endpoint is to assess the microbiological response to bisphosphocin™ 
Nu-3 as determined by reduction of pathogenic bacteria following Nu-3 treatment. The 
microbiological assessments from the local lab will be used to measure aerobic and anaerobic 
culture and sensitivity at screening, after a single dose treatment (Day 1), after a second dose 
treatment (Day 2), after twice daily for 7 day treatment, at Day 15.  
 
14.2 Secondary Efficacy Endpoint Assessment 
The primary efficacy endpoint is to assess the clinical response to bisphosphocin™ Nu-3 as 
determined by visual evaluation of the ulcer following Nu-3 treatment.  The measurement of 
this variable will be determined by the DUSS score at screening, after a single dose treatment 
(Day 1), after a second dose treatment (Day 2), after twice daily for 7 day treatment, at Day 
15. 
 
15 PHARMACOKINETIC ASSESSMENT 
There are no pharmacokinetic assessments being conducted in this study. 
 
16 STATISTICAL CONSIDERATIONS 
This study is designed to assess the safety and efficacy of Nu-3. Safety will be assessed by 
monitoring clinical laboratory parameters and treatment emergent adverse events. Efficacy 
will be assessed using endpoints of the clinical and microbiological response to Nu-3   
 
Safety data including laboratory evaluations and vital signs assessments are summarized by 
dose group and time point of collection. Descriptive statistics are calculated for quantitative 
safety data, and frequency counts are compiled for classification of qualitative safety data. In 
addition, a mean change from baseline table is provided for vital signs and a shift table 
describing out of normal range shifts is provided for clinical laboratory results. 
 
For all parameters, a comparison will be made of each active treatment group versus placebo 
using the appropriate statistical test. Unless otherwise indicated, all statistical tests will be 


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two-sided and differences will be considered statistically significant if the associated p-value 
is less than 0.05. All data will be presented in data listings. 
 
A formal statistical analysis plan will be provided in a separate document. The Statistical 
Analysis Plan will contain additional details of the planned analysis along with tables, listings 
and figures that will become part of the Clinical Study Report. The Statistical Analysis Plan 
will be finalized prior to the database lock. 
 
16.1 Justification of Sample Size and Power Analysis 
This is a pilot dose-ranging study.  No sample size calculations were conducted.  
 
16.2 Analysis Populations 
All randomized subject will be accounted for in a listing for subject disposition. All 
randomized subjects will be included in the intent-to-treat (ITT) population. All subjects 
administered any amount of investigational product will be included in the Safety population. 
The per-protocol (PP) population will include all subjects who meet enrollment criteria and 
receive all doses of investigational product as required by the protocol and who have no 
major protocol violations.  Major protocol violations are defined as follows: 
1. Failure to properly obtain consent or subject not consented prior to dosing. 
2. Subject not meeting the entry criteria and enrolled without a written waiver. 
3. Subject randomized in the incorrect treatment arm. 
4. Subject given incorrect dose (overdose/under-dose). 
5. Data not available from the post-dose central laboratory assessment. 
6. Failure to complete the study. 
7. Performing study procedure(s) not described in the approved protocol. 
All safety and efficacy analyses will be performed on all populations.  
 
16.3 Interim Analysis 
A safety review will be conducted after each cohort to confirm proceeding to the next dose 
level.  
 
16.4 Analysis of Baseline Data 
Demographic and baseline characteristics will be summarized using descriptive statistics. 
Continuous variables will be summarized by mean, median, standard deviation (SD), 
minimum and maximum. Categorical variables will be summarized by counts and 
percentages.  
 


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16.5 Analysis of Primary Outcome Measure 
The microbiological response of Nu-3 to eliminate pathogenic bacteria will be determined for 
each group.  Aerobic and anaerobic culture and sensitivity will be assessed at screening, after 
a single dose treatment (Day 1), after a second dose treatment (Day 2), after twice daily for 7 
day treatment, at Day 15. An uncorrected chi-square analysis or a Fisher’s Exact test will be 
used to compare each treatment group to the placebo group. An associated p-value less than 
0.05 will be used to define statistical significance.  An associated p-value less than 0.016 
(0.05 ÷ 5; Bonferroni correction) will be considered highly conclusive. 
 
The clinical response of Nu-3 via visual evaluation of the ulcer will be determined for each 
group.  DUSS Score will be assessed at screening, after a single dose treatment (Day 1), after 
a second dose treatment (Day 2), after twice daily for 7 day treatment, and at Day 15. An 
uncorrected chi-square analysis or a Fisher’s Exact test will be used to compare each 
treatment group to the placebo group. An associated p-value less than 0.05 will be used to 
define statistical significance.  An associated p-value less than 0.016 (0.05 ÷ 5; Bonferroni 
correction) will be considered highly conclusive. 
 
17 REGULATORY AND ADMINISTRATIVE ISSUES 
17.1 
Subject Information and Informed Consent 
The risks and benefits of participating in the study, including their right to withdraw at any 
time, will be verbally explained to each potential subject prior to entering the study. Prior to 
any screening tests or procedures for the study, the subject must sign and date the IRB/IEC 
approved informed consent form (which also defines the risk and benefits of participating in 
the study).  
 
The investigator shall seek consent only under circumstances that provide the subject 
sufficient opportunity to consider whether or not to participate and that minimize the 
possibility of coercion or undue influence. The information that is given to the subject shall 
be in language understandable to the subject. No informed consent may include any 
exculpatory language through which the subject is made to waive or appear to waive any of 
their legal rights, or releases or appears to release the investigator, Lakewood Amedex, the 
institution, or its agents from liability for negligence. The subject, or legal guardian, must be 
able to comprehend the informed consent form and sign prior to subject enrollment. 
 
The study sponsor must agree with the final IRB/IEC-approved consent form prior to 
initiation of the study. The original signed consent forms will be retained by the Investigator 
and a copy provided to each subject.  
 
If any significant new information develops during the conduct of the study which may affect 
the subject’s willingness to continue participation, written consent for continued participation 
in the study must be obtained from each subject. 
 
17.2 
Pre-study Documentation 
The following documentation must be received by the Sponsor prior to the initiation of the 
trial: 
 Qualified Investigator Study Undertaking form/Statement of Investigator (FDA Form 
1572 or QIU (Canada)) 
 Certification of Financial Disclosure 


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 Curricula vitae and current medical licenses of the Investigator and all Sub-
Investigators 
 Completed “Investigator Approval” 
 Written documentation of Institutional Review Board (IRB)/Independent Ethics 
Committee (IEC) approval for the protocol and informed consent form, signed by the 
IRB/IEC chairperson or designee. The protocol and the informed consent form should 
be clearly identified by protocol number or title and date of approval. 
 Copy of the informed consent form that was reviewed and approved by the IRB/IEC 
 Current normal laboratory values for all tests required in the protocol and current 
laboratory certification 
 
17.3 
Independent/ Institutional Ethics Committee Approval  
The study protocol, the Investigator's Brochure, the informed consent form, advertising and 
subject recruitment procedures must be approved by an appropriately comprised IRB/IEC 
according to the Declaration of Helsinki, ICH Guidelines and other applicable regulations 
before the study is initiated at the investigator site.   
 
Any protocol modifications or revisions to the informed consent form require prior written 
approval by Lakewood Amedex. No deviations from or changes to protocol or informed 
consent form can be initiated without prior IRB/IEC approval of the appropriate amendment, 
except when necessary to eliminate immediate harm to the subjects or when the change(s) 
involves only logistical or administrative aspects of the study.  
 
The Investigator is required to report the following to the IRB/IEC to the extent necessary 
under local requirements: 
 Periodic reports on the progress of the study 
 Deviations from, or changes to, the protocol to eliminate immediate harm to the study 
subjects 
 Change that increases the risk to the subject or significantly affects the conduct of the 
study 
 All serious adverse events 
 New information that may adversely affect the safety of the subjects or the conduct of 
the study 
 Final study report 
 
In addition, updates to the Investigator’s Brochure during the study must be submitted to the 
IRB/IEC for approval or acknowledgement (as appropriate). 
 
17.4 
Ethical Conduct of the Study 
The study will be conducted in accordance with the ethical principles with origin in the 
Declaration of Helsinki and are consistent with Good Clinical Practice, applicable regulatory 
requirements, protocol, in accordance with the sponsor and CRO’s standard operating 
procedures (SOPs). Any significant deviation from the protocol must be reported 
immediately to the Study Medical Monitor. 


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Subject identity will be kept confidential and to the extent permitted by applicable laws and 
regulations will not be made publicly available. If the results of the study are published, 
subject identity will remain confidential. 
 
 
18 DATA RECORDING AND COLLECTION 
18.1 Source Documents 
Source documents are records maintained at the investigator site through the electronic 
source documentation SureSource™ Source documents include signed informed consent 
forms, written progress notes, laboratory reports, etc. All source documents and subject 
assessments must be maintained at the investigator site. The site staff will use caution to 
avoid any unblinding through normal reading lab reports, clinical notes, pharmacy reports, 
etc. 
 
Any corrections should be made by a single line drawn through the entry, adding the correct 
information, initialing and dating by the person making the change, and preferably indicating 
why the change was required. 
 
18.2 
Case Report Forms (CRFs) 
Case Report Forms will not be utilized for LAI2014-1. All study information will be captured 
directly in the electronic source documentation system SureSource™. The Principal 
Investigator is responsible for all information collected on subjects enrolled at their 
investigational site. All data collected during the course of this study must be reviewed and 
verified for completeness and accuracy by the investigator.  A copy of each subjects enrolled 
at a specific investigational site will be provided to the Principal Investigator at the 
completion of the study. 
 
19 MONITORING, AUDIT, RETENTION AND CONFIDENTIALITY OF STUDY 
RECORDS  
The Investigator is required to prepare and maintain adequate individual eCRFs provided for 
each subject and accurate case histories (e.g., medical records) designed to record all 
observations and other data pertinent to the study for each study participant. This also 
includes accurate documentation of investigational product accountability. 
 
All case report forms (eCRFs) will be kept up to date and the Principal Investigator will be 
responsible for reviewing each subject’s completed eCRFs. These will be reviewed by the 
sponsor/CRO at regular intervals to ensure protocol compliance, compare eCRFs with 
original source documents for accuracy and completeness of data recording, review study 
logs to assess drug accountability, and ensure that the study is being conducted according to 
pertinent regulatory requirements. Periodically, the facilities used in the study may be 
reviewed. The review of the subject’s medical records will be performed in a manner that 
subject confidentiality is maintained. 
 
In addition, a study center may be audited in depth for study quality assurance by Lakewood 
Amedex, a CRO or an external auditor on behalf of Lakewood Amedex, or inspected by a 
national regulatory authority (such as the FDA or its equivalent) with or without prior 
notification, at random or for cause. The audit may include review of all source documents, 


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drug records, original clinic notes, some or all of the facilities, etc. Direct access to original 
source data will be required for inspections/audits, which will be carried out giving due 
consideration to data protection and subject confidentiality. 
 
The Investigator must arrange for retention of study documents according to specific country 
requirements/regulations, after the last approval of a marketing application and until there are 
no pending or contemplated marketing applications in the ICH region or at least two years 
have elapsed since the formal discontinuation of clinical development of the investigational 
product. These documents may be retained for a longer period if required by the applicable 
regulatory requirements or by an agreement with Lakewood Amedex. It is Lakewood 
Amedex’s responsibility to inform Investigator/Institution as to when these documents no 
longer need to be retained. Regardless of the above time frame, destruction of study records 
at any time requires confirmation by Lakewood Amedex. Lakewood Amedex shall retain 
ownership of all CRFs, data analyses, and reports that result from this study. 
 
Monitors, auditors and other authorized agents of Lakewood Amedex, the CRO, the IRB/IEC 
approving this research, the FDA, and others (as applicable) will be granted direct access to 
the study subjects’ original medical records for verification of clinical study procedure and 
data, without violating the confidentiality of the subjects, to the extent permitted by the law 
and regulations. Any presentations of the results of the study at meetings or in publications 
will not contain subject names. 
 
 
20 STUDY TERMINATION 
The study may be terminated by the investigator or the Sponsor. If, in the opinion of the 
investigator, clinical observations made during the study suggest that it may be unwise to 
continue, he or she may stop the study.  A study termination by the investigator will be 
reported to the sponsor. 
 
In addition, a written statement fully documenting the reasons for this action will be 
submitted to the Sponsor by the Investigator within five working days. 
 
In the event that the Sponsor chooses to discontinue or terminate the study, appropriate 
notification will be given to the Investigator. 
 
 
21 CONFIDENTIALITY AND PUBLICATION POLICY 
The investigator agrees, by signing the protocol, to keep all information provided by 
Lakewood Amedex in strict confidence and to request similar confidentiality from his/her 
staff and the IRB/IEC. Study documents provided by Lakewood Amedex (such as protocol, 
Investigator’s Brochures, CRFs, etc.) will be stored appropriately to ensure confidentiality. 
The information provided to the investigator may not be disclosed to others without direct 
written authorization from Lakewood Amedex, except to the extent required by local 
regulations and necessary to obtain informed consent from subjects who wish to participate in 
the study.   
 
Investigators may publish results generated from this study provided the publication is 
reviewed and approved by Lakewood Amedex and a copy of the proposed oral or written 
publication is submitted to Lakewood Amedex for review at least 60 days prior to submission 


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for publication or presentation.  Lakewood Amedex will respond to the investigator within 30 
days with any changes or deletions in technical or confidential information, and investigator 
agrees to make such changes or deletions prior to publication or presentation.  
 
No patent application based on the results of the study may be made by the Investigator nor 
may assistance be given to any third party to make such an application without the written 
authorization of the Study Sponsor. 
 
 
22 REFERENCES 
ICH E6 Guidelines for Good Clinical practice 
ICH E2A Clinical Safety Data Management: Definitions and Standards for Expedited Reporting 
 
 


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23 APPENDICES 
 


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Appendix A:  Use of the Aranz Medical Silhouette™ System for Photo documentation 
of the DFU and Calculation of Area and Depth, and Volume. 
 
Capturing a Wound Image 
 
1. Log in to Silhouette Connect and Select the Patient to be imaged. 
2. Review the patient details and select Capture Images Button.  The laser lines should 
come on from the Silhouette Star Camera. 
3. To capture the wound image position the Silhouette Star Camera directly above the 
wound and move the camera up or down so that the laser lines form a star.  The center 
of the laser line star should be over the deepest part of the wound. 
4. Capture an image of the wound by pressing the bottom on the camera.   
5. Repeat the process to capture multiple images. 
6. All images will be uploaded to the Silhouette Connect Database. 
 
Calculating the Ulcer Area, Volume, and Depth 
 
1. Select the Ulcer image to be used for assessment on the tablet. 
2. Enlarge the image so the perimeter of the ulcer is easily visible 
3. Outline the perimeter of the ulcer by either clicking around the perimeter so that the 
entire ulcer is surrounded by dots or tracing the perimeter of the ulcer with a pointer 
or finger.   
4. Clicking on the circular target to initiate calculation of the area, volume, and depth of 
the ulcer by the SilhouetteConnect software. 
5. Click on the Notes(?) button to enter comments on the ulcer margins, appearance, and 
surrounding skin 
6. Click Generate pdf report and Save to Store it to the database. 
7. Upload .pdf report to the SureSource™ Database. 
 
 


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Appendix B: Subject Home Treatment Log:  
 
Subject ID No.: 
 
 
 
 
 
 
 
 
 
Study Drug Number: 
 
 
 
 
 
 
 
Issue Date: 
 
 
 
 
 
Return Date:  
 
 
 
 
Instructions:  Subjects/caregivers should log both the date and approximate time of each 
treatment and the person administering the study drug should initial in the corresponding box.  
A space is also provided for the subject/caregiver to write any comments concerning the 
administration or observations of the ulcer. 
 
Day 
Treatment 
1 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Treatment 
2 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Comments 
1 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
2 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
3 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
4 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
5 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
6 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
7 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
☐   Yes        
☐   No        
☐1-4 hrs.      
☐ 5-8 hrs.     
☐ 9-12 hrs.   
 
 
 


Study Title: 
Sponsor: 
IND No.: 
EudraCT Number 
Indication: 
Protocol ID: 
Protocol Coordinator: 
Medical Monitor 
Protocol Version/Date: 
LAKE,WOOD 
AME DEX 
CLINICAL STUDY PROTOCOL 
A Phase I/Ila, Randomized Double Blind, Placebo-Controlled, Dose 
Escalating Study to Evaluate the Safety and Tolerability of Topically 
Applied Bisphosphocin Nu-3 on Infected Diabetic Ulcers of Subjects 
With Type I or II Diabetes Mellitus 
Lakewood-Amedex, Inc. 
3030 University Pkwy 
Sarasota, FL 34243 
Not Applicable 
Diabetic Foot Infections 
LAI2014-1 ----
V2.0 
24 September 2015 
CONFIDENTIALITY STATEMENT 
The information contained in this document, particularly unpublished data, is the property or under control of 
Lakewood-Arnedex, and is provided to you in confidence as an investigator, potential investigator, or consultant, for 
review by you, your staff, and an applicable Institutional Review Board or Independent Ethics Committee. The 
information is only to be used by you in connection with authorized clinical studies of the investigational drng 
described in the protocol. You will not disclose any of the infonnation to others without written authorization from 
Lakewood-Arnedex, Inc., except to the extent necessary to obtain infonned consent from those persons to whom the 
drug may be administered. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Revised – 24SEP2015 
 
Page 2 
 
 
 
SIGNATURE PAGE 
 
 
 
 
 
 
 
Lakewood-Amedex Bisphosphocin NU-3 Clinical Protocol 
 
 
A PHASE I/IIa, RANDOMIZED DOUBLE BLIND, PLACEBO CONTROLLED, DOSE 
ESCALATING STUDY TO EVALUATE THE SAFETY AND TOLERABILITY OF 
TOPICALLY APPLIED BISPHOSPHOCIN™ NU-3 ON INFECTED DIABETIC 
ULCERS OF SUBJECTS WITH TYPE I OR II DIABETES MELLITUS 
 
 
Version 2.0 
 
24 September 2015 
 
 
 
Approved by: 
 
 
 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Revised – 24SEP2015 
 
Page 3 
 
STUDY ACKNOWLEDGEMENT 
 
A  Phase I/IIa, Randomized Double Blind, Placebo-Controlled, Dose Escalating Study to 
Evaluate the Safety and Tolerability of Topically Applied Bisphosphocin™ Nu-3 on Infected 
Diabetic Ulcers of Subjects With Type I or II Diabetes Mellitus 
Version 2.0:  24 September 2015 
 
INVESTIGATOR STATEMENT 
 
I have read the protocol and Investigator’s Brochure, including all appendices, and I agree that it 
contains all necessary details for me and my staff to conduct this study as described.  I will 
conduct this study as outlined herein and will make a reasonable effort to complete the study 
within the time designated. 
I will provide all study personnel under my supervision copies of the protocol and access to all 
information provided by Lakewood-Amedex.  I will discuss this material with them to ensure 
that they are fully informed about the drugs and the study. 
 
 
 
 
 
 
Principal Investigator Name (Printed) 
 
Signature 
 
 
 
Date 
 
 
 
 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Revised – 24SEP2015 
 
Page 4 
 
GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
 C  
degrees Celsius 
 F 
degrees Fahrenheit 
AE 
Adverse Event/Adverse Experience 
ALT 
Alanine Aminotransferase 
API 
Active Pharmaceutical Ingredient 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Unit 
CLSI 
Clinical and Laboratory Standards Institute 
CMP 
Comprehensive Metabolic Panel 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTCAE 
Common Terminology Criteria for Adverse Events v4.0 
DFI 
Diabetic Foot Infection 
DSMB 
Data and Safety Monitoring Board 
DUSS 
Diabetic Ulcer Severity Score 
ELSD 
Evaporative Light Scatter Detection 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
hCG 
Human Chorionic Gonadotropin 
HCV 
Hepatitis C Virus 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HPLC 
High Performance Liquid Chromatography 
IB 
Investigator’s Brochure 
ICH 
International Committee on Harmonization 
ICMJE 
International Committee of Medical Journal Editors 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IR 
Infrared Spectroscopy 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
LAI 
Lakewood-Amedex, Inc 
LCMS 
Liquid Chromatography Mass Spectrometry 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Revised – 24SEP2015 
 
Page 5 
 
LLN 
Lower Limit of Normal 
MAD 
Multiple Ascending Dose 
MBC 
Minimum Bactericidal Concentration 
MIC 
Minimum Inhibitory Concentration 
MOA 
Mechanism of Action 
MRSA 
Methicillin Resistant Staphylococcus aureus 
MS 
Mass Spectroscopy 
NDM-1 
New Delhi Metallo-Beta Lactamase-1 
NMR 
Nuclear Magnetic Resonance 
PBS 
Phosphate Buffered Saline 
PI 
Principal Investigator 
RBCs 
Red Blood Cells 
SAD 
Single Ascending Dose 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
TS 
Trypic Soy 
TSBD 
Trypic Soy Broth with 1% Dextrose 
ULN 
Upper Limit of Normal 
US 
United States 
WBC 
White Blood Cell 
 
 


Bisphosphocin NU-3 
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Page 6 
 
TABLE OF CONTENTS 
TITLE PAGE ................................................................................................................................................................. 1 
SIGNATURE PAGE ..................................................................................................................................................... 2 
STUDY ACKNOWLEDGEMENT ............................................................................................................................... 3 
GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS ...................................................................... 4 
TABLE OF CONTENTS .............................................................................................................................................. 6 
PROTOCOL SUMMARY............................................................................................................................................. 9 
1 
INTRODUCTION .............................................................................................................................................. 11 
1.1 
Background Information ............................................................................................................................ 11 
1.2 
Bisphosphocin Nu-3 .................................................................................................................................. 13 
1.2.1 
General Information .................................................................................................................. 13 
1.2.2 
Pre-Clinical Pharmacology and Toxicology .............................................................................. 14 
1.2.3 
Clinical Trials of Bisphosphocin Nu-3 ...................................................................................... 21 
1.3 
Rationale for Current Study ....................................................................................................................... 21 
2 
OBJECTIVES ..................................................................................................................................................... 23 
3 
STUDY DESIGN ................................................................................................................................................ 25 
3.1 
Selection and Exclusion of Subjects .......................................................................................................... 25 
3.2 
Inclusion Criteria ....................................................................................................................................... 26 
3.3 
Exclusion Criteria ...................................................................................................................................... 26 
4 
STUDY DRUGS ................................................................................................................................................. 27 
4.1 
Randomization, Blinding, and Drug Product/Placebo Packaging and Labeling ........................................ 27 
4.2 
Description and Handling of Bisphosphocin Nu-3 .................................................................................... 27 
4.3 
Dosage and Administration of Bisphosphocin Nu-3 or Placebo ................................................................ 28 
4.4 
Prior and Concomitant Medications .......................................................................................................... 28 
5 
STUDY PROCEDURES .................................................................................................................................... 29 
5.1 
Subject Enrollment and Treatment Assignment ........................................................................................ 29 
5.2 
Pre-treatment Assessments ........................................................................................................................ 29 
5.2.1 
Screening Visits ......................................................................................................................... 29 
5.3 
Enrollment/Baseline in Single Ascending Dose Arm (SAD) .................................................................... 30 
5.4 
Follow-up Single Ascending Dose Arm .................................................................................................... 31 
5.5 
Enrollment/Baseline in Multiple Ascending Dose (MAD) Arm ............................................................... 32 
5.6 
Subjects will be provided a Subject Home Treatment Log (Appendix B) and instructed in the proper 
method of documenting the timing of treatments and recording of any observations. .............................. 32 
5.7 
Final Study Visit – Visit 5 (7-Day MAD) ................................................................................................. 33 
5.8 
Early Termination Visit ............................................................................................................................. 33 
5.9 
Unscheduled Visit ...................................................................................................................................... 34 
6 
ADVERSE EVENTS AND TOXICITY MANAGEMENT ............................................................................... 35 
6.1 
Specification of Safety Parameters ............................................................................................................ 35 
6.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters .................................. 35 
6.2.1 
Adverse Events .......................................................................................................................... 35 
6.2.2 
Reactogenicity ........................................................................................................................... 36 
6.2.3 
Serious Adverse Events ............................................................................................................. 37 
6.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or Abnormal 
Clinical Findings ........................................................................................................................ 38 


Bisphosphocin NU-3 
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Revised – 24SEP2015 
 
Page 7 
 
6.3 
Serious Advent Event Reporting Procedures ............................................................................................. 38 
6.3.1 
Reporting Pregnancy ................................................................................................................. 39 
6.4 
Safety Oversight ........................................................................................................................................ 40 
6.4.1 
Independent Safety Monitor Committee .................................................................................... 40 
6.4.2 
Study Termination ..................................................................................................................... 41 
6.4.3 
Removal of Subjects from the Trial or Study Drug ................................................................... 42 
6.4.4 
Handling Withdrawals ............................................................................................................... 42 
7 
STATISTICAL CONSIDERATIONS ................................................................................................................ 43 
7.1 
Study Hypothesis ....................................................................................................................................... 43 
7.2 
Sample Size Considerations ....................................................................................................................... 43 
7.3 
Planned Interim Analysis ........................................................................................................................... 43 
7.3.1 
Safety Review ............................................................................................................................ 44 
7.4 
Statistical Analysis Overview .................................................................................................................... 44 
8 
RESPONSIBILITIES.......................................................................................................................................... 45 
8.1 
Investigator Responsibilities ...................................................................................................................... 45 
8.1.1 
Good Clinical Practice ............................................................................................................... 45 
8.1.2 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) Approval.................. 45 
8.1.3 
Informed Consent ...................................................................................................................... 46 
8.1.4 
Confidentiality ........................................................................................................................... 46 
8.1.5 
Study Files and Retention of Records ........................................................................................ 46 
8.1.6 
Case Report Forms .................................................................................................................... 47 
8.1.7 
Drug Accountability .................................................................................................................. 48 
8.1.8 
Inspections ................................................................................................................................. 48 
8.1.9 
Protocol Compliance ................................................................................................................. 48 
8.2 
Sponsor Responsibilities ............................................................................................................................ 49 
8.2.1 
Protocol Modifications .............................................................................................................. 49 
8.2.2 
Study Report and Publications ................................................................................................... 49 
8.3 
Joint Investigator/Sponsor Responsibilities ............................................................................................... 50 
8.3.1 
Access to Information for Monitoring ....................................................................................... 50 
8.3.2 
Access to Information for Auditing or Inspections .................................................................... 50 
8.3.3 
Study Discontinuation ............................................................................................................... 50 
9 
REFERENCES ................................................................................................................................................... 51 
10 
APPENDICES .................................................................................................................................................... 53 
Appendix A. 
Project Timeline and Flow Chart ......................................................................................... 54 
Appendix B:  
Schedule of Events ............................................................................................................... 56 
Appendix C:  
Use of the Aranz Medical Silhouette™ System for Photo documentation of the DFU 
and Calculation of Area and Depth, and Volume. ................................................................ 57 
Appendix D: 
Subject Home Treatment Log: ............................................................................................. 58 
 
 
 


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LIST OF IN-TEXT TABLES 
 
Table 1. 
Bisphosphocin Nu-3 Activity in a Minimum Inhibitory Assay according to CLSI 
Guidelines ............................................................................................................................ 14 
Table 2. 
Anti-fungal Activity of Bisphosphocin Nu-3 ....................................................................... 15 
Table 3. 
Bactericidal activity of Nu-3 against bacteria established in biofilm ................................... 16 
Table 4.   
Time and concentration dependent activity of Nu-3 against biofilm forming bacterial 
strains. .................................................................................................................................. 17 
Table 5. 
Log reduction in bacterial cell counts in surgical wound model .......................................... 18 
Table 6. 
Mouse Superficial Skin Polymicrobial Infection Bacterial Counts ...................................... 19 
Table 7. 
Summary of Nu-3 Preclinical Toxicology Data ................................................................... 20 
Table 8. 
Product Label Specifications ................................................................................................ 27 
 
 
 
LIST OF IN-TEXT FIGURES 
 
Figure 1. 
Structure, Chemical Name and Chemical Formula of Bisphosphocin™ Nu-3 .................... 13 
 
 


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PROTOCOL SUMMARY 
Title: 
A  Phase I/IIa, Randomized Double Blind, Placebo-Controlled, 
Dose Escalating Study to Evaluate the Safety and Tolerability of 
Topically Applied Bisphosphocin™ Nu-3 on Infected Diabetic 
Ulcers of Subjects With Type I or II Diabetes Mellitus 
Phase: 
I/IIa 
Population: 
Up to 30 subjects over 18 years of age suffering from diabetes 
mellitus and a chronic infected diabetic ulcer(s). 
Number of Sites: 
2 
Study Duration: 
6 months 
Subject Participation Duration: 
44 days (includes screening visit, study treatment and all follow-ups 
visits through Day 15 post treatment. 
Description of Agent or 
Intervention: 
Bisphosphocin™ Nu-3 to be applied as a topical solution to the 
ulcer and covered by a bandage.  This study includes three dose 
ascending cohorts.  Within each cohort there are two phases with 
both a single ascending dose and multiple ascending dose arms. The 
three dose levels (cohorts) are 0.1% (1mg/ml, anticipated low dose), 
1% (10mg/ml, anticipated therapeutic dose, and 2% (20mg/ml, 
anticipated high dose). 
Objectives: 
Primary:  To assess the safety and tolerability of escalating doses 
of topically applied bisphosphocin™ Nu-3, 
 
, to an open infected ulcer.  Assessment of safety will be 
determined by visual evaluation of the ulcer, vital signs, CBC 
analysis, CMP analysis, and physical examination, as well as the 
incidence and severity of emergent adverse events that occur during 
the study participation.  The study will be monitored by the Safety 
Monitoring Committee (SMC). 
Secondary: To assess the clinical and microbiological response to 
bisphosphocin™ Nu-3 administered topically to the ulcer, including 
the improvement of wound appearance and elimination of the 
pathogenic bacteria.   
Estimated Time to Complete 
Enrollment: 
24 Weeks 


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Study Design: 
Allocation: Randomized, Placebo-Controlled 
Endpoint: Safety and Tolerability 
Enrollment Model: Sequential Assignment 
Masking: Double Blind (Subject, Caregiver, Investigator) 
Primary Purpose: Safety 
Description of Study Design 
Phase I/IIa, three cohort ascending dose with two dosing arms per 
cohort, study in Type I or II diabetes mellitus subjects with a 
chronic infected diabetic ulcer defined as having a DUSS score of 
0 to 3 and DFI wound score of 1 to 9.  The study proposes to use 
the same subject in SAD Arm 1 and MAD Arm 2 of each cohort 
The study is designed to run the cohorts in series with the 
completion of the first cohort before initiating the next dosing level.  
At all study visits the ulcer will be visually examined for any 
changes and photographed using the Aranz Medical Silhouette™ 
system that will calculate area and depth of the ulcer. 
In Arm 1, eligible subjects will be treated with a single application 
of Nu-3 or placebo in 4 to 1 ratio to judge the initial safety of Nu-3 
over a brief one (1) hour interval and 24-hr interval post 
application. Bisphosphocin Nu-3 will be applied topically to the 
chronic infected ulcer, covered with a non-abrasive bandage 
following the initial observation period.  The subject will be 
released with verbal instructions to leave the bandage on the wound 
and return for a follow up visit within 24h ± 2h.  At the follow up 
visit, the bandage will be removed, the ulcer visually examined and 
the subject cleared for the MAD Arm 2 based on the 
recommendation of the PI and absence of any SAEs.   
In Arm 2, eligible subjects which are those who have been 
approved by the PI after the Visit 2 examination will be instructed 
in the proper application of bisphosphocin Nu-3.  The subjects will 
be observed applying the first dose in the clinic to ensure 
compliance. Subjects will then be given a 7 day supply and sent 
home to continue treatment.  Visit 4 or earlier in the case of any 
adverse events, subjects will return to the clinic for an examination, 
including visual examination of the ulcer, vital signs, adverse 
events, photo documentation, collection of a sample for 
microbiology and concomitant medication use.  A final follow up 
visit will be scheduled +7 days after last dose of study medication 
(Day 15) for a complete examination as described above. 


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1 
INTRODUCTION 
1.1 
Background Information 
Infectious disease represents a growing threat to world health due to the emergence of new 
viruses and the increase in bacterial resistance to antibiotics1,2.  It is now estimated that almost 
one-third of the world population or 2 billion people are infected with Mycobacterium 
tuberculosis with between 3-5% of these cases being multidrug or extremely drug resistant3.  In 
addition, bacteria have repeatedly demonstrated the ability to become resistant to antibiotics 
much faster than new compounds or sub-classes can be identified.  The NDM-1 resistance gene 
represents a perfect example of the ability of bacteria to not only acquire resistance but broad 
spectrum resistance on a single plasmid that provides resistance against most of the major or 
widely used classes of antibiotics.  This highlights the shortcomings of current and past antibiotic 
research which has relied on derivatives of existing classes that all share a common or similar 
core structure making them susceptible to inactivation by a single enzyme.  In addition, 
traditional antibiotics share a related mechanism of action in that they all function through a 
target protein or receptor molecule to inhibit a cellular process to kill the bacteria allowing the 
bacteria to rapidly become resistant by acquiring a beneficial mutation in the target molecule4.   
Over the past ten years, the amount of research and development invested by large 
pharmaceutical companies in the discovery of novel antibiotics has steadily declined while 
bacteria have become increasingly resistant to the currently available classes of antibiotics.  
According to the Centers for Disease Control and Prevention, approximately 2 million people 
each year are infected with bacteria resistant to at least one antibiotic which results in around 
23,000 deaths5.  Methicillin resistant Staphylococcus aureus (MRSA) has become a major public 
health issue resulting in the closing of several public schools for cleaning and demonstrating that 
this is a problem no longer restricted to hospitals.  No other factor highlights the need for a 
greater effort into the research and development of novel anti-bacterial compounds than the ever 
increasing ability of bacteria to rapidly acquire resistance to current and new derivatives of 
existing antibiotics.  The recent identification of a strain of Streptococcus resistant to more than 
18 different antibiotics highlights this fact and the obvious need for novel anti-infective 
compounds like Bisphosphocins.   
Lakewood Amedex has discovered a new class of broad spectrum antimicrobial, termed 
bisphosphocins™, formerly known as nubiotics, that have been proven effective in vitro at 
killing 70 different strains of bacteria, including all Category A pathogens and in vivo against the 
difficult to treat infections caused by Francisella tularensis, Helicobacter pylori, and 
Pseudomonas aeruginosa.  Bisphosphocins are a new class of synthetic broad spectrum 
antimicrobial that are characterized by a core structure bridging two phosphate groups attached 
to even chain alkyl or alcohol groups.  The compounds are activated through an acidification 
process which is reversible by exposing the compounds to a buffer or base.  The compounds 
were found to be bactericidal and effective in killing slow growing or stationary bacteria.  
Preclinical studies have demonstrated the effectiveness of these compounds in the treatment of a 
number of serious bacterial infections6,7,8.  In addition, the compounds were initially developed 
as a homeopathic treatment and were found to reduce the severity of several common infection 


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associated with acne, diabetic foot infection, and skin infections9.  The compounds function 
through a unique mechanism of action, are amendable to multiple formulations, and routes of 
administrations enabling a clinical development plan distinct from most traditional antibiotics.  
The current clinical development plan entails development of a topical formulation of 
bisphosphocin™ Nu-3 for a number of topical infections such as diabetic foot infections, chronic 
urinary tract infections, and pulmonary infections using direct delivery of the compound to the 
site of the infection.  This clinical approach will demonstrate initial safety in humans and keep 
delivery of the antimicrobial localized to the site of infection and thus decrease the chance of 
bacteria becoming resistant.  This clinical study will also yield important data for planning 
follow on clinical trials, including those involving Nu-3 for systemic administration to treat 
serious gram negative infections such as complicated urinary tract infections.   
Diabetic foot infections are one of the most common complications for people who suffer from 
diabetes and a frequent cause of hospitalization10.  According to the American Diabetes 
Association approximately 8.3% of the US population suffers from diabetes and in 2006 there 
were 65,700 non-traumatic amputations performed on diabetics.  In addition, almost half of 
diabetic patients that have an amputation will develop an ulcer on the remaining limb within 
18 months after surgery.  The treatment of a diabetic foot infection is complicated as it requires 
both antimicrobial therapy to cure the infection and proper wound care management to heal the 
ulcer.  Curing the infection is particularly challenging since the infection tends to be 
polymicrobial in nature requiring a broad spectrum antibiotic, the ulcer is prone to re-infection, 
and the pathogenic bacteria are increasingly becoming resistant to most front line therapies.  In 
addition, recent studies have estimated that approximately 60-80% of chronic infections involve 
biofilm formation which makes the bacteria more resistant to traditional antibiotics11.  Therefore, 
proper management of an infected diabetic foot ulcer requires an antimicrobial therapy to cure or 
clear the infection to allow proper wound management or therapy to heal the ulcer and prevent 
relapse. 
Bisphosphocins™, a new class of broad spectrum antimicrobials, possess a number of 
characteristics that make these compounds extremely well-suited to the management of diabetic 
foot infections.  First, bisphosphocins™ are rapidly and directly bactericidal functioning through 
a unique mechanism of action providing the ability to quickly cure an infection.  The compounds 
are fairly low in molecule weight, have an excellent safety profile, and have shown good 
penetration into wounds.  In addition, there are no known bacterial mechanisms of resistance and 
no resistant bacteria have been identified to date eliminating a common cause of treatment 
failure7. Finally, bisphosphocins have been demonstrated highly effective at killing bacteria 
established in biofilms12 which is increasingly become associated with many chronic infections.  
The preclinical studies conducted on the bisphosphocin™ class clearly support their evaluation 
in the proposed Phase I/IIa safety study in diabetic foot infections. 


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1.2 
Bisphosphocin Nu-3 
1.2.1 General Information 
Bisphosphocins™ were originally discovered by 
 while conducting research in 
the field of antisense technology and attempting to kill bacteria using gene silencing.  The initial 
compounds identified were protonated/acidified oligonucleotides that demonstrated bactericidal 
activity independent of nucleotide sequence.  Further experimentation expanded the class to 
protonated/acidified nucleotides and derivative molecules that exhibit chemical stability, acid pH 
resistance, and nuclease resistance enabling formulations for oral, intravenous, pulmonary, and 
topical delivery.  The lead compound, designated Nu-3, is a thymidine derivative that would be 
considered to be in the nucleotide analogue class of drugs (Figure 1).  Experimental data has 
elucidated a non-receptor dependent mechanism of action involving disruption of the bacterial 
cell membrane causing depolarization and rapid cell death.  Bisphosphocins™ have an extremely 
broad spectrum of activity in vitro testing against over 70 different bacterial strains carrying 
various antibiotic resistant markers.  There is currently no known mechanism of resistance to the 
bisphosphocin class and because they were synthetically derived there is no natural pool of 
resistant bacteria.  In addition, panelling the compounds against a number of strains with known 
antibiotic resistance or clinical isolates has failed to identify any cross-resistance or resistance 
mechanisms.   The following preclinical pharmacological and toxicology data is provided as a 
brief summary of the activity of bisphosphocin Nu-3 to support its potential as a treatment for 
diabetic foot infections and this clinical protocol should be read in conjunction with the 
Investigator Brochure on the product. 
Figure 1. 
Structure, Chemical Name and Chemical Formula of Bisphosphocin™ Nu-3 
 


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1.2.2 Pre-Clinical Pharmacology and Toxicology 
Specific Pharmacology 
Initial experiments revealed that bisphosphocins™ had an antimicrobial activity distinct from 
any currently existing antibiotic class.  Extensive in vitro and in vivo experiments have been 
conducted to further define their activity, identify their mechanism of action, and begin to 
establish a dose range to allow entry in human clinical trials.  An important factor to consider in 
the review of the following experimental results is that it is now known bisphosphocins™ do not 
follow the simple inhibitory kinetic model of one compound binding to one target molecule of 
the current classes of antibiotics but a more complex kinetic model involving the binding of 
multiple molecules to a bacterium resulting in direct bacteria cell death.  For this reason, the 
current hypothesis is bisphosphocin™ dosing in vivo will be based on total dose delivered and 
not on maintaining a circulating level above the minimum inhibitory concentration or at the site 
of infection to achieve an efficacy outcome because the molecules rapidly bind and kill bacteria 
as opposed to inhibiting growth.  Therefore, the therapeutic effect of bisphosphocins™ can be 
manipulated by not only controlling the dose but frequency and duration of treatment not 
because of the pharmacokinetics of the compound but due to the fact that each dose will result in 
a reduction of bacterial load. The following experiments are presented to further define, 
distinguish and compare the activity of the bisphosphocin class from traditional antibiotics in 
order to develop a comprehensive clinical plan to enable evaluation in humans.  The majority of 
the experimental data presented in the following sections was performed in the laboratory of 
outside collaborators or researchers who then published their results6,7,8,12 or by independent 
Contract Research Organization following the current guidelines for antimicrobial testing.  Due 
to the unique MOA of the bisphosphocin™ class some standard assays were modified to enable 
the evaluation which has been noted in the protocol. Table 1 and Table 2 summarizes the activity 
of Nu-3 in the standard MIC assay against bacterial pathogens commonly found in diabetic foot 
infections and against common fungal pathogens. 
Table 1. 
Bisphosphocin Nu-3 Activity in a Minimum Inhibitory Assay according to 
CLSI Guidelines 
Organism 
Number of Strains 
MIC  
mg/ml 
Acinetobacter baumannii 
N=15 
5-10 
Enterococcus faecalis, VRE 
N=15 
5 
Enterococcus faecalis, VSE 
N=15 
5 
Enterococcus faecium, VRE 
N=15 
5 
Enterococcus faecium, VRE 
N=15 
5 
Escherichia coli 
N=15 
10 
Proteus mirabilis 
N=15 
5-10 
Proteus vulgaris 
N=15 
5-10 
Pseudomonas aeruginosa 
N=5 
2.5-5 


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Organism 
Number of Strains 
MIC  
mg/ml 
Pseudomonas aeruginosa 
N=15 
5-10 
Serratia marcescens 
N=15 
10 
Staphylococcus aureus, MSSA 
N=15 
5-10 
Staphylococcus aureus, MRSA 
N=15 
5-10 
Staphylococcus epidermidis, MRSE 
N=15 
10 
Staphylococcus epidermidis, MSSE 
N=15 
10 
Streptococcus agalactiae 
N=15 
10 
 
 
Table 2. 
Anti-fungal Activity of Bisphosphocin Nu-3 
Yeast Strain 
ATCC # 
Drug Resistance 
MIC mg/ml 
 
 
 
Nu3 
Candida albicans 
44373 
5-Fluorocytosine 
≤0.53 
Candida albicans 
44374 
 
≤0.53 
Saccharomyces pastorius 
2366 
 
≤0.07 
Trichophytan mentagrophytes var. interdigitale 
200099 
 
5.0 
 
Evaluation of Nu-3 Activity against Bacteria Established in Biofilm 
The following studies were performed by 
 
 and were designed to evaluate the activity of Nu-3 
against bacteria established in biofilms which is increasing becoming associated with chronic 
infections12,unpublished.  Biofilm was formed in borosilicate glass tubes by standard methods and 
then treated with Nu-3 or kept as untreated controls.  As shown in Table 3, Nu-3 was able to 
penetrate the biofilm and kill the bacteria as judged by the lack of growth in Nu-3 treated tubes.  


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Table 3. 
Bactericidal activity of Nu-3 against bacteria established in biofilm 
Bacterial Strain 
Designation 
Assessment 
Treatment 
 
 
Nu-3 Exposure 
(10mg/ml) 
Media 
Control 
10min 
30min 
Klebsiella pneumoniae Kp-S1 
Clinical 
Growth 
N.G. 
1.1 x 109 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Acinetobacter baumannii 
BAA-1605 
Growth 
N.G. 
2 x 109 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus epidermidis 
35984 
Growth 
N.G. 
2.7 x 107 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus epidermidis 
PCI1200 
Growth 
N.G. 
5.2 x 108 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus aureus 
NRS684 
Growth 
N.G. 
4.3 x 108 
cfu/ml 
5 x 108 
cfu/ml 
8.6 x 108 
cfu/ml 
Biofilm 
 
 
 
 
Staphylococcus aureus 
NRS732 
Growth 
N.G 
7.6 x 108 
cfu/ml 
N.G 
N.G 
Biofilm 
 
 
 
 
 


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Assessment of the rate of kill of Nu-3 in 0.85% normal saline: 
 
Bacteria in the log phase of growth were collected by centrifugation, re-suspended in sterile 
saline (0.85% NaCl) at 1 x 105 CFU/ml. Nu-3 in sterile saline was added to a final concentration 
of 0.2 to 10mg/ml to the bacteria and the bacteria/Nu-3 mixture incubated at room temperature. 
At time points ranging from 5 to 60 minutes, aliquots were removed, diluted and plated onto TS 
agar plates for quantitation of the number of colony forming units (CFU's).  After 24h at 37°C, 
the number of CFU's were determined and the Nu-3 concentrations and exposure times resulting 
in 100% kill were determined (Table 4). 
Table 4.   
Time and concentration dependent activity of Nu-3 against biofilm forming 
bacterial strains. 
Bacterial Strain 
Nu-3 Concentration 
0.625 (mg/ml) 
1 (mg/ml) 
5 (mg/ml) 
10 (mg/ml) 
K. pneumoniae Sp1 
15 min 
 
5 min 
 
K. pneumoniae Sp5 
 
15 min 
5 min 
 
A. baumannii BAA-1605 
30 min 
 
10 min 
5 min 
S. aureus NRS684 
 
 
 
60 min 
S. aureus NRS732 
 
 
 
60 min 
 
 
Mouse Suture Wound Infection Model 
Briefly, a 10cm length of suture was threaded onto a needle and soaked for 45 minutes in 
undiluted cultures of S. aureus cvcc2248 or P. aeruginosa cvcc 5668 at 35°C for 8 hours.  The 
needle and sutures were removed, placed on filter paper, and dried at 4°C until implantation.  A 
1-cm long piece of suture was implanted under the skin in the shaved and washed mid-back of 
anesthetized animals using knots to secure it in place.  An incision was made between the knots 
on top of the suture line with a scalpel.  The animals were then divided into groups and treated as 
indicated at 4h and 8h post-surgery and then 3 times daily for 3 days with either placebo, Nu-3 or 
ciprofloxacin ointment.  On Day 5 the animals were euthanized and a 1cm2 area surrounding and 
including the wound was harvest for homogenization and CFU counts (Table 5).  The initial 
inoculum (i.e. CFU of bacteria in 1 cm length of suture) was determined by vortexing 1-cm of 
suture in bacterial growth media and plating serial dilutions to obtain bacteria counts. 


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Table 5. 
Log reduction in bacterial cell counts in surgical wound model 
Treatment  
Mean Bacterial Count ± SD (Log10 CFU/Wound) 
P. aeruginosa 
S. aureus 
Initial Inoculation  
4.93 ± 0.33 
4.79 ± 0.31 
Untreated  
7.52 ± 0.57 
7.64 ± 0.58 
Glycerin  
7.34 ± 0.27 
7.47 ± 0.34 
5% Nu-3 in Saline  
5.62 ± 0.61 
5.23 ± 0.5 
5% Nu-3 in Glycerin  
5.07 ± 0.53 
4.51 ± 0.55 
Ciprofloxacin HCl Ointment  
4.82 ± 0.58 
5.63 ± 0.31 
 
Mouse Superficial Skin Polymicrobial Infection Model 
Briefly, mice are pre-treated with cyclophosphamide four day prior to initiating the infection to 
suppress the immune system.  The mice are anesthetized and kept sedated while the fur on the 
back dorsal surface is shaved using electric clippers followed by ‘wet shaving’ with a disposable 
razor. The skin is then sterilized with a betadine wash followed by alcohol swab and abraded 
with a +50 grit emery board that results in a smooth, red, shiny appearance with no bleeding.  A 
droplet of the bacterial inoculum is placed directly on the abraded skin that contains 
Pseudomonas aeruginosa UNT034-1 (ATCC 27853) 6.35 Log10 CFU/ mouse and S. aureus 
UNT006-4 (smith) 5.65 Log10 CFU/ mouse.  The infected area is treated with 50 microliters of 
varying concentrations of bisphosphocin Nu-3, gentamycin, mupirocin, or normal saline twice 
daily for 3 days.  The mice are then euthanized, the area of infected skin excised, and the CFU of 
both bacteria is determined by culture or serial diluted samples of homogenized tissue (Table 6).  
The data shows that the top 5% dose of bisphosphocin Nu-3 results in a 1 log reduction in the 
CFU count of both S. aureus and P. aeruginosa.   


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Table 6. 
Mouse Superficial Skin Polymicrobial Infection Bacterial Counts 
S. aureus 
P. aeruginosa 
Compound 
Dose 
CFU 
SD 
CFU 
SD 
NU3 
1.0 mg/mL 
9.22 
0.19 
6.35 
0.21 
10 mg/mL 
8.78 
0.24 
6.81 
0.23 
20 mg/mL 
9.00 
0.27 
7.08 
0.19 
50 mg/mL 
7.61 
1.35 
5.65 
1.16 
Gentamicin 
0.3% 
4.40 
0.85 
5.17 
0.87 
Mupirocin 
2.0% 
6.32 
1.01 
6.95 
0.51 
Vehicle 
saline 
8.73 
0.25 
6.3 
0.42 
Infection Control 
Day 4 
8.72 
0.52 
6.68 
0.69 
4 hr 
5.40 
0.45 
4.33 
0.83 
 
 
Preclinical Toxicology 
Summary of Preclinical Toxicology Data 
Nu-3 is the first in a new class of broad spectrum antimicrobials, termed bisphosphocins, being 
developed to treat a wide range of serious bacterial infections.  Chemically Nu-3 would be 
considered to be in the nucleotide analog class of drugs and the following set of preclinical 
studies was designed based on the preclinical and clinical data collected over the past 50 years on 
this class.  The nucleotide analog class of drugs have a good safety profile even when 
administered long term at low doses and toxicities are generally associated with their 
interference of normal DNA/RNA synthesis.   
Nu-3 or 
 is a thymidine analog with both 
the 3’ and 5’ sites blocked by butyl phosphate which prevents it from causing early chain 
termination by incorporation by the polymerase unlike most compounds in this class.  Nu-3 is 
activated through an acidification step that results in an acidic or protonated compound that when 
dissolved in water has a pH between 2 and 2.5.  The compound can be neutralized or unactivated 
by a strong base or buffer such as sodium bicarbonate.  In addition, the molecule was designed to 
minimize the potential for toxic side effects by utilizing a thymidine core molecule, natural 
phosphate linkages, and even carbon butyl blocking groups, all of which are either found in the 
body and/or feed into natural recycling pathways. 
A summary of the preclinical toxicology studies completed with Nu-3 is presented in Table 7.   
The data indicates that Nu-3 is safe and well-tolerated when administered systemically or 


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topically and no specific toxicities were identified that would be cause for concern for the 
proposed Phase I/IIa study in subjects with infected diabetic foot ulcers.  In general, the data 
indicates that Nu-3 functions through a membrane depolarization mechanism of action that is 
highly specific for bacterial cell membranes.   
Table 7. 
Summary of Nu-3 Preclinical Toxicology Data 
Study 
Test Article/Dose 
Species 
Lot # 
Result 
Genotoxicity 
Bacterial Reverse Mutation 
Assay (GLP) 
Nu-3 ACT 
In vitro 
LWA-01R-14 
Non-mutagenic 
Bacterial Reverse Mutation 
Assay (GLP) 
Nu-3 NEUT 
In vitro 
LWA-01R-14 
Non-mutagenic 
Systemic Toxicity Study 
7-Day Daily Repeat IV 
Toxicology (GLP) 
0, 2, 10, 50mg/kg 
Rats 
CCS-
2009/ST-
03/00114 
 NOAEL at 50mg/kg 
No drug related findings 
IV Max Tolerated Dose 
(GLP) 
0, 250, 500, 
1000mg/kg 
Rats 
LWA-01-14 
NOEL and NOEAL at 250mg/kg 
LD50 ≥ 500mg/kg 
IV Max Tolerated Dose 
Study  (Non-GLP) 
0, 250, 500, 750, 
1000mg/kg 
Mice 
LWA-01-14 
LD50 ≥ 1000mg/kg 
Oral Max Tolerated Dose 
Study (Non-GLP) 
1000, 1256, 1580, 
1988, 2500mg/kg 
Mice 
PN 1127 
LD50 2001mg/kg according to 
the Karber method 
Acute Single IV Dose 
(Non-GLP) 
50, 200mg/kg 
Rat 
PN 1127 
No observable effect on blood 
chemistry (CBC, metabolic 
panel) 
Local Tolerance Studies 
Primary Dermal Irritation 
(GLP) 
10% Nu-3 
Rabbit 
LWA-01R-14 
Not a primary irritant 
MapTek EpiOcular™ 
Toxicity (GLP) 
1%, 2.5%, 5% 
Nu-3 
In vitro 
LWA-01-14 
1% - Non-irritating, Minimal; 
2.5%, 5% - Moderate irritants; 
No significant IL1α stimulation 
for all doses 
In vitro Sensitization 
(GLP) 
10%, 6.8%, 4.6%, 
3.2%, 2.2%, 
1.5%, 1%, 0.68% 
In vitro 
LWA-01-14 
4.6% Sensitizer, All other 
concentrations are Non-
sensitizers 
3 Day Ophthalmic 
Irritation Study (Non-GLP) 
1% 
Rat 
PN 1127 
No sign of irritation 
28 Day Repeat Dermal 
Toxicity (Non-GLP) 
10% 
Rat 
PN1127 
No pathological changes in skin, 
no clinical observations 
 


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1.2.3 Clinical Trials of Bisphosphocin Nu-3 
Bisphosphocin™ Nu-3 is a new chemical entity that has not been evaluated under a formal IND 
application in human clinical studies.  The compound was initial developed as a homeopathic 
treatment for a variety of topical infections as it was considered to fall under the HPUS DNA 
guidelines9.  As a homeopathic treatment, Nu-3 was used topically to treat a variety of infections 
including acne, urethritis, ear infections, diabetic foot infections, and certain fungal infections.  
The information collected from these homeopathic treatments revealed that Nu-3 applied 
topically in solution for up to 14 days was able to improve the severity of an infection based 
visual observation.   
1.3 
Rationale for Current Study 
The proposed clinical study is designed to demonstrate the safety and tolerability of increasing 
doses of topically applied bisphosphocin™ Nu-3 on a chronic diabetic foot infection.  In 
addition, the study is designed to collect data on the efficacy of Nu-3 as it relates to eradication 
of the infection and wound healing to enable planning of future trials.  The 3 dosing levels and 
length of treatment were selected based on in vitro, in vivo, and toxicology studies of Nu-3 and 
its unique mechanism of action.  While the current trial is considered a first-in-human trial, the 
design also has taken into consideration data collected from the development of the compound 
initially as a homeopathic treatment where the use as a topical solution was able to reduce the 
severity of several common topical infections.  Recent studies have shown that a high percentage 
of chronic wounds have biofilm associated which interferes with wound healing.  This trial is 
intended to evaluate the efficacy of an antimicrobial with known activity against biofilm encased 
bacteria to not only eliminate the infection but potentially improve wound healing.  For this 
reason, bisphosphocin Nu-3 is being formulated in a normal saline solution so that a clearer 
picture can be gained of its effect on the wound without the interference of an ointment base.  
Bisphosphocins have been shown to effectively reduce the severity of the infection in preclinical 
animal model testing, but these models have limits as they do not accurately mimic the complex 
environment of a chronic wound infection and in many cases rely on immunosuppression to 
maintain the infection.  This first in human study is designed to collect both safety and efficacy 
data so that the parameters such as dose, duration and frequency of administration can be refined 
for future more intensive studies.  Based on the data collected from preclinical studies, it is 
anticipated that a therapeutic effect may be observed in the middle (1% w/v, or 10 mg/ml) and 
high dose  (2% w/v, or 20 mg/ml) cohorts that could justify extending the treatment course an 
additional seven (7) days.  An extension of the treatment duration to fourteen (14) days would 
only be initiated after five subjects in Cohorts 2 and 3 completed the seven (7) day treatment and 
approval was given by the SMC.  The decision to extend treatment of individual subjects 
showing a clinical response would then be made by the principal investigator. 
Potential Risks 
This clinical trial is a first in human trial so there is a potential risk of an adverse reaction from 
the study drug.  Bisphosphocin Nu-3, however would be considered a nucleotide analog which is 
a class of drugs with a strong safety record so the likelihood of this particular risk is considered 


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minimal.  The major distinction between bisphosphocin Nu-3 and the nucleotide analog class are 
that both 3’ and 5’ sites are blocked and a novel acid activation step that is reversed when the 
compounds are exposed to buffers or base.  Therefore, it is anticipated that any reaction would be 
localized since the sodium bicarbonate in the blood would neutralize the compound which is 
supported by inhibitory effect of blood in MIC assays.  In addition to a reaction from the study 
drug, there is also the possibility that the drug is ineffective and the infection worsens which 
could necessitate more aggressive therapy or treatment. 
Potential Benefits 
The major benefit of the proposed study will be an improvement in the wound due to the 
elimination of the pathogenic bacteria that is inhibiting healing.  Bisphosphocin™ Nu-3 is a new 
class of broad spectrum antimicrobial so it is unlikely that the pathogenic bacteria will be outside 
its spectrum of activity or resistant, which is a major cause of treatment failure in this indication.  
The dose levels have been selected so that two cohorts are likely to be receiving efficacious dose 
levels of drug with a satisfactory safety margin so that some improvement in wound appearance 
is anticipated in some subjects in these cohorts.  In addition to directly eliminating the 
pathogenic bacteria associated with the chronic ulcer, it is anticipated that a second potential 
benefit would be the elimination of any biofilm encased bacteria that would then make the 
infection more susceptible to traditional therapy as a follow up therapy.  Any of these outcomes 
would benefit the subject as it would reduce the risk of limb loss due to a worsening infection 
and the pain. 
 


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2 
OBJECTIVES 
Objective/Purpose: The primary objective of this study is to assess the safety and tolerability of 
Nu-3 when applied topically to a chronically infected diabetic ulcer. 
A secondary objective of this study is to obtain preliminary data on the microbiological activity 
of Nu-3 as measured by clinical wound assessments and presence of pathogenic bacteria.  Based 
on preclinical data, it is anticipated that improvement in the clinical wound assessment may be 
observed in the middle and high dose (1% and 2%) cohorts. 
Statement of Hypothesis:  Nu-3 is a broad spectrum antimicrobial with a unique mechanism of 
action that involves cell membrane depolarization and rapid bacterial cell death.  As an 
antimicrobial it is particularly well suited for development as a topical for Diabetic Foot 
Infections for the following reasons: 
1. These infections tend to be polymicrobial in nature, involve biofilm, and require an 
antibiotic(s) with a broad spectrum of activity. 
2. Many of the bacteria in these infected wounds are resistant to one or more classes of 
antibiotics making treatment difficult 
3. The directly bactericidal mode of action of Nu-3 should translate into an immediate 
reduction in the severity of the infections and offers new options in dosing. 
4. Research has shown that many chronic infections are due to the formation of biofilm 
which is inherently resistant to treatment by traditional antibiotics but susceptible to 
treatment by bisphosphocins such as Nu-3 
 
The proposed clinical trial design is aimed at defining the initial parameters for safety and 
tolerability in an infected wound based on the currently available preclinical data on Nu-3. The 
trial is focused on DFIs of mild to moderate severity and an area of 0.5 to 6 cm2 as a population 
of subjects that will provide the data across a range of pathogens and ulcers of varying duration.  
In addition, the middle and high dose (1% and 2% Nu-3) cohorts were selected so that some 
clinical improvement in wound assessment be observed which will enable better planning of 
following clinical trials.   
Route of Administration: Study medication will be applied topically directly to the infected 
ulcer 
Dosage:  The study includes a sequential ascending dose protocol with Cohort 1 at 0.1% w/v or 
1mg/ml, Cohort 2 at 1% w/v or 10mg/ml, and Cohort 3 at 2% w/v or 20mg/ml.  In addition, two 
subjects in each cohort will be randomized to placebo.  The proposed doses and treatment 
duration were based on data from in vitro antimicrobial assays, preclinical animal efficacy 
models, and toxicology studies and were selected to demonstrate safety at a high dose that 
provides a significant therapeutic margin lethal to all tested bacteria and fungi to date while 
proven safe in animal toxicology studies.   


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Dosing Regime:  To establish the initial safety and tolerability of Nu-3, a twice daily application 
of the compound was selected for this clinical trial based on preclinical studies on efficacy and 
toxicology.  Subjects will be required to keep a daily log of when treatments are applied and 
contacted daily by a member of the study team. 
Ulcer Imaging and Area Calculations:  Photographs of the ulcer(s) will be taken at study visits 
using the Aranz Medical Silhouette™ system which allows capture of a color image and 
computer calculation of ulcer(s) area, depth, and volume with more consistent and greater 
accuracy than traditional methods. 
Randomization: Eligible subjects will be randomly assigned in a 4:1 ratio to Nu-3 (n=8) or 
matching placebo (n=2) in each cohort. 
Treatment Period:  Day 1 (in clinic): Day 2 (end of SAD/Beginning of MAD) through Day 8  
Follow-Up Period: Day 15  
Selection of Study Population:  Type I or II diabetes mellitus subjects with a chronic infected 
lower extremity ulcer defined as having a DUSS score of 0 to 3 and a DFI wound score 1 to 9 as 
this is the most appropriate population to assess the safety of a new drug aimed at treating 
infected wounds.  The proposed inclusion criteria have been selected to define a subject 
population that will have a diverse spectrum of pathogens and range of chronic ulcers to ensure a 
proper assessment of safety and tolerability as well as data to better define follow on clinical 
studies.  The DFI wound score range is intended to select subjects with mild to moderate 
infection which have the best potential to respond to the treatment.  A radiograph of the affected 
area will be performed during the screening period to rule out the possibility that the ulcer is due 
to an underlying osteomyelitis.  Preclinical studies have demonstrated the safety of Nu-3 on 
mammalian cells, intact and damaged skin, and when injected intravenously, therefore exposure 
of healthy subject to Nu-3 would provide little if any additional safety information. 
 
 


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3 
STUDY DESIGN 
Phase I/IIa, three cohort double-blind ascending dose with two dosing phases per cohort, study in 
diabetes mellitus subjects with a chronic infected diabetic ulcer defined as having a DUSS score 
between 0 and 3 and a DFI wound score between 1 and 9.  The study proposes to use the same 
subject(s) in SAD Phase 1 and MAD Phase 2 of each cohort. 
The study is designed to conduct the cohorts sequentially with the completion of the first cohort 
before initiating the next dosing level.    
In Phase 1, eligible subjects will be treated with a single application of Nu-3 or placebo in a 4 to 
1 ratio to judge the initial safety of Nu-3 over a brief 15-30 minute interval and 24 hour interval.  
Bisphosphocin Nu-3 will be applied topically to the chronic infected ulcer (DUSS score: 1-3; 
DFI wound score: 1-9), and the area observed over a 15-30 minute time period and 
photographed.  If no visible reaction is observed, the ulcer is covered with a bandage.  The 
subject will be released with verbal instructions to leave the nonabrasive, hypoallergenic 
bandage on the wound, avoid getting the bandage wet, and return for a follow up visit within 
24 ± 2h.  At the follow up visit, the bandage will be removed, the ulcer visually examined, 
photographed using the Aranz Medical Silhouette™ system, and the subject cleared for the 
MAD Phase 2 based on the recommendation/decision of the PI and on the absence of any drug 
related SAEs.   
In Phase 2, eligible subjects, which are those who have been approved by the PI after the Visit 2 
examination, will be instructed in the proper application of bisphosphocin Nu-3 or placebo.  In 
the clinic, the subjects will be observed applying the first dose to ensure compliance.  The 
subject will then be given a 7-day supply of study drug, non-abrasive bandages, a home diary, 
written instructions for care and hygiene, and sent home to continue treatment.  On Day 9 ± 2h or 
earlier in the case of any adverse events, subjects will return to the clinic for an examination, 
including visual evaluation of the ulcer, vital signs, adverse events, photo documentation using 
the Aranz Medical Silhouette™ system, collection of a sample for microbiological assessment, 
and concomitant medication use.  A final follow up visit will be scheduled for Day 15 for a 
complete examination as described above.  Based on the data collected from preclinical studies, 
it is anticipated that a therapeutic effect may be observed in the middle (1% w/v, or 10 mg/ml) 
and high-dose (2% w/v, or 20 mg/ml) cohorts. 
SUBJECT POPULATION 
3.1 
Selection and Exclusion of Subjects 
Subjects, greater than 18 years of age, will be recruited from the area surrounding the Phase I/IIa 
Clinical Trial Centers as well as existing subjects of the center.  Strategies for recruiting subjects 
will include web based advertising and social media sites, or traditional advertising in local 
newspapers all using IRB-approved materials.  It is anticipated that 50-60 subject will be 
screened to identify the 30 subject needed for the proposed study.  Subjects will be allowed as 


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much time as necessary to review and give informed signed prior to any study related procedures 
being performed.   
3.2 
Inclusion Criteria 
1. Signed Informed Consent Form must be obtained for the subject and if necessary the 
subject’s caregiver prior to any study related procedures being performed 
2. Non-hospitalized ambulatory subjects suffering from Diabetes mellitus, Type I or II 
3. Male or Female older than 18 yrs 
4. Diabetic foot ulcer(s) with a DUSS Score of 0 to 3 
5. A radiograph, MRI, and/or CAT scan evaluation within the last seven (7) days to 
determine the ulcer is not caused by an osteomyelitic bone infection 
6. Infection as defined by the IDSA as ≥ 2 classic findings of inflammation or purulence 
7. Ulcerated area(s) of not more than two (2) ulcers between 0.5 to 6 cm2 as calculated by 
the Aranz Medical Silhouette™ system 
8. Infection must be localized to the area of the ulcer and defined as mild (superficial and 
limited in size and depth) with a DFI Wound Score between 1 and 6 
9. Subject must agree to be treated as an outpatient, follow all protocol procedures, return 
for all schedule visits, and provide informed consent 
10. Any female of child bearing age must consent to use medically acceptable birth control 
for the duration of the study 
 
3.3 
Exclusion Criteria 
1. A DUSS Score above 3 
2. Any ulcer caused by an underlying osteomyelitic bone infection 
3. Assessed with a Moderate to Severe Infection, including abscesses, extensive gangrene, 
symptoms of systemic infection, or a limb threatening infection.  DFI Wound Score 
above 6 
4. Hemoglobin A1c (HbA1c) level ≥ 9.0% 
5. An ulcer area(s) greater than 6 cm2 or more than two (2) ulcers 
6. Any subject that has received systemic or topical antibiotics within the last seven (7) days 
7. Any subject on topical antimicrobial treatment for their infected diabetic foot ulcer whose 
ulcer is responding to treatment 
8. Any subject that would be unable to follow the protocol procedures, safely monitor the 
infection status at home, and return for schedule visits 
9. Positive pregnancy test at Screening or Visit 2 
10. Positive drug or alcohol test at Screening Visit or Visit 2, unless determined by the 
Principal Investigator (PI) the positive result would not impair full participation in the 
clinical research study 
 


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4 
STUDY DRUGS 
4.1 
Randomization, Blinding, and Drug Product/Placebo Packaging and Labeling 
The drug product kits will be labeled as shown in Table 8 to ensure blinding of both the Principal 
Investigator and Subject.  The kits will contain a 0.5 oz dropper bottle containing either 15 ml of 
Bisphosphocin™ Nu-3 or Placebo packaged in a cardboard a carton with a foam insert for 
protection.  Both the carton and dropper bottle will be labelled. 
The randomization will consist of two materials that have been allocated within 3 Cohorts. 
 
Material 1: Bisphosphocin™ Nu-3 at 0.1%, 1%, or 2% w/v 
 
Material 2: Matching Placebo  
Within each Cohort, a material blocking ratio of 4:1 has been utilized with three (3) blocks per 
cohort.  All Cohorts utilize a different bottle number range with ranges set at Cohort 1 from 
0101 to 0115, Cohort 2 from 0201-0215, and Cohort 3 from 0301 to 0315.  All cohorts will have 
a different starting Randomization Specification Seed value which will result in a different 
statistical outcome. 
Table 8. 
Product Label Specifications 
GC: X; LID: X; CARTON 
Protocol #: 
LAI2014-1 
Product: 
Bisphosphocin Nu-3 topical antibiotic or placebo 
Bottle Number: 
#### 
Lot Number*: 
14K### 
Directions for Use: 
As directed, apply twice daily to the affected area 
Storage: 
Room Temperature^ 
Caution: 
New Drug-Limited by federal law to Investigational Use 
Keep out of reach of children 
Manufacturer: 
Lakewood Amedex Inc, Sarasota, FL (941)225-2515 
*Lot number represents the group of kits being assembled for each cohort. 
^Room temperature is defined according to the USP as the temperature that is prevailing in a work area. 
 
 
4.2 
Description and Handling of Bisphosphocin Nu-3 
Nu-3 or 
 is a thymidine analog with both 
the 3’ and 5’ sites blocked by butyl phosphate which prevents it from causing early chain 
termination by incorporation by the polymerase unlike most compounds in this class.  Nu-3 is 


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activated through an acidification step that results in an acidic or protonated compound that when 
dissolved in water has a pH between 2 and 2.5.  The antibacterial activity can be neutralized by a 
strong base or buffer.  Prior non-human investigations indicate that all doses being used in this 
study are likely to be safe, medical personnel and caregivers should use caution when handling 
the drug to avoid exposure of sub-MIC levels through inadvertent application to the skin and 
normal skin flora that could potentially lead to resistant strains.   
4.3 
Dosage and Administration of Bisphosphocin Nu-3 or Placebo 
Bisphosphocin Nu-3 will be dosed at three different concentrations of 0.1% w/v (1mg/ml), 1% 
w/v (10mg/ml), and 2% w/v (20mg/ml) in 0.85% normal saline.  Placebo will be 0.85% normal 
saline solution.  The drug or placebo will be administered as a single dose and then twice daily 
for 7 days.  The drug or placebo will be administered to the infected diabetic foot ulcer and 
surrounding intact skin at a dose of 1-3 drops from a dropper bottle per 1 cm2 of ulcer area. 
4.4 
Prior and Concomitant Medications 
Subjects are allowed to continue on any concomitant prescribed medication for medical 
conditions not related to the treatment of the infected diabetic ulcer.  All systemic and topical 
antibiotics must be stopped seven (7) days prior to admission to the proposed study. 
 


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5 
STUDY PROCEDURES 
5.1 
Subject Enrollment and Treatment Assignment 
Screened subjects who sign the Informed Consent and meet all the inclusion criteria and none of 
the exclusion criteria will be considered eligible for study participation.  Eligible subjects will be 
assigned to Cohort 1 until a total of 10 subjects are enrolled, followed by Cohort 2, and Cohort 3.  
There will be a minimum of 7 days between the last subject completing the Day 9 assessment 
from each cohort and the first subject enrolled into the next cohort.  All subjects who are eligible 
for enrollment will be randomly assigned in a 4:1 ratio to NU-3 (n=8) or matching placebo (n=2) 
in each cohort.  The dose escalation will proceed following the Safety Monitoring Committee 
recommendation/approval.   
5.2 
Pre-treatment Assessments 
The schedule of evaluations and procedures that must be performed at specific time points is 
described in the following sections.  The Time and Event Schedule (Appendix B) summarizes 
the frequency and timing of various safety evaluations and clinical microbiological sampling.  As 
soon as a subject is considered to be a potential subject for this study and prior to any other study 
procedures, the nature of the study will be explained to him/her by the study investigator or 
designee and the potential subject will be asked to provide written informed consent.  Informed 
consent must be obtained prior to any study procedures being conducted. 
5.2.1 Screening Visits 
Screening Visit/Visit 1  
Subjects diagnosed with diabetes mellitus and suffering from a chronic infected diabetic foot 
ulcer will be evaluated for entry criteria during a screening period conducted within the 30 days 
prior to bisphosphocin Nu-3 or placebo treatment.  After informed consent has been obtained, the 
following procedures will be completed for each subject prior to inclusion in the study.   
 Medical History 
 Physical Examine with vital signs 
 Concomitant medications 
 Urine Pregnancy tests (HCG), if applicable 
 Drug and alcohol blood toxicity screen 
 Draw blood for CBC and CMP 
 Review of shoe gear worn 
 Radiograph, MRI, and/or CAT scan evaluations of the infected ulcer within the last seven 
(7) days to confirm the absence of osteomyelitis 
 Scoring of the diabetic ulcer using the DUSS Scoring system 


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 Rating of the infection using the DFI Wound Scoring  system 
 Examination of Chronic Ulcer(s)  
 Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ as described in Appendix C. 
 
Diabetic Ulcer Wound Scoring System (DUSS) 
Parameter 
Score 
Palpable Pedal Pulses 
Presence = 0 
Absence = 1 
Probing to Bone 
No = 0 
Yes = 1 
Location of Ulcer 
Toe = 0 
Foot = 1 
Number of Ulcerations 
Single = 0 
Multiple = 1 
Score Range  
0 
4 
 
Diabetic Foot Ulcer Wound Infection Score 
 
Parameter 
Wound Infection Score 
0 
1 
2 
3 
Purulent discharge 
Absent 
 
 
Present 
Nonpurulant drainage (serious, 
sanguinous) 
Absent 
Mild 
 
 
Erythema 
None 
Mild pink, 
barely 
perceptible 
Moderate pale 
red, defined 
edges 
Severe red to 
dark red 
Induration 
None 
Mild 
Moderate 
Severe 
Tenderness (sign) 
None 
Mild 
Moderate 
Severe 
Pain (symptoms) 
None 
Mild 
Moderate 
Severe 
Local Warmth (relative to uninfected 
contralateral foot) 
Same 
Mildly increased 
Moderately 
increased 
Severely 
increased 
 
5.3 
Enrollment/Baseline in Single Ascending Dose Arm (SAD) 
Treatment Visit/Visit 2 (Day 1) 
 A urine pregnancy test must be performed prior to starting Nu-3 treatment in all women 
of child-bearing potential and a negative result must be documented. 
 A urine drug and alcohol toxicology screen must be performed before starting Nu-3 
treatment for each subject and a negative result must be documented. 
 Draw blood for CBC and CMP 


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 Study subject will be assigned to the open cohort and treatment number per standard 
procedure.  The treatment number will correspond with the dropper bottle number that 
will be used for treatment. 
 Before the treatment begins, the following data/samples will be collected and procedures 
completed: 
o Concomitant medications 
o Vital signs 
o Debridement, if deemed necessary by the PI 
o Collection of  sample from the ulcer for microbiological assessment 
 Study Treatment for SAD Arm will be applied topically to the chronic ulcer and the area 
observed over a fifteen minute period for any acute reactions prior to being covered by a 
non-abrasive hypoallergenic bandage.  The Nu-3 or placebo solution will be applied from 
a pre-packaged dropper bottle at a rate of 1-3 drops per 1 cm2 of ulcer making sure to 
cover the entire ulcer as well as up to 0.5 cm of surrounding tissue. 
 One hour after the treatment the following data will be collected: 
o Vital signs 
o Signs and symptoms of adverse events (e.g. redness of the ulcer, swelling, fever, 
chills) 
 One hour post-treatment the study subject will be allowed to leave the clinic and 
instructed to call or return to the office if any of the following occur: 
o Increased pain or swelling of the infected ulcer or surrounding area. 
o Any observed increase in redness or swelling of the ulcer. 
 
5.4 
Follow-up Single Ascending Dose Arm 
Post Treatment Visits/Visit 3 Day 2 (24+/-2 h from Visit 2) 
Study subjects will return to the clinical site for a follow-up evaluation 24±2h post treatment 
prior to starting the multiple ascending dose arm (MAD).  At this follow-up visit, the subject will 
undergo the following procedures and evaluations to determine if they can be cleared for 
enrollment in the multiple ascending dose arm (MAD). 
 Vital sign measurements including, weight, temperature, resting blood pressure, heart 
rate, and respiratory rate. 
 Assessment of adverse events since the previous study visit 
 Visual examination of the ulcer  
 Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ system 
 


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5.5 
Enrollment/Baseline in Multiple Ascending Dose (MAD) Arm 
Treatment Visit/Visit 3 (Day 2)  
 Subjects will be instructed on the proper procedure for applying study treatment for the 
MAD Arm and observed performing the first application to the chronic ulcer and 
applying the non-abrasive hypoallergenic bandage.  The Nu-3 or placebo solution will be 
applied from a pre-packaged dropper bottle at a rate of 1-3 drops per 1 cm2 of ulcer 
making sure to cover the entire ulcer as well as up to 0.5cm of surrounding tissue.  The 
subject will be provided the Subject Treatment Log in Appendix D. 
 The study subject will be discharged from Visit 3 with a seven-day supply of study 
medication and supplies.  The study subject will be instructed to continue treatment until 
the night before their next Visit. 
 The study subject will be allowed to leave the clinic and instructed to call or return to the 
clinic if any of the following occur: 
o Increased pain or swelling of the infected ulcer or surrounding area. 
o Any observed increase in redness or swelling of the ulcer. 
 
5.6 
Subjects will be provided a Subject Home Treatment Log (Appendix B) and 
instructed in the proper method of documenting the timing of treatments and 
recording of any observations.  
Follow-Up 7 Day Multiple Ascending Dose Arm Visit/Visit 4 (Day 9+1) 
The study subject will return for a follow-up evaluation to the clinical site on Day 9 (or the next 
day) following the final MAD treatment.  At this follow-up visit, the subject will undergo the 
following procedures and evaluations. 
 Vital sign measurements including weight, temperature, resting blood pressure, heart rate, 
and respiratory rate. 
 Radiograph, MRI, and/or CAT evaluations of the infected ulcer to rule out the 
development of osteomyelitis, if determined by the Principal Investigator to be necessary 
 Concomitant medication use 
 Draw blood for CBC and CMP. 
 Assessment of adverse events and interim history since the previous study visit 
 Visual examination of the ulcer  
 Scoring of the Ulcer(s) using both the DUSS and DFI Wound Scoring Systems 
 Photo documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ system 
 Collection of a sample from the ulcer for microbiological assessment 
 Subjects will be given a 7 day supply of non-abrasive bandage and written instructions on 
the proper care and hygiene to include keeping the ulcer clean and bandage dry until the 
Day 15 follow up visit.  In addition, subjects will be told to call if there is any worsening 
of the ulcer with regard to pain, infection, or swelling.  


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Subjects will be given a 7 day supply of non-abrasive bandage and given written 
instructions on proper care and hygiene which includes keeping the ulcer clean and 
bandaged dry until the Day 15 Final Study Visit.  In addition, subjects will be told to call 
if there is any worsening of the ulcer with regard to pain, infection, or swelling.  
 
5.7 
Final Study Visit – Visit 5 (7-Day MAD)  
Post Treatment Final Visit (Day 15) 
All study subjects will have the following procedures performed at their final visit (Day 15) 
 Vital sign measurements including weight, temperature, resting blood pressure, heart rate 
and respiratory rate 
 Physical examinations 
 Visual examination of the chronic ulcer 
 Scoring of the Ulcer(s) using both the DUSS and DFI Wound Scoring Systems  
 Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ System 
 Assessment of adverse events and interim history since the previous study visit 
 Collection of a sample from the ulcer for microbiological evaluation 
 
5.8 
Early Termination Visit 
If a subject withdraws prior to completing the study, the reason for withdrawal will be 
documented on source documentation and in the CRF.  If a subject withdraws early due to an 
adverse event, he/she will be followed until resolution/stabilization of the adverse event. 
If a subject prematurely withdraws from the study they will be asked to complete the study 
procedures and evaluations performed in the final study visit at the time of withdrawal from the 
study (i.e. Study subject withdraws at the time of visit and consents to having 
procedures/evaluations done): 
 Vital sign measurements including weight, temperature, resting blood pressure, heart rate 
and respiratory rate 
 Physical examinations 
 Visual examination of the chronic ulcer   
 Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ system 
 Concomitant medication use 
 Assessment of adverse events and interim history since the previous study visit 
 Collection of a sample from the ulcer for microbiological evaluation 


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Administrative withdrawals will be replaced to ensure that each cohort includes a total of 10 
subjects who have completed the entire treatment.  The study sponsor has designated the 
following person to be in charge of the shipping of study drug as they will be unblinded and able 
to match the proper replacement kit. 
 
 
 
  
 
 
 
  
 
 
 
  
 
 
Subjects withdrawn from the study due to an AE/SAE related to the study drug will not be 
replaced. 
 
5.9 
Unscheduled Visit 
Unscheduled visits will be documented using the CRFs used for the Day 1 follow-up visit 
determined to be required by the PI, except in the case of photo documentation which must be 
performed.  If the unscheduled visit occurs as a result of an event, additional testing consistent 
with the event will be performed. 


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6 
ADVERSE EVENTS AND TOXICITY MANAGEMENT 
6.1 
Specification of Safety Parameters 
The primary endpoint safety variables will be the incidence of localized swelling surrounding the 
ulcer, development of a rash, joint pain, and any adverse changes in the appearance of the ulcer.   
The Safety Monitoring Committee (SMC) will receive safety reports outlining the number of 
subjects enrolled in each cohort to date, summary of the safety data Day 0 – 15 for the most 
recent cohort, adverse events, and serious adverse events listed by grade as well as all laboratory 
tests data.  These reports will present the data blinded and will be unblinded only if necessary to 
determine the cause of an AE or SAE at the request of the SMC.  In addition, safety data from 
completed cohorts will be summarized in the same manner and included in the report.  All 
information on adverse events will be provided in a table.   
6.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters 
6.2.1 Adverse Events 
Adverse Event: According to the ICH E6 guidelines, an AE is defined as any untoward medical 
occurrence in a subject/subjects in a clinical study regardless of its relationship to the 
administration of the study drug.  Therefore, an AE is any unintended, unfavorable, or 
unexpected sign, laboratory test, symptom, reaction, or disease temporarily associated with a 
clinical subject/subjects during the duration of the study.  The occurrence of an AE may come to 
the attention of study personnel at a study visit and during interviews of subjects seeking medical 
care, or upon reviews conducted by study monitors.  During all visits and interviews after 
administration of the study drug, clinical staff will inquire about any anticipated adverse events 
or symptoms using standardized questions.  The data will be recorded on the appropriate CRFs, 
All AEs whether local or systemic reactions not meeting the criteria for “serious adverse events” 
should be documented on the appropriate CRF.  The information to be recorded should include a 
description of the event, time of onset, clinician’s assessment of the severity, relationship to the 
study drug (assessment to be conducted only by those with the qualified training and authority to 
make a diagnosis and include the CMO, MD, PA, Nurse Practitioner, DO, DPM in podiatry and 
DDS), and time to resolution or stabilization. All AEs that occur during the study regardless of 
their association with the study drug must be documented appropriately and followed to adequate 
resolution. 
Any AE (i.e. a new event or an exacerbation of a pre-existing condition) with an onset date after 
the screening visit up to the last day on study (including follow-up), should be recorded as an AE 
on the appropriate CRF page(s). 
 
 


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An AE does not include: 
 Medical or surgical procedures) e.g. surgery, endoscopy, tooth extraction, transfusion); 
the condition that leads to the procedure is an adverse event. 
 Pre-existing diseases or conditions or laboratory abnormalities present or detected prior 
to the screening visit, that do not worsen. 
 Situations where an untoward medical occurrence has not occurred (e.g. hospitalization 
for elective surgery, social and/or convenience admissions). 
 Overdose of either study drug or concomitant medication without any signs or symptoms 
unless the subject is hospitalized for observation. 
Severity of Event: All AEs will be assessed by the investigator and recorded on the appropriate 
CRF page, including the date of onset and resolution, severity, relationship to study drug or study 
procedures, outcome and action taken with study medication.  The clinician will assess all AEs 
using a protocol defined grading system as follows. 
Scale 
Definition 
Grade 1 
No intervention required because the event is mild or asymptomatic.  A clinical or diagnostic 
observation. 
Grade 2 
Minimal to moderate intervention and/or localized event that requires non-invasive treatment 
Grade 3 
A debilitating or incapacitating condition, severe or medically significant that is not life threatening 
that requires hospitalization or prolonged treatment 
Grade 4 
Requiring urgent intervention because the event is life threatening 
Grade 5 
Death of a subject due to an adverse event. 
 
Relationship to Study Products: 
6.2.2 Reactogenicity 
Adverse events associated with the study product may include vesicle (blistering) development, 
burning sensation at the ulcer site (may be masked by diabetic neuropathy), increase in local 
temperature, and expansion of necrotic tissue surrounding the ulcer. 
 


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6.2.3 Serious Adverse Events 
A serious adverse event (SAE) is defined as follows: 
 Any adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
 Death; 
 Life-threatening situation (subject is at immediate risk of death); 
 In-patient hospitalization or prolongation of existing hospitalization (excluding those for 
study therapy or placement of an indwelling catheter, unless associated with other serious 
events); 
 Persistent or significant disability/incapacity; 
 Congenital anomaly/birth defect in the offspring of a subject who received study drug; 
 Other: medically significant events that may not result in death, be immediately life-
threatening, or require hospitalization, may be considered an SAE when, based upon 
appropriate medical judgment, they jeopardize the Subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
Examples of such events are: 
 Intensive treatment in an emergency room or at home for allergic bronchospasm 
 Blood dyscrasias or convulsions that do not result in hospitalization 
 Development of drug dependency or drug abuse  
 
Clarification of Serious Adverse Events 
 Death is an outcome of an adverse event, and not an adverse event in itself.  In reports of 
death due to “Disease Progression”, where no other information is provided, the death 
will be assumed to have resulted from progression of the disease being treated with the 
study drug(s). 
 All deaths, regardless of cause or relationship, must be reported for subjects on study and 
for deaths occurring within 30 days of last study drug dose or within 30 days of last study 
evaluation, whichever is longer. 
 “Occurring at any dose” does not imply that the subject is receiving study drug at the 
time of the event.  Dosing may have been given as treatment cycles or interrupted 
temporarily prior to the onset of the SAE, but may have contributed to the event. 
 “Life-threatening” means that the subject was at immediate risk of death from the event 
as it occurred.  This does not include an event that might have led to death, if it had 
occurred with greater severity. 
 Complications that occur during hospitalizations are AEs.  If a complication prolongs 
hospitalization, it is an SAE. 


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 “In-patient hospitalization” means that the subject has been formally admitted to a 
hospital for medical reasons, for any length of time.  This may or may not be overnight.  
It does not include presentation and care within an emergency department. 
 The investigator should attempt to establish a diagnosis of the event based on signs, 
symptoms and/or other clinical information.  In such cases the diagnosis should be 
documented as the AE and/or SAE and not the individual signs/symptoms.    
 
A distinction should be drawn between serious and severe AEs.  An AE that is assessed as Grade 
4 (potentially life-threatening) should not be confused with a SAE.  Severity is a category 
utilized for rating the intensity of an event; and both AEs and SAEs can be assessed as Grade 4.  
An event is defined as “serious” when it meets one of the pre-defined outcomes as described 
above in Section 6.2.3.  
6.2.4 Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
Laboratory abnormalities are usually not recorded as adverse events or serious adverse events 
unless they are associated with clinical signs and/or symptoms. However, laboratory 
abnormalities (e.g. clinical chemistry, hematology, urinalysis, etc.) independent from the 
underlying medical condition, that require medical or surgical intervention, or lead to study drug 
interruption or discontinuation must be recorded as an AE, as well as an SAE, if applicable.  In 
addition, laboratory or other abnormal assessments (e.g. electrocardiogram, X-rays, vital signs) 
that are associated with sign and/or symptoms must be recorded as an AE or SAE if they meet 
the definition of an adverse event (or serious adverse event) as described in the protocol.  If the 
laboratory abnormality is part of a syndrome, record the syndrome or diagnosis.   
Severity should be recorded and graded according to Common Terminology Criteria for Adverse 
Events v4.0 (CTCAE).  For adverse events associated with laboratory abnormalities, the event 
should be graded based on the clinical severity in the context of the underlying conditions, which 
may or may not be in agreement with the grading of the laboratory abnormality. 
6.3 
Serious Advent Event Reporting Procedures 
The study sponsor is required to expedite to worldwide regulatory authorities reports of Serious 
Adverse Drug Reactions or Suspected Unexpected Serious Adverse Reactions (SUSARs); 
therefore, the study sponsor or its delegate must be notified immediately regarding the 
occurrence of any SAE that occurs after the subject consents to participate in the study, including 
SAEs resulting from protocol-associated procedures performed from screening onwards. The 
procedures for reporting all SAEs, regardless of causal relationship, are as follows: 
 Record the SAE on the AE CRF and complete the “Serious Adverse Event Report” form. 
 E-mail or fax the SAE form to the attention of the Drug Safety Manager within 24 hours 
of the investigator’s knowledge of the event. Contact information is as follows: 


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 
For fatal or life-threatening events, also e-mail or fax copies of hospital case reports, autopsy reports, 
and other documents when requested and applicable. Transmission of such documents should occur 
with Personal Subject Details de-identified, without losing the traceability of a document to the 
Subject Identifiers. 
 
The study sponsor or its delegate may request additional information from the investigator to ensure 
the timely completion of accurate safety reports. 
The investigator must take all therapeutic measures necessary for resolution of the SAE. Any 
medications necessary for treatment of the SAE must be recorded onto the concomitant 
medication section of the subject’s CRF and the event description section of the SAE form. 
Follow-up of adverse events will continue through the last day on study (including the follow-up 
off-study medication period of the study) and/or until the investigator and/or study sponsor 
determine that the subject’s condition is stable.  The study sponsor or its delegate may request 
that certain adverse events be followed until resolution. 
6.3.1 Reporting Pregnancy 
The risks of treatment with bisphosphocin during pregnancy has not been evaluated. 
The subject must be instructed to discontinue all study drugs and inform the investigator 
immediately if she becomes pregnant during the study. 
The investigator should report all pregnancies to Lakewood-Amedex within 24 hours of 
becoming aware of the pregnancy.  The investigator should counsel the subject regarding the 
possible effects of prior study drug exposure on the fetus and the need to inform the study site of 
the outcome of the pregnancy.   
Any pregnancy complication or elective termination of a pregnancy for medical reasons will be 
recorded as an AE or an SAE. 
A spontaneous abortion is always considered to be a SAE and will be reported as described in 
the adverse and Serious Adverse Events section.  Furthermore, any SAE occurring as an adverse 
pregnancy outcome post-study must be reported to Lakewood-Amedex. 
Lakewood-Amedex 
Inc.  Drug Safety 
Manager 
    
 
      
 
    
  
 
        
 
 
   
   
 
Medical Monitor: 
 
  
 
  
 
 
 
 
 
 
 


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Additionally, all pregnancies that occur during the study should be reported using the Pregnancy 
Report CRF page.  Monitoring of the subject should continue until the conclusion of the 
pregnancy.  The outcome should be reported to Lakewood-Amedex using the Pregnancy 
Outcome CRF page(s).  If the end of the pregnancy occurs after the study has been complete the 
outcome should be reported directly to Lakewood-Amedex.  Pregnancies that occur after the 
subject has discontinued study drugs do not require monitoring.  
6.4 
Safety Oversight 
6.4.1 Independent Safety Monitor Committee 
Clinical Response Evaluations: 
The clinical outcome will be based on the Principle Investigator’s judgment as to the 
improvement of the wound status from Visit 1 to Visit 4 (7-Day MAD) and Visits 5 and 6.  The 
PI will score the ulcer(s) using both the DUSS and DFI Wound Scoring Systems. 
Diabetic Ulcer Wound Scoring System (DUSS) 
Parameter 
Score 
Palpable Pedal Pulses 
Presence = 0 
Absence = 1 
Probing to Bone 
No = 0 
Yes = 1 
Location of Ulcer 
Toe = 0 
Foot = 1 
Number of Ulcerations 
Single = 0 
Multiple = 1 
Score Range  
0 
4 
 
Diabetic Foot Ulcer Wound Infection Score 
Parameter 
Wound Infection Score 
0 
1 
2 
3 
Purulent discharge 
Absent 
 
 
Present 
Nonpurulant drainage (serious, 
sanguinous) 
Absent 
Mild 
 
 
Erythema 
None 
Mild pink, 
barely 
perceptible 
Moderate pale 
red, defined 
edges 
Severe red to 
dark red 
Induration 
None 
Mild 
Moderate 
Severe 
Tenderness (sign) 
None 
Mild 
Moderate 
Severe 
Pain (symptoms) 
None 
Mild 
Moderate 
Severe 
Local Warmth (relative to uninfected 
contralateral foot) 
Same 
Mildly 
increased 
Moderately 
increased 
Severely 
increased 


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An improvement in the DFI Wound Score will be used to indicate a clinical improvement of the 
wound and a change in the DUSS Score wound be used to indicate the healing of a small 
ulceration. 
Microbiological Response Evaluations: 
This assessment will be conducted on samples collected by the investigator on Visit 2, Visit 4, 
Visit 5, and Visit 6.of the study which will be cultured to determine: microbial load and identity 
of the infected pathogens or wound flora.  The microbiological response will be determined by a 
comparison of the results pre- and post-treatment. 
SMC Safety Reports: 
Safety reports will be written by the SMC three times during the course of the study.  The first 
will follow the first interim analysis (once the first SAD/MAD Cohort 1 reaches Day 9), the 
second report will follow the second interim analysis (once the second SAD/MAD Cohort 2 
reaches Day 9), and the third report will follow the third interim analysis (once the third 
SAD/MAD Cohort 3 reaches Day 9).  The study reports will be presented to the Principal 
Investigator at each clinical site and Lakewood-Amedex Inc.    
6.4.2 Study Termination 
This study may be terminated at any time by Lakewood-Amedex Inc.  The PI and SMC may 
provide Lakewood-Amedex with recommendations regarding termination of the trial, however, 
Lakewood-Amedex ultimately makes the final decision.  Reasons for terminating the study 
include: 
 If a serious or unexpected adverse event occurs and the event is judged to be probably or 
definitively related to exposure to Nu-3, the study will be immediately suspended by the 
Principal Investigator pending review of all appropriate safety data.  The event will be 
reported to the SMC, IRB, and Sponsor Medical Monitor within 24 hours or notification 
of its occurrence.  No additional subjects will receive Nu-3 until a joint decision is 
reached between the Sponsor, PI, and SMC as to whether further doses can be given or 
the trial is terminated.  Subjects currently in a MAD Arm will be contacted to ascertain 
treatment status and if any AEs have occurred.  Absent any findings, subjects in the 
ongoing MAD arms will be allowed to complete treatment. 
 Safety Monitoring Committee terminates the study based on interim safety review 
 In the event of a clinical hold being placed on the study following a report of an AE to the 
FDA 
 At the discretion of Lakewood-Amedex. 
 


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6.4.3 Removal of Subjects from the Trial or Study Drug 
Subjects will be free to withdraw at any time for any reason, or they may be withdrawn if 
necessary, to protect their health and safety or the integrity of the study data.  A subject will be 
removed from participation in the study if any of the following occur: 
 Any clinical adverse event (AE), laboratory abnormality, intercurrent illness, or other 
medical condition or situation arises such that in the judgment of the PI continued 
participation in the study would not be in the best interest of the subject 
 A protocol violation/deviation occurs that might compromise the integrity of the data, 
compliance, or subject safety 
 Informed consent is withdrawn 
 Subject is lost to follow-up 
 
6.4.4 Handling Withdrawals 
If a subject is withdrawn prematurely from the study after receiving Nu-3, the reason for 
withdrawal is to be documented in the source document and in the CRF. If the withdrawal of a 
subject is due to an AE, the subject will be followed until resolution/stabilization of the adverse 
event. 
In the case of a subject prematurely withdrawing from the study, the following procedures and/or 
evaluations will be performed if possible at the time of withdrawal or as soon thereafter as 
possible (i.e. study subject withdraws at time of visit and consents to the procedures or consents 
to come in for a visit and the procedures) 
 Photo documentation of ulcer site using the Aranz Medical Silhouette™ system. 
 Collection of a sample for microbiological assessment 
 Assessment of adverse events 
 
In the event a subject is lost to follow-up, the site staff must make reasonable attempts to contact 
the subject. A minimum of two documented phone calls followed by a certified mailed letter is 
considered reasonable. The measures taken to follow up must be documented. 
Administrative withdrawals will be replaced in order to achieve the objective of 10 subjects per 
cohort who receive a complete treatment regime.  Subjects withdrawn from the study due to an 
AE/SAE related to the study drug will not be replaced. 


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7 
STATISTICAL CONSIDERATIONS 
7.1 
Study Hypothesis 
Study Hypothesis – Bisphosphocin Nu-3 solution applied topically to a chronic infected diabetic 
ulcer will be safe and well tolerated in subjects suffering from diabetes mellitus.  In addition, the 
Nu-3 MAD Arm subjects may show improvement in the ulcer appearance, reduction in size, and 
improvement in both the DUSS score and DFI Wound Score. 
The study hypothesis will be judged by the following two study objectives: 
 Primary objective: To assess the safety and tolerability of escalating single and multiple 
doses of Nu-3, a novel antimicrobial of the bisphosphocin class, topically to an open 
wound/diabetic ulcer 
 Secondary objectives: To assess clinical response of subjects of the ascending multiple 
dose arm of Nu-3 applied topically to an open wound/diabetic ulcer to determine clinical 
and microbiological response.  
 
7.2 
Sample Size Considerations 
The number of treated subjects is based on the desire to gain preliminary safety, tolerability and 
efficacy information to support future work, while exposing a minimal number of subjects to the 
study procedures and medication.   Inferential statistical testing is not the primary intent of the 
study, therefore, no formal sample size calculation was considered.  Ten subjects per Cohort (1, 
2, and 3), for a total not to exceed 30, is considered sufficient to show clinical significance for 
safety and preliminary efficacy analysis.  The sample sizes for this study were set based on the 
trial design for dose escalation and safety evaluation requirements and not for statistical analysis. 
7.3 
Planned Interim Analysis 
There will be an interim analysis to review safety data on each cohort of subjects when all 
members of a cohort complete Visit 4 (7-Day MAD Follow Up).  In addition, an interim analysis 
will be conducted after the first 5 subjects of Cohort 2 and 3 complete Visit 4 (7-Day MAD 
Follow Up).  The interim analysis will be performed by the Safety Monitoring Committee 
(SMC) which will be composed of three physicians and may include one infectious disease 
specialist, one diabetologist, and one orthopedic surgeon.  The SMC will communicate by email 
or teleconference on at least 3 occasions during the study and more frequently if necessary. 
No formal statistical interim analyses are planned for this Phase I/IIa study since it is primarily 
focused on evaluating safety and tolerability.  Interim safety data on each cohort will be provided 
to the SMC when all members of a cohort have reached Day 3.  The SMC will decide if the 
study can proceed to the next dose cohort (majority vote).  The SMC will also determine if the 
study may proceed after receiving a report about a study related Grade 3 or 4 adverse event 


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(majority vote).  The Chairman of the SMC will share their findings and recommendations with 
Lakewood Amedex and Principal Investigators.  
7.3.1 Safety Review 
Subjects will be monitored closely for the occurrence of study related adverse events during their 
24-hr admission in the SAD Arm and for up to 7 days following their last Nu-3 treatment in the 
study.  If any grade 3 or 4 adverse events occur and the event is judged to be probably or 
definitely related to having received the Nu-3, the study will be immediately suspended by the 
Principal Investigator pending review of all safety data.  Adverse events determined to be related 
to the study drug will be reported to the SMC, Lakewood-Amedex, and second PI within 
24 hours of notification of its occurrence.  No additional subjects will receive Nu-3 depending on 
the joint decision of the SMC, Principal Investigator, and Lakewood Amedex Project 
Coordinator and CMO as to whether further doses can be given or the entire study should be 
terminated.  In addition, the SMC will review all interim safety data prior to the next cohort 
beginning treatment.  At each review, the SMC will determine whether the study may proceed to 
the next dose cohort (majority vote). 
7.4 
Statistical Analysis Overview 
This study is designed to evaluate the safety, tolerability, and preliminary efficacy of a minimum 
of three dose levels of Nu-3 relative to placebo.  Data summaries will be presented in order to 
assess the safety of Nu-3 and each Nu-3 dose group will be compared with placebo to determine 
the presence or absence of a treatment effect.  Evaluation across all dose levels of Nu-3 will be 
conducted to determine if there is evidence of a dose response to any treatment effect noted.   All 
subjects treated with placebo from all cohorts will be combined for any analysis.  Evaluations 
will also be conducted within each subject over time to assess the rate of change (if any) in study 
outcome measures over the full course of the study treatment and follow-up periods.  Inferential 
statistical testing is not the primary interest of this study. 
All subjects receiving any amount of study treatment will be evaluated for safety, tolerability, 
and efficacy.  An “intent to treat”  approach will be followed in all data summaries, using all 
available data in the summaries and analysis for an assessment of safety and tolerability while an 
“as treated” approach will be used for microbiological and clinical wound assessment data 
summaries and reports.  
All descriptive summaries and analyses will be presented in tabular and/or graphical form from 
all subjects as appropriate.  These summaries and analysis will be described in detail in the 
Statistical Analysis Plan (SAP) which will be produced prior to breaking the study blind.  All 
statistical summaries and analysis will be conducted using SAS®. 
 
 


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8 
RESPONSIBILITIES 
8.1 
Investigator Responsibilities 
8.1.1 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of the 
“Declaration of Helsinki” (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonization (ICH) guidelines, or with the laws and 
regulations of the country in which the research is conducted, whichever affords the greater 
protection to the study subject. For studies conducted under a United States IND, the investigator 
will ensure that the basic principles of “Good Clinical Practice,” as outlined in 21 CFR 312, 
subpart D, “Responsibilities of Sponsors and Investigators,” 21 CFR, part 50, 1998, and 21 CFR, 
part 56, 1998, are adhered to. 
Since this is a “covered” clinical trial, the investigator will ensure that 21 CFR, Part 54, 1998, is 
adhered to; a “covered” clinical trial is any “study of a drug or device in humans submitted in a 
marketing application or reclassification petition subject to this part that the applicant or FDA 
relies on to establish that the product is effective (including studies that show equivalence to an 
effective product) or that make a significant contribution to the demonstration of safety.” This 
requires that investigators and all sub-investigators must provide documentation of their financial 
interest or arrangements with Lakewood-Amedex or proprietary interests in the drug being 
studied. This documentation must be provided before participation of the investigator and any 
sub-investigator. The investigator and sub-investigator agree to notify Lakewood-Amedex of any 
change reportable interests during the study and for one year following completion of the study. 
Study completion is defined as the date that the last subject has completed the protocol defined 
activities. 
This study is also subject to and will be conducted in accordance with 21 CFR, part 320, 1993, 
“Retention of Bioavailability and Bioequivalence Testing Samples.” 
8.1.2 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) 
Approval 
This protocol and any accompanying material to be provided to the subject (such as 
advertisements, subject information sheets, or descriptions of the study used to obtain informed 
consent) will be submitted by the investigator to an IRB in compliance with FDA regulations for 
IRBs (21CFR Part 56). Approval from the IRB must be obtained before starting the study and 
must be documented in a letter to the investigator specifying the protocol number, protocol 
version, protocol date, documents reviewed, and date on which the committee met and granted 
the approval. 
Any modifications made to the protocol after receipt of IRB approval must also be submitted to 
the IRB for approval before implementation. 


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8.1.3 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must utilize an IRB approved consent form for documenting written informed 
consent. Each informed consent will be appropriately signed and dated by the subject or the 
subject’s legally authorized representative and the person obtaining consent.  The information in 
the informed consent must comply with 21CFR Part 50, Subpart B.  The elements of the 
informed consent must be consistent with the requirements of 21CFR Part 50.25. 
8.1.4 
Confidentiality 
The investigator must assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, and an 
identification code (i.e., not names) should be recorded on any form or biological sample 
submitted to the Sponsor, IRB, or laboratory. The investigator must keep a screening log 
showing codes, names, and addresses for all subjects screened and for all subjects enrolled in the 
trial. 
The investigator agrees that all information received from  Lakewood-Amedex, including but not 
limited to the Investigator Brochure, this protocol, CRFs, the investigational new drug, and any 
other study information, remain the sole and exclusive property of  Lakewood-Amedex during 
the conduct of the study and thereafter. This information is not to be disclosed to any third party 
(except employees or agents directly involved in the conduct of the study or as required by law) 
without prior written consent from Lakewood-Amedex. The investigator further agrees to take 
all reasonable precautions to prevent the disclosure by any employee or agent of the study site to 
any third party or otherwise into the public domain. 
8.1.5 
Study Files and Retention of Records 
The investigator must maintain adequate and accurate records to enable the conduct of the study 
to be fully documented and the study data to be subsequently verified. These documents should 
be classified into at least the following two categories: (1) investigator’s study file, and 
(2) subject clinical source documents. 
The investigator’s study file will contain the protocol/amendments, CRF and query forms, IRB 
and governmental approval with correspondence, informed consent, drug records, staff 
curriculum vitae and authorization forms, and other appropriate documents and correspondence. 
The required source data are listed in the Source Data verification Plan, and should include 
sequential notes containing at least the following information for each subject: 
 subject identification (name, date of birth, gender); 
 documentation that subject meets eligibility criteria, i.e., history, physical examination, 
and confirmation of diagnosis (to support inclusion and exclusion criteria); 


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 participation in trial (including trial number); 
 trial discussed and date of informed consent; 
 dates of all visits; 
 documentation that protocol specific procedures were performed; 
 results of efficacy parameters, as required by the protocol; 
 start and end date (including dose regimen) of trial medication (preferably drug 
dispensing and return should be documented as well); 
 record of all adverse events and other safety parameters (start and end date, and 
preferably including causality and intensity); 
 concomitant medication (including start and end date, dose if relevant; dose changes 
should be motivated); 
 date of trial completion and reason for early discontinuation, if applicable. 
 
All clinical study documents must be retained by the investigator until at least 2 years after the 
last approval of a marketing application in an ICH region (i.e., United States, Europe, or Japan) 
and until there are no pending or contemplated marketing applications in an ICH region; or, if no 
application is filed or if the application is not approved for such indication, until 2 years after the 
investigation is discontinued and regulatory authorities have been notified. Investigators may be 
required to retain documents longer if required by applicable regulatory requirements, by local 
regulations, or by an agreement with Lakewood-Amedex. The investigator must notify 
Lakewood-Amedex before destroying any clinical study records. 
Should the investigator wish to assign the subject records to another party or move them to 
another location, Lakewood-Amedex should be notified in advance. 
If the investigator cannot guarantee this archiving requirement at the study site for any or all of 
the documents, special arrangements must be made between the investigator and  Lakewood-
Amedex to store these in sealed containers outside of the site so that they can be returned sealed 
to the investigator in case of a regulatory audit. When source documents are required for the 
continued care of the subject, appropriate copies should be made for storage outside of the site. 
8.1.6 
Case Report Forms 
For each subject enrolled, a CRF must be completed and signed by the principal investigator or 
sub-investigator (as appropriate) within a reasonable time period after data collection. This also 
applies to records for those subjects who fail to complete the study (even during a pre-
randomization screening period if a CRF was initiated). If a subject withdraws from the study, 
the reason must be noted on the CRF. If a subject is withdrawn from the study because of a 
treatment-limiting adverse event, thorough efforts should be made to clearly document the 
outcome. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
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Revised – 24SEP2015 
 
 
Page 48 
 
8.1.7 
Drug Accountability 
The investigator or designee (i.e., pharmacist) is responsible for ensuring adequate accountability 
of all used and unused investigational medicinal product, placebos, and comparators. This 
includes acknowledgment of receipt of each shipment of study product (quantity and condition), 
subject dispensing records, and returned or destroyed study product. Dispensing records will 
document quantities received from  the study sponsor and quantities dispensed to subjects, 
including kit number, date dispensed, subject identifier number, subject initials, and the initials 
of the person dispensing the medication. 
At study initiation, the monitor will evaluate the site’s standard operating procedure for 
investigational medicinal product disposal/destruction in order to ensure that it complies with 
Lakewood-Amedex requirements. Drug may be returned on an ongoing basis during the study, if 
appropriate. At the end of the study, following final drug inventory reconciliation by the monitor, 
the study site will ship any remaining investigational product (to include empty containers) to 
for disposal.   
All drug supplies and associated documentation will be periodically reviewed and verified by the 
study monitor over the course of the study. 
8.1.8 
Inspections 
The investigator understands that source documents for this trial must be made available to 
appropriately qualified personnel from Lakewood-Amedex or its representatives, to IRBs or to 
regulatory authority or health authority inspectors. 
8.1.9 
Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements.  The noncompliance maybe either on the part of 
the subject, the investigator, or the study site staff.  As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly. 
These practices are consistent with ICH E6: 
 Compliance with Protocol sections 4.5.1, 4.5.2, and 4.5.3 
 Quality Assurance and Quality Control, section 5.1.1 
 Noncompliance sections 5.20.1, and 5.20.2 
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations 
within 5 working days of identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity.  All significant deviations must be promptly reported to 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 49 
 
Lakewood Amedex Inc. to the attention of 
 via fax.  Reports can be sent to 
Lakewood -Amedex as specified below copying the CRA assigned to your site: 
 Via the web-based PD forms submission 
 FAX submission (1
): utilizing the Protocol Deviation form and the 
email/fax transmittal form, or 
 E-mail submission (
) utilizing the Protocol Deviation 
form and the email/fax transmittal form. 
 
All deviations from the protocol must be addressed in the study subject source documents.  A 
completed copy of the LAI Protocol Deviation form must be maintained in the regulatory file, as 
well as in the subject’s source document.  Significant protocol deviations must be sent to the 
local IRB/EC per their guidelines.  The site PI/study staff is responsible for knowing and 
adhering to their IRB/EC requirements. 
8.2 
Sponsor Responsibilities 
8.2.1 
Protocol Modifications 
Protocol modifications, except those intended to reduce immediate risk to study subjects, may be 
made only by Lakewood-Amedex. All protocol modifications must be submitted to the IRB in 
accordance with local requirements. Approval must be obtained before changes can be 
implemented. 
8.2.2 
Study Report and Publications 
A clinical study report will be prepared by Protocol Coordinator and provided to the regulatory 
agency(ies).  Lakewood-Amedex will ensure that the report meets the standards set out in the 
ICH Guideline for Structure and Content of Clinical Study Reports (ICH E3). Note that an 
abbreviated report may be prepared in certain cases. 
Lakewood-Amedex Inc. will have the sole decision on whether or not to publish the results of 
this research.  The Company encourages its scientists and collaborators to published the results 
of any research sponsored by the Company but only after a review of the data by the Company to 
ensure that any discoveries or trade secrets are properly protected by issued, pending, or newly 
filed patents or patents applications.   
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials registration policy as a condition for publication.  This policy requires that all clinical 
trials except Phase I studies be registered in a public trials registry such as ClinicalTrials.gov, 
which is sponsored by the National Library of Medicine.  Other biomedical journals are 
considering adopting similar policies.  Lakewood-Amedex, Inc. will be responsible for 
registering the clinical trial with an acceptable registry.  Any clinical trial starting enrollment 
after 01 July 2005 must be registered on or before subject enrollment. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 50 
 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subject to intervention or comparison groups to study the cause-and-effect relationship between 
medical intervention and a health outcome.  Studies designed for other purposes, such as to study 
pharmacokinetics or major toxicity (e.g. Phase I trials) would be exempt from this policy. 
8.3 
Joint Investigator/Sponsor Responsibilities 
8.3.1 
Access to Information for Monitoring 
In accordance with ICH Good Clinical Practice (ICH GCP) guidelines, the study monitor must 
have direct access to the investigator’s source documentation in order to verify the data recorded 
in the CRFs for consistency. 
The monitor is responsible for routine review of the CRFs at regular intervals throughout the 
study to verify adherence to the protocol and the completeness, consistency, and accuracy of the 
data being entered on them. The monitor should have access to any subject records needed to 
verify the entries on the CRFs. The investigator agrees to cooperate with the monitor to ensure 
that any problems detected in the course of these monitoring visits are resolved. 
8.3.2 
Access to Information for Auditing or Inspections 
Representatives of regulatory authorities or of Lakewood-Amedex may conduct inspections or 
audits of the clinical study. If the investigator is notified of an inspection by a regulatory 
authority the investigator agrees to notify the Lakewood-Amedex medical monitor immediately. 
The investigator agrees to provide to representatives of a regulatory agency or Lakewood-
Amedex access to records, facilities, and personnel for the effective conduct of any inspection or 
audit. 
8.3.3 
Study Discontinuation 
Both the sponsor and the investigator reserve the right to terminate the study at any time. Should 
this be necessary, both parties will arrange discontinuation procedures and notify the appropriate 
regulatory authority(ies), and IRB/IECs. In terminating the study, Lakewood-Amedex and the 
investigator will assure that adequate consideration is given to the protection of the subjects’ 
interests. 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 51 
 
9 
REFERENCES 
1. 
National intelligence estimate: the global infectious disease threat and its 
implications for the United States. Environ Change Secur Proj Rep. 2000 
Summer;(6):33-65. Publication NIE 99-17D. 
2. 
Institute of Medicine, America’s Vital Interest in Global Health: Protecting Our 
People, Enhancing Our Economy, and Advancing our International Interests 
(National Academy Press, Washington, DC, 1997); 
http//www.nap.edu/readingroom/books/avi/. 
3. 
World Health Organization 2007 Tuberculosis Facts. and 2014 TB Facts 
http://www.who.int/features/factfiles/tuberculosis/en/. 
4. 
Lewis, K.. Platforms for antibiotic discovery.  Nature Reviews Drug Discovery 
2013 12:p371-387 
5. 
U.S. Department of Health and Human Services, Centers for Disease Control and 
Prevention. Antibiotic resistant threats in the United States, 2013. 
6. 
Dale, R.M.K., Schnell, G., Zhang, R.J.D., and Wong, J.P..  Therapeutic efficacy of 
aerosolized liposome-encapsulated nubiotic against pulmonary Pseudomonas 
aeruginosa infection. Therapy (2007) vol 4 (4) p441-9.   
7. 
Dale, R.M.K., Schnell, G., and Wong. J.P..  Therapeutic Efficacy of “Nubiotics” 
against Burn Wound Infections by Pseudomonas aeruginosa. Antimicrobial 
Agents and Chemotherapy  Aug (2004) vol 48 (8), p2918-2923.   
8. 
Cao, S., Sun, L.Q., and Wang, M... Antimicrobial activity and mechanism of 
action of Nu-3, a protonated modified nucleotide. Annals of Clinical Microbiology 
And Antimicrobials 2011 10(1) p1-9. 
9. 
Roderic M.K. Dale, United States Patent No. 7,868,162 B1 “ Antimicrobial and 
Antiviral Compounds and Methods of Their Use”  Jan 11, 2011 
10. 
Lipsky, B., Berendt, A.,Cornia, P., Pile, J., Peters, E., Armstrong, D.,Deery, H., 
Embil, J., Joseph, W., Karchimer, Pinzor, M., and Sennerville, E.. 2012 Infectious 
Diseases Society of America Clinical Practice Guideline for the Diagnosis and 
Treatment of Diabetic Foot Infections. IDSA Guidelines for Diabetic Foot 
Infections. CID 2:254p 1679-1684 
11. 
Chen., L. and Wen, Y. 2011 The role of bacterial biofilm in persistent infections 
and control strategies. Int. J. Oral Sci. 3:p66-73. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 52 
 
12. 
Akiyoshi, D., Dilo, J., and DiTullio, P., Novel Bisphosphocin Nu-3 Demonstrates 
Rapid Killing of Bacteria Encased in Biofilm In vitro.  Abstract 2013, 53rd ICAAC 
Sept. 10-13, Denver, CO. 
 
 
 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 53 
 
10 
 APPENDICES 
 


Bisphosphocin NU-3 
Protocol #LAI2014- l 
Lakewood Amedex 
Appendix A. Project Timeline and Flow Chart 
Obtain Infonned Consent 
Screen Subject 
Assign to Open Cohort 
SAD Ann 1: Cohort 1 - 0.1% or 
lmg/ml 
(n=lO) 
(2 Placebo; 8 Nu-3) 
Perform Infonned Consent and 
Docwnent 
Pregnancy and Drug Screen 
<24h Before Treatment 
Apply Treatment 
Follow up Visit 3 
PI Review 
... SAD Ann 1 :Cohort 2 - 1 % or 
at 24hr Examination 
lOmg/ml 
No Safety Issues. Proceed to MAD 
~= 10) 
Ann - Cohort 1 
(2 Placebo; 8 Nu-3) 
Perform Informed Consent and 
Docwnent 
Instruct on Treatment 
Observe Subject Apply 1st Treatment 
Pregnancy and Drug Screen 
Follow up Visit 5 
<24h Apply Treatment 
Follow up Visit 3 
SMC Review After All Subject 
PI Review at 24hr Examination 
Have Completed Cohort 1 
No Safety Issues. Proceed to MAD 
No Safety Issues. Proceed to Next 
Cohort 
Ann Cohort 2 
,_ 
Instruct on Treatment 
Observe Subject Apply 1st 
Treatment 
Follow up Visit 5 
SMC Review After All Subject 
Have Completed Cohort 2 
No Safety Issues. Proceed to 
Next Cohort 
Page 54 
Revised - 24SEP2015 
r+ SAD Ann 1: Cohort 3 - 2% or 20mg/ml 
(n=lO) 
(2 Placebo; 8 Nu-3) 
Perform Informed Consent and 
Document 
Pregnancy and Drug Screen 
<24h Before Treatment 
Review Informed Consent & 
Document 
t, Apply Treatment 
Follow up Day 2 
PI Review at 24hr Examination 
No Safety Issues. Proceed to MAD 
1-
Ann 2 Cohort 3 


Bisphosphocin NU-3 
Protocol #LAI2014-l 
Lakewood Amedex 
Page 55 
Revised - 24SEP2015 
Instruct on Treatment 
Observe Subject Apply 151 Treatment 
Follow up Visit 5 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Revised – 24SEP2015 
 
 
Page 56 
 
Appendix B:  Schedule of Events 
Key 
a Visit window 24 hours ± 2 hours post Visit 2 
b Visit window 8 days + 1 day post Visit 2 
c Visit window 14 days ± 1 day post Visit 2  
 
1 Height and weight (screening, Day 1, follow-up visit and ET), temperature, resting blood pressure (BP), heart and respiration rate 
2 Resting BP, heart and respiration rate to be obtained prior to and 1 hour post SAD Treatment 
3 Urine pregnancy test for all female subjects of childbearing potential 
4  Radiography, MRI and/or CT evaluation required within 2 weeks of Visit 1 (Screening Visit), at all other visits only necessary if determined by PI 5 
 
Upon SMC review and safety approval of 1st five (5) subjects in Cohorts 2 and 3, Principal Investigator (PI) may elect to extend treatment to fourteen (14) days if in the opinion 
of the PI the subject is showing improvement at Visit 4. 
5 Signs and symptoms of adverse events to be obtained 1 hour post SAD Treatment 
Event 
Visit 1 
Screening 
Visit  
Visit 2 
SAD 
Treatment  
Visit 
3a 
 
 Visit 
4b   
 
Visit 
5c  
 
Early 
Termination 
(ET) Visit 
Informed Consent Process 
X 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
Physical Exam 
X 
 
 
 
X 
X 
Vital Signs1  
X 
X2 
X 
X 
X 
X 
CBC, CMP Blood Draw 
X 
X 
 
X 
X 
X 
Collect ulcer tissue sample for microbiological evaluation 
X 
X 
 
X 
X 
X 
Urine Pregnancy Test (HCG)3 
X 
X 
 
 
 
Drug and ETOH toxicity screen 
X 
X 
 
 
 
 
Visual Examination of Chronic Ulcer 
X 
X 
X 
X 
X 
X 
Radiograph, MRI and/or CT evaluation of the infected ulcer4 
X 
 
 
X 
 
 
Photo Documentation and Calculation of target ulcer area and depth – 
ARANZ Silhouette™ Medical System 
X 
X 
X 
X 
X 
X 
Score target ulcer using DUSS Scoring System 
X 
X 
 
X 
X 
 
Rating of the infected ulcer using DFI Wound Scoring system 
X 
 
 
X 
X 
 
Investigational Product 
 
X 
X 
 
 
 
Concomitant Medication 
X 
X 
X 
X 
X 
X 
Adverse Event 
X 
X5 
X 
X 
X 
X 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Original – 31Oct2014 
 
 
Page 57 
 
Appendix C:  Use of the Aranz Medical Silhouette™ System for Photo documentation of 
the DFU and Calculation of Area and Depth, and Volume. 
 
Capturing a Wound Image 
 
1. Log in to Silhouette Connect and Select the Patient to be imaged. 
2. Review the patient details and select Capture Images Button.  The laser lines should 
come on from the Silhouette Star Camera. 
3. To capture the wound image position the Silhouette Star Camera directly above the 
wound and move the camera up or down so that the laser lines form a star.  The center of 
the laser line star should be over the deepest part of the wound. 
4. Capture an image of the wound by pressing the bottom on the camera.   
5. Repeat the process to capture multiple images. 
6. All images will be uploaded to the SilhouetteConnect Database on the tablet. 
 
Calculating the Ulcer Area, Volume, and Depth 
 
1. Select the Ulcer image to be used for assessment on the tablet. 
2. Enlarge the image so the perimeter of the ulcer is easily visible 
3. Outline the perimeter of the ulcer by either clicking around the perimeter so that the 
entire ulcer is surrounded by dots or tracing the perimeter of the ulcer with a pointer or 
finger.   
4. Clicking on the circular target to initiate calculation of the area, volume, and depth of the 
ulcer by the SilhouetteConnect software. 
5. Click on the Notes(?) button to enter comments on the ulcer margins, appearance, and 
surrounding skin 
6. Click Generate pdf report and Save to Store it to the database. 
7. Click Send to export to the ClinicalInk Database. 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood Amedex 
Original – 31Oct2014 
 
 
Page 58 
 
Appendix D: Subject Home Treatment Log:  
 
Subject ID No.: 
 
 
 
 
 
 
 
 
 
Study Drug Number: 
 
 
 
 
 
 
 
Issue Date: 
 
 
 
 
 
Return Date:  
 
 
 
 
Instructions:  Subjects/caregivers should log both the date and approximate time of each 
treatment and the person administering the study drug should initial in the corresponding box.  A 
space is also provided for the subject/caregiver to write any comments concerning the 
administration or observations of the ulcer. 
 
Day 
Treatment 
1 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Treatment 
2 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Comments 
1 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
2 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
3 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
4 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
5 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
6 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
7 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
 


Study Title: 
Sponsor: 
IND No.: 
EudraCT Number 
Indication: 
Protocol ID: 
Protocol Coordinator: 
Medical Monitor 
Protocol Version/Date: 
~,J LAKEWoool 
Jjl1 AMEDEX j 
CLINICAL STUDY PROTOCOL 
A Phase I/Ila, Randomized Double Blind, Placebo-Controlled, Dose 
Escalating Study to Evaluate the Safety and Tolerability of Topically 
Applied Bisphosphocin Nu-3 on Infected Diabetic Ulcers of Subjects 
With Type I or II Diabetes Mellitus 
Lakewood-Amedex, Inc. 
9015 Town Center, Pkwy, Unit 110 
Bradenton, FL 34202 
Not Applicable 
Diabetic Foot Infections 
LAI2014-1 
vl.O 
31October 2014 
CONFIDENTIALITY STATEMENT 
The info1mation contained in this document, particularly unpublished data, is the property or under control of 
Lakewood-Amedex, and is provided to you in confidence as an investigator, potential investigator, or consultant, for 
review by you, yom· staff, and an applicable Institutional Review Board or Independent Ethics Conunittee. The 
info1mation is only to be used by you in com1ection with authorized clinical studies of the investigational drug 
described in the protocol. You will not disclose any of the infonnation to others without written authorization from 
Lakewood-Ame.dex, Inc., except to the extent necessruy to obtain infonned consent from those persons to whom the 
dmg may be administered. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood-Amedex 
Original - 310ct2014 
Lakewood-Amedex Bisphosphocin NU-3 Clinical Protocol 
A PHASE I/Ila, RANDOMIZED DOUBLE BLIND, PLACEBO CONTROLLED, DOSE 
ESCALATING STUDY TO EVALUATE THE SAFETY AND TOLERABILITY OF 
TOPICALLY APPLIED BISPHOSPHOCIN™ NU-3 ON INFECTED DIABETIC 
ULCERS OF SUBJECTS WITH TYPE I OR II DIABETES MELLITUS 
Version 1.0 
31October2014 
Approved by: 
II 
Page 2 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Original – 31Oct2014 
 
 
 
Page 3 
 
STUDY ACKNOWLEDGEMENT 
 
A  Phase I/IIa, Randomized Double Blind, Placebo-Controlled, Dose Escalating Study to 
Evaluate the Safety and Tolerability of Topically Applied Bisphosphocin™ Nu-3 on Infected 
Diabetic Ulcers of Subjects With Type I or II Diabetes Mellitus 
Version 1.0:  31 October 2014 
 
INVESTIGATOR STATEMENT 
 
I have read the protocol and Investigator’s Brochure, including all appendices, and I agree that it 
contains all necessary details for me and my staff to conduct this study as described.  I will 
conduct this study as outlined herein and will make a reasonable effort to complete the study 
within the time designated. 
I will provide all study personnel under my supervision copies of the protocol and access to all 
information provided by Lakewood -Amedex.  I will discuss this material with them to ensure 
that they are fully informed about the drugs and the study. 
 
 
 
 
 
______________________________ 
______________________________ 
Principal Investigator Name (Printed) 
Signature 
 
______________________________ 
 
Date 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Original – 31Oct2014 
 
 
 
Page 4 
 
GLOSSARY OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
° C  
degrees Celsius 
° F 
degrees Fahrenheit 
AE 
Adverse Event/Adverse Experience 
ALT 
Alanine Aminotransferase 
API 
Active Pharmaceutical Ingredient 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Unit 
CLSI 
Clinical and Laboratory Standards Institute 
CMP 
Comprehensive Metabolic Panel 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTCAE 
Common Terminology Criteria for Adverse Events v4.0 
DFI 
Diabetic Foot Infection 
DSMB 
Data and Safety Monitoring Board 
DUSS 
Diabetic Ulcer Severity Score 
ELSD 
Evaporative Light Scatter Detection 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
hCG 
Human Chorionic Gonadotropin 
HCV 
Hepatitis C Virus 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HPLC 
High Performance Liquid Chromatography 
IB 
Investigator’s Brochure 
ICH 
International Committee on Harmonization 
ICMJE 
International Committee of Medical Journal Editors 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IR 
Infrared Spectroscopy 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
LAI 
Lakewood-Amedex, Inc 
LCMS 
Liquid Chromatography Mass Spectrometry 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Original – 31Oct2014 
 
 
 
Page 5 
 
LLN 
Lower Limit of Normal 
MAD 
Multiple Ascending Dose 
MBC 
Minimum Bactericidal Concentration 
MIC 
Minimum Inhibitory Concentration 
MOA 
Mechanism of Action 
MRSA 
Methicillin Resistant Staphylococcus aureus 
MS 
Mass Spectroscopy 
NDM-1 
New Delhi Metallo-Beta Lactamase-1 
NMR 
Nuclear Magnetic Resonance 
PBS 
Phosphate Buffered Saline 
PI 
Principal Investigator 
RBCs 
Red Blood Cells 
SAD 
Single Ascending Dose 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
TS 
Trypic Soy 
TSBD 
Trypic Soy Broth with 1% Dextrose 
ULN 
Upper Limit of Normal 
US 
United States 
WBC 
White Blood Cell 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
Lakewood-Amedex 
Original – 31Oct2014 
 
 
 
Page 6 
 
TABLE OF CONTENTS 
 
1 
INTRODUCTION ....................................................................................................................................... 12 
1.1 
Background Information ..................................................................................................................... 12 
1.2 
Bisphosphocin Nu-3 ........................................................................................................................... 14 
1.2.1 
General Information............................................................................................................ 14 
1.2.2 
Pre-Clinical Pharmacology and Toxicology ....................................................................... 15 
1.2.3 
Clinical Trials of Bisphosphocin Nu-3 ............................................................................... 22 
1.3 
Rationale for Current Study ................................................................................................................ 22 
2 
OBJECTIVES .............................................................................................................................................. 24 
3 
STUDY DESIGN ......................................................................................................................................... 26 
3.1 
Selection and Exclusion of Subjects ................................................................................................... 27 
3.2 
Inclusion Criteria ................................................................................................................................ 27 
3.3 
Exclusion Criteria ............................................................................................................................... 27 
4 
STUDY DRUGS .......................................................................................................................................... 28 
4.1 
Randomization, Blinding, and Drug Product/Placebo Packaging and Labeling ................................. 28 
4.2 
Description and Handling of Bisphosphocin Nu-3 ............................................................................. 28 
4.3 
Dosage and Administration of Bisphosphocin Nu-3 or Placebo ......................................................... 29 
4.4 
Prior and Concomitant Medications ................................................................................................... 29 
5 
STUDY PROCEDURES ............................................................................................................................. 30 
5.1 
Subject Enrollment and Treatment Assignment ................................................................................. 30 
5.2 
Pre-treatment Assessments ................................................................................................................. 30 
5.2.1 
Screening Visits .................................................................................................................. 30 
5.3 
Enrollment/Baseline in Single Ascending Dose Arm (SAD) ............................................................. 31 
5.4 
Follow-up Single Ascending Dose Arm ............................................................................................. 32 
5.5 
Enrollment/Baseline in Multiple Ascending Dose (MAD) Arm ........................................................ 33 
5.6 
Final Study Visit – Visit 5 (7-Day MAD) or Visit 6 (14-Day MAD) ................................................. 34 
5.7 
Early Termination Visit ...................................................................................................................... 35 
5.8 
Unscheduled Visit ............................................................................................................................... 36 
6 
ADVERSE EVENTS AND TOXICITY MANAGEMENT ........................................................................ 37 
6.1 
Specification of Safety Parameters ..................................................................................................... 37 
6.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters ........................... 37 
6.2.1 
Adverse Events ................................................................................................................... 37 
6.2.2 
Reactogenicity .................................................................................................................... 38 
6.2.3 
Serious Adverse Events ...................................................................................................... 39 
6.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values 
or Abnormal Clinical Findings ........................................................................................... 40 
6.3 
Serious Advent Event Reporting Procedures ...................................................................................... 40 
6.3.1 
Reporting Pregnancy .......................................................................................................... 41 
6.4 
Safety Oversight ................................................................................................................................. 42 
6.4.1 
Independent Safety Monitor Committee ............................................................................. 42 
6.4.2 
Study Termination .............................................................................................................. 43 
6.4.3 
Removal of Subjects from the Trial or Study Drug ............................................................ 44 
6.4.4 
Handling Withdrawals ........................................................................................................ 44 


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7 
STATISTICAL CONSIDERATIONS ......................................................................................................... 46 
7.1 
Study Hypothesis ................................................................................................................................ 46 
7.2 
Sample Size Considerations ................................................................................................................ 46 
7.3 
Planned Interim Analysis .................................................................................................................... 46 
7.3.1 
Safety Review ..................................................................................................................... 47 
7.4 
Statistical Analysis Overview ............................................................................................................. 47 
8 
RESPONSIBILITIES ................................................................................................................................... 48 
8.1 
Investigator Responsibilities ............................................................................................................... 48 
8.1.1 
Good Clinical Practice ........................................................................................................ 48 
8.1.2 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) 
Approval ............................................................................................................................. 48 
8.1.3 
Informed Consent ............................................................................................................... 49 
8.1.4 
Confidentiality .................................................................................................................... 49 
8.1.5 
Study Files and Retention of Records ................................................................................. 49 
8.1.6 
Case Report Forms ............................................................................................................. 50 
8.1.7 
Drug Accountability ........................................................................................................... 51 
8.1.8 
Inspections .......................................................................................................................... 51 
8.1.9 
Protocol Compliance .......................................................................................................... 51 
8.2 
Sponsor Responsibilities ..................................................................................................................... 52 
8.2.1 
Protocol Modifications ....................................................................................................... 52 
8.2.2 
Study Report and Publications ............................................................................................ 52 
8.3 
Joint Investigator/Sponsor Responsibilities ........................................................................................ 53 
8.3.1 
Access to Information for Monitoring ................................................................................ 53 
8.3.2 
Access to Information for Auditing or Inspections ............................................................. 53 
8.3.3 
Study Discontinuation ........................................................................................................ 53 
9 
REFERENCES ............................................................................................................................................ 54 
10 
APPENDICES ............................................................................................................................................. 56 
 
Appendix A. 
Project Timeline and Flow Chart .................................................................................. 57 
Appendix B:  
Schedule of Events ........................................................................................................ 59 
Appendix C:  
Use of the Aranz Medical Silhouette™ System for Photo 
documentation of the DFU and Calculation of Area and Depth, and 
Volume. ......................................................................................................................... 60 
Appendix D: 
Subject Home Treatment Log: ...................................................................................... 61 
 
 
 


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Page 8 
 
LIST OF IN-TEXT TABLES 
 
Table 1. 
Bisphosphocin Nu-3 Activity in a Minimum Inhibitory Assay 
according to CLSI Guidelines ....................................................................................... 15 
Table 2. 
Anti-fungal Activity of Bisphosphocin Nu-3 ................................................................ 16 
Table 3. 
Bactericidal activity of Nu-3 against bacteria established in biofilm ............................ 17 
Table 4.   
Time and concentration dependent activity of Nu-3 against biofilm 
forming bacterial strains. ............................................................................................... 18 
Table 5. 
Log reduction in bacterial cell counts in surgical wound model ................................... 19 
Table 6. 
Mouse Superficial Skin Polymicrobial Infection Bacterial Counts ............................... 20 
Table 7. 
Summary of Nu-3 Preclinical Toxicology Data ............................................................ 21 
Table 8. 
Product Label Specifications ......................................................................................... 28 
 
 
 
LIST OF IN-TEXT FIGURES 
 
Figure 1. 
Structure, Chemical Name and Chemical Formula of 
Bisphosphocin™ Nu-3 .................................................................................................. 14 
 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood-Amedex 
Title: 
Phase: 
Population: 
Number of Sites: 
Study Duration: 
Subject Participation Duration: 
Description of Agent or 
Intervention: 
Objectives: 
Estimated Time to Complete 
Enrollment: 
Original - 310ct2014 
PROTOCOL SUMMARY 
A 
Phase I/Ila, Randomized Double Blind, Placebo-Controlled, 
Dose Escalating Snidy to Evaluate the Safety and Tolerability of 
Topically Applied Bisphosphocin™ Nu-3 on Infected Diabetic 
Ulcers of Subjects With Type I or II Diabetes Mellinis 
I/Ila 
Up to 30 subjects over 18 years of age suffering from diabetes 
mellinis and a chronic infected diabetic ulcer(s). 
2 
6 months 
44 days (includes screening visit, snidy treatment and all follow-ups 
visits through Day 15 or Day 22 post treatment. 
Bisphosphocin™ Nu-3 to be applied as a topical solution to the 
ulcer and covered by a bandage. This snidy includes three dose 
ascending coho1ts. Within each coho1t there are two phases with 
both a single ascending dose and multiple ascending dose aims. The 
three dose levels (coho1ts) ai·e 0.1 % (lmg/ml, anticipated low dose), 
1 % (1 Omg/ml, anticipated therapeutic dose, and 2% (20mg/ml, 
anticipated high dose). 
to an open 
ecte u cer. Assessment of safety w1 
e 
etennmed by visual evaluation of the ulcer, vital signs, CBC 
analysis, CMP analysis, ai1d physical exainination, as well as the 
incidence and severity of emergent adverse events that occur during 
the study paiticipation. The study will be monitored by the Safety 
Monitoring Committee (SMC). 
Secondary: To assess the clinical and rnicrobiological response to 
bisphosphocin™ Nu-3 administered topically to the ulcer, including 
the improvement of wound appearance and elimination of the 
pathogenic bacte1ia. 
24 Weeks 
Page 9 


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Original – 31Oct2014 
 
 
 
Page 10 
 
Study Design: 
Allocation: Randomized, Placebo-Controlled 
Endpoint: Safety and Tolerability 
Enrollment Model: Sequential Assignment 
Masking: Double Blind (Subject, Caregiver, Investigator) 
Primary Purpose: Safety 
Description of Study Design 
Phase I/IIa, three cohort ascending dose with two dosing arms per 
cohort, study in Type I or II diabetes mellitus subjects with a 
chronic infected diabetic ulcer defined as having a DUSS score of 0 
to 3 and DFI wound score of 1 to 9.  The study proposes to use the 
same subject in SAD Arm 1 and MAD Arm 2 of each cohort 
The study is designed to run the cohorts in series with the 
completion of the first cohort before initiating the next dosing level.  
Prior to initiating treatment of the infected diabetic ulcer, subject 
will be evaluated for skin sensitivity to Nu-3 by applying 1 drop to 
the forearm and monitoring the site for any acute reaction over a 
10-15 minute interval for any sign of reddening, swelling, and/or 
irritation.  In addition, at all study visits the ulcer will be visually 
examined for any changes and photographed using the Aranz 
Medical Silhouette™ system that will calculate area and depth of 
the ulcer. 
In Arm 1, eligible subjects will be treated with a single application 
of Nu-3 or placebo in 4 to 1 ratio to judge the initial safety of Nu-3 
over a brief 15-30 minute interval and 24-hr interval. 
Bisphosphocin Nu-3 will be applied topically to the chronic 
infected ulcer, covered with a non-abrasive bandage following the 
initial observation period.  The subject will be released with verbal 
instructions to leave the bandage on the wound and return for a 
follow up visit within 24h ± 2h.  At the follow up visit, the bandage 
will be removed, the ulcer visually examined and the subject 
cleared for the MAD Arm 2 based on the recommendation of the PI 
and absence of any SAEs.   
In Arm 2, eligible subjects which are those who have been 
approved by the PI after the Visit 2 examination will be instructed 
in the proper application of bisphosphocin Nu-3.  The subjects will 
be observed applying the first dose in the clinic to ensure 
compliance. Subjects will then be given a 7 day supply and sent 
home to continue treatment.  Visit 4 or earlier in the case of any 
adverse events, subjects will return to the clinic for an examination, 
including visual examination of the ulcer, vital signs, adverse 
events, photo documentation, collection of a sample for 
microbiology and concomitant medication use. For Cohorts 2 and 3, 
an extension of the treatment duration to fourteen (14) days would 
only be initiated after five subjects in Cohorts 2 and 3 completed 


Bisphosphocin NU-3 
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Original – 31Oct2014 
 
 
 
Page 11 
 
the seven (7) day treatment and approval was given by the SMC.  
The decision to extend treatment of individual subjects showing a 
clinical response would then be made by the Principal Investigator. 
A final follow up visit will be scheduled +7 days after last dose of 
study medication (Day 15 or Day 22) for a complete examination as 
described above. 


Bisphosphocin NU-3 
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Original – 31Oct2014 
 
 
 
Page 12 
 
1 
INTRODUCTION 
1.1 
Background Information 
Infectious disease represents a growing threat to world health due to the emergence of new 
viruses and the increase in bacterial resistance to antibiotics1,2.  It is now estimated that almost 
one-third of the world population or 2 billion people are infected with Mycobacterium 
tuberculosis with between 3-5% of these cases being multidrug or extremely drug resistant3.  In 
addition, bacteria have repeatedly demonstrated the ability to become resistant to antibiotics 
much faster than new compounds or sub-classes can be identified.  The NDM-1 resistance gene 
represents a perfect example of the ability of bacteria to not only acquire resistance but broad 
spectrum resistance on a single plasmid that provides resistance against most of the major or 
widely used classes of antibiotics.  This highlights the shortcomings of current and past antibiotic 
research which has relied on derivatives of existing classes that all share a common or similar 
core structure making them susceptible to inactivation by a single enzyme.  In addition, 
traditional antibiotics share a related mechanism of action in that they all function through a 
target protein or receptor molecule to inhibit a cellular process to kill the bacteria allowing the 
bacteria to rapidly become resistant by acquiring a beneficial mutation in the target molecule4.   
Over the past ten years, the amount of research and development invested by large 
pharmaceutical companies in the discovery of novel antibiotics has steadily declined while 
bacteria have become increasingly resistant to the currently available classes of antibiotics.  
According to the Centers for Disease Control and Prevention, approximately 2 million people 
each year are infected with bacteria resistant to at least one antibiotic which results in around 
23,000 deaths5.  Methicillin resistant Staphylococcus aureus (MRSA) has become a major public 
health issue resulting in the closing of several public schools for cleaning and demonstrating that 
this is a problem no longer restricted to hospitals.  No other factor highlights the need for a 
greater effort into the research and development of novel anti-bacterial compounds than the ever 
increasing ability of bacteria to rapidly acquire resistance to current and new derivatives of 
existing antibiotics.  The recent identification of a strain of Streptococcus resistant to more than 
18 different antibiotics highlights this fact and the obvious need for novel anti-infective 
compounds like Bisphosphocins.   
Lakewood Amedex has discovered a new class of broad spectrum antimicrobial, termed 
bisphosphocins™, formerly known as nubiotics, that have been proven effective in vitro at 
killing 70 different strains of bacteria, including all Category A pathogens and in vivo against the 
difficult to treat infections caused by Francisella tularensis, Helicobacter pylori, and 
Pseudomonas aeruginosa.  Bisphosphocins are a new class of synthetic broad spectrum 
antimicrobial that are characterized by a core structure bridging two phosphate groups attached 
to even chain alkyl or alcohol groups.  The compounds are activated through an acidification 
process which is reversible by exposing the compounds to a buffer or base.  The compounds 
were found to be bactericidal and effective in killing slow growing or stationary bacteria.  
Preclinical studies have demonstrated the effectiveness of these compounds in the treatment of a 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
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Original – 31Oct2014 
 
 
 
Page 13 
 
number of serious bacterial infections6,7,8.  In addition, the compounds were initially developed 
as a homeopathic treatment and were found to reduce the severity of several common infection 
associated with acne, diabetic foot infection, and skin infections9.  The compounds function 
through a unique mechanism of action, are amendable to multiple formulations, and routes of 
administrations enabling a clinical development plan distinct from most traditional antibiotics.  
The current clinical development plan entails development of a topical formulation of 
bisphosphocin™ Nu-3 for a number of topical infections such as diabetic foot infections, chronic 
urinary tract infections, and pulmonary infections using direct delivery of the compound to the 
site of the infection.  This clinical approach will demonstrate initial safety in humans and keep 
delivery of the antimicrobial localized to the site of infection and thus decrease the chance of 
bacteria becoming resistant.  This clinical study will also yield important data for planning 
follow on clinical trials, including those involving Nu-3 for systemic administration to treat 
serious gram negative infections such as complicated urinary tract infections.   
Diabetic foot infections are one of the most common complications for people who suffer from 
diabetes and a frequent cause of hospitalization10.  According to the American Diabetes 
Association approximately 8.3% of the US population suffers from diabetes and in 2006 there 
were 65,700 non-traumatic amputations performed on diabetics.  In addition, almost half of 
diabetic patients that have an amputation will develop an ulcer on the remaining limb within 
18 months after surgery.  The treatment of a diabetic foot infection is complicated as it requires 
both antimicrobial therapy to cure the infection and proper wound care management to heal the 
ulcer.  Curing the infection is particularly challenging since the infection tends to be 
polymicrobial in nature requiring a broad spectrum antibiotic, the ulcer is prone to re-infection, 
and the pathogenic bacteria are increasingly becoming resistant to most front line therapies.  In 
addition, recent studies have estimated that approximately 60-80% of chronic infections involve 
biofilm formation which makes the bacteria more resistant to traditional antibiotics11.  Therefore, 
proper management of an infected diabetic foot ulcer requires an antimicrobial therapy to cure or 
clear the infection to allow proper wound management or therapy to heal the ulcer and prevent 
relapse. 
Bisphosphocins™, a new class of broad spectrum antimicrobials, possess a number of 
characteristics that make these compounds extremely well-suited to the management of diabetic 
foot infections.  First, bisphosphocins™ are rapidly and directly bactericidal functioning through 
a unique mechanism of action providing the ability to quickly cure an infection.  The compounds 
are fairly low in molecule weight, have an excellent safety profile, and have shown good 
penetration into wounds.  In addition, there are no known bacterial mechanisms of resistance and 
no resistant bacteria have been identified to date eliminating a common cause of treatment 
failure7. Finally, bisphosphocins have been demonstrated highly effective at killing bacteria 
established in biofilms12 which is increasingly become associated with many chronic infections.  
The preclinical studies conducted on the bisphosphocin™ class clearly support their evaluation 
in the proposed Phase I/IIa safety study in diabetic foot infections. 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood-Amedex 
1.2 
Bisphosphocin Nu-3 
1.2.1 
General Information 
Original - 310ct2014 
Bisphosphocins™ were originally discovered by-
while conducting research in 
the field of antisense technology and attempting t~mg gene silencing. The initial 
compounds identified were protonated/acidified oligonucleotides that demonstrated bactericidal 
activity independent of nucleotide sequence. Fmiher experimentation expanded the class to 
protonated/acidified nucleotides and derivative molecules that exhibit chemical stability, acid pH 
resistance, and nuclease resistance enabling fonnulations for oral, intravenous, pulmonaiy, and 
topical delive1y. The lead compound, designated Nu-3, is a thymidine derivative that would be 
considered to be in the nucleotide analogue class of mugs (Figure 1). Experimental data has 
elucidated a non-receptor dependent mechanism of action involving disrnption of the bacterial 
cell membrane causing depolarization and rapid cell death. Bisphosphocins™ have an extremely 
broad spectrum of activity in vitro testing against over 70 different bacterial sti·ains canying 
various antibiotic resistant mai·kers. There is cmTently no known mechanism of resistance to the 
bisphosphocin class and because they were synthetically derived there is no natural pool of 
resistant bacteria. In addition, panelling the compounds against a number of strains with known 
antibiotic resistance or clinical isolates has failed to identify any cross-resistance or resistance 
mechanisms. 
The following preclinical phannacological and toxicology data is provided as a 
brief smnmaiy of the activity of bisphosphocin Nu-3 to support its potential as a ti·eatinent for 
diabetic foot infections and this clinical protocol should be read in conjunction with the 
Investigator Brochure on the product. 
Figure 1. 
Structure, Chemical Name and Chemical Formula of Bisphosphocin™ Nu-3 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
 
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Original – 31Oct2014 
 
 
 
Page 15 
 
1.2.2 Pre-Clinical Pharmacology and Toxicology 
Specific Pharmacology 
Initial experiments revealed that bisphosphocins™ had an antimicrobial activity distinct from 
any currently existing antibiotic class.  Extensive in vitro and in vivo experiments have been 
conducted to further define their activity, identify their mechanism of action, and begin to 
establish a dose range to allow entry in human clinical trials.  An important factor to consider in 
the review of the following experimental results is that it is now known bisphosphocins™ do not 
follow the simple inhibitory kinetic model of one compound binding to one target molecule of 
the current classes of antibiotics but a more complex kinetic model involving the binding of 
multiple molecules to a bacterium resulting in direct bacteria cell death.  For this reason, the 
current hypothesis is bisphosphocin™ dosing in vivo will be based on total dose delivered and 
not on maintaining a circulating level above the minimum inhibitory concentration or at the site 
of infection to achieve an efficacy outcome because the molecules rapidly bind and kill bacteria 
as opposed to inhibiting growth.  Therefore, the therapeutic effect of bisphosphocins™ can be 
manipulated by not only controlling the dose but frequency and duration of treatment not 
because of the pharmacokinetics of the compound but due to the fact that each dose will result in 
a reduction of bacterial load. The following experiments are presented to further define, 
distinguish and compare the activity of the bisphosphocin class from traditional antibiotics in 
order to develop a comprehensive clinical plan to enable evaluation in humans.  The majority of 
the experimental data presented in the following sections was performed in the laboratory of 
outside collaborators or researchers who then published their results6,7,8,12 or by independent 
Contract Research Organization following the current guidelines for antimicrobial testing.  Due 
to the unique MOA of the bisphosphocin™ class some standard assays were modified to enable 
the evaluation which has been noted in the protocol. Table 1 and Table 2 summarizes the activity 
of Nu-3 in the standard MIC assay against bacterial pathogens commonly found in diabetic foot 
infections and against common fungal pathogens. 
Table 1. 
Bisphosphocin Nu-3 Activity in a Minimum Inhibitory Assay according to 
CLSI Guidelines 
Organism 
Number of Strains 
MIC  
mg/ml 
Acinetobacter baumannii 
N=15 
5-10 
Enterococcus faecalis, VRE 
N=15 
5 
Enterococcus faecalis, VSE 
N=15 
5 
Enterococcus faecium, VRE 
N=15 
5 
Enterococcus faecium, VRE 
N=15 
5 
Escherichia coli 
N=15 
10 
Proteus mirabilis 
N=15 
5-10 
Proteus vulgaris 
N=15 
5-10 


Bisphosphocin NU-3 
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Organism 
Number of Strains 
MIC  
mg/ml 
Pseudomonas aeruginosa 
N=5 
2.5-5 
Pseudomonas aeruginosa 
N=15 
5-10 
Serratia marcescens 
N=15 
10 
Staphylococcus aureus, MSSA 
N=15 
5-10 
Staphylococcus aureus, MRSA 
N=15 
5-10 
Staphylococcus epidermidis, MRSE 
N=15 
10 
Staphylococcus epidermidis, MSSE 
N=15 
10 
Streptococcus agalactiae 
N=15 
10 
 
Table 2. 
Anti-fungal Activity of Bisphosphocin Nu-3 
Yeast Strain 
ATCC # 
Drug Resistance 
MIC mg/ml 
 
 
 
Nu3 
Candida albicans 
44373 
5-Fluorocytosine 
≤0.53 
Candida albicans 
44374 
 
≤0.53 
Saccharomyces pastorius 
2366 
 
≤0.07 
Trichophytan mentagrophytes var. interdigitale 
200099 
 
5.0 
 
Evaluation of Nu-3 Activity against Bacteria Established in Biofilm 
The following studies were performed by 
 
 and were designed to evaluate the activity of Nu-3 
against bacteria established in biofilms which is increasing becoming associated with chronic 
infections12,unpublished.  Biofilm was formed in borosilicate glass tubes by standard methods and 
then treated with Nu-3 or kept as untreated controls.  As shown in Table 3, Nu-3 was able to 
penetrate the biofilm and kill the bacteria as judged by the lack of growth in Nu-3 treated tubes.  


Bisphosphocin NU-3 
Protocol # 
Lakewood Amedex 
 
Page 17 
 
Table 3. 
Bactericidal activity of Nu-3 against bacteria established in biofilm 
Bacterial Strain 
Designation 
Assessment 
Treatment 
 
 
Nu-3 Exposure 
(10mg/ml) 
Media 
Control 
10min 
30min 
Klebsiella pneumoniae Kp-S1 
Clinical 
Growth 
N.G. 
1.1 x 109 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Acinetobacter baumannii 
BAA-1605 
Growth 
N.G. 
2 x 109 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus epidermidis 
35984 
Growth 
N.G. 
2.7 x 107 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus epidermidis 
PCI1200 
Growth 
N.G. 
5.2 x 108 
cfu/ml 
N.G. 
N.G. 
Biofilm 
 
 
 
 
Staphylococcus aureus 
NRS684 
Growth 
N.G. 
4.3 x 108 
cfu/ml 
5 x 108 
cfu/ml 
8.6 x 108 
cfu/ml 
Biofilm 
 
 
 
 
Staphylococcus aureus 
NRS732 
Growth 
N.G 
7.6 x 108 
cfu/ml 
N.G 
N.G 
Biofilm 
 
 
 
 
 


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Protocol #LAI2014-1 
 
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Original – 31Oct2014 
 
 
Page 18 
 
Assessment of the rate of kill of Nu-3 in 0.85% normal saline: 
 
Bacteria in the log phase of growth were collected by centrifugation, re-suspended in sterile 
saline (0.85% NaCl) at 1 x 105 CFU/ml. Nu-3 in sterile saline was added to a final concentration 
of 0.2 to 10mg/ml to the bacteria and the bacteria/Nu-3 mixture incubated at room temperature. 
At time points ranging from 5 to 60 minutes, aliquots were removed, diluted and plated onto TS 
agar plates for quantitation of the number of colony forming units (CFU's).  After 24h at 37°C, 
the number of CFU's were determined and the Nu-3 concentrations and exposure times resulting 
in 100% kill were determined (Table 4). 
Table 4.   
Time and concentration dependent activity of Nu-3 against biofilm forming 
bacterial strains. 
Bacterial Strain 
Nu-3 Concentration 
0.625 (mg/ml) 
1 (mg/ml) 
5 (mg/ml) 
10 (mg/ml) 
K. pneumoniae Sp1 
15 min 
 
5 min 
 
K. pneumoniae Sp5 
 
15 min 
5 min 
 
A. baumannii BAA-1605 
30 min 
 
10 min 
5 min 
S. aureus NRS684 
 
 
 
60 min 
S. aureus NRS732 
 
 
 
60 min 
 
 
Mouse Suture Wound Infection Model 
Briefly, a 10cm length of suture was threaded onto a needle and soaked for 45 minutes in 
undiluted cultures of S. aureus cvcc2248 or P. aeruginosa cvcc 5668 at 35°C for 8 hours.  The 
needle and sutures were removed, placed on filter paper, and dried at 4°C until implantation.  A 
1-cm long piece of suture was implanted under the skin in the shaved and washed mid-back of 
anesthetized animals using knots to secure it in place.  An incision was made between the knots 
on top of the suture line with a scalpel.  The animals were then divided into groups and treated as 
indicated at 4h and 8h post-surgery and then 3 times daily for 3 days with either placebo, Nu-3 or 
ciprofloxacin ointment.  On Day 5 the animals were euthanized and a 1cm2 area surrounding and 
including the wound was harvest for homogenization and CFU counts (Table 5).  The initial 
inoculum (i.e. CFU of bacteria in 1 cm length of suture) was determined by vortexing 1-cm of 
suture in bacterial growth media and plating serial dilutions to obtain bacteria counts. 


Bisphosphocin NU-3 
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Original – 31Oct2014 
 
 
Page 19 
 
Table 5. 
Log reduction in bacterial cell counts in surgical wound model 
Treatment  
Mean Bacterial Count ± SD (Log10 CFU/Wound) 
P. aeruginosa 
S. aureus 
Initial Inoculation  
4.93 ± 0.33 
4.79 ± 0.31 
Untreated  
7.52 ± 0.57 
7.64 ± 0.58 
Glycerin  
7.34 ± 0.27 
7.47 ± 0.34 
5% Nu-3 in Saline  
5.62 ± 0.61 
5.23 ± 0.5 
5% Nu-3 in Glycerin  
5.07 ± 0.53 
4.51 ± 0.55 
Ciprofloxacin HCl Ointment  
4.82 ± 0.58 
5.63 ± 0.31 
 
Mouse Superficial Skin Polymicrobial Infection Model 
Briefly, mice are pre-treated with cyclophosphamide four day prior to initiating the infection to 
suppress the immune system.  The mice are anesthetized and kept sedated while the fur on the 
back dorsal surface is shaved using electric clippers followed by ‘wet shaving’ with a disposable 
razor. The skin is then sterilized with a betadine wash followed by alcohol swab and abraded 
with a +50 grit emery board that results in a smooth, red, shiny appearance with no bleeding.  A 
droplet of the bacterial inoculum is placed directly on the abraded skin that contains 
Pseudomonas aeruginosa UNT034-1 (ATCC 27853) 6.35 Log10 CFU/ mouse and S. aureus 
UNT006-4 (smith) 5.65 Log10 CFU/ mouse.  The infected area is treated with 50 microliters of 
varying concentrations of bisphosphocin Nu-3, gentamycin, mupirocin, or normal saline twice 
daily for 3 days.  The mice are then euthanized, the area of infected skin excised, and the CFU of 
both bacteria is determined by culture or serial diluted samples of homogenized tissue (Table 6).  
The data shows that the top 5% dose of bisphosphocin Nu-3 results in a 1 log reduction in the 
CFU count of both S. aureus and P. aeruginosa.   


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood Amedex 
Original - 310ct2014 
Table 6. 
Mouse Superficial Skin Polymicrobial Infection Bacterial Counts 
S. aureus 
P. aeruginosa 
Compound 
Dose 
CFU 
SD 
CFU 
SD 
1.0 tng/mL 
9.22 
0.19 
6.35 
0.21 
10 mg/mL 
8.78 
0.24 
6.81 
0.23 
NU3 
2 0 mg/mL 
9.00 
0.27 
7.08 
0.19 
5 0 mg/mL 
7.61 
1.35 
5.65 
1.16 
Gentatnicin 
0.3% 
4.40 
0.85 
5.17 
0.87 
Mupirocin 
2.0% 
6.32 
1.01 
6.95 
0.51 
Vehicle 
saline 
8.73 
0.25 
6.3 
0.42 
Day4 
8.72 
0.52 
6.68 
0.69 
Infection Control 
4 ln· 
5.40 
0.45 
4.33 
0.83 
Preclinical Toxicology 
Summary of Preclinical Toxicology Data 
Nu-3 is the first in a new class of broad spectnnn antimicrobials, te1med bisphosphocins, being 
developed to treat a wide range of serious bacterial infections. Chemically Nu-3 would be 
considered to be in the nucleotide analog class of drngs and the following set of preclinical 
studies was designed based on the preclinical and clinical data collected over the past 50 years on 
this class. 
The nucleotide analog class of mu gs have a good safety profile even when 
adininistered long te1m at low doses and toxicities are generally associated with their 
interference of n01mal DNA/RNA synthesis. 
is a thymidine analog with both 
t e 3' an 5' sites 
oc e 
y uty p osp ate w ic prevents it from causing early chain 
te1mination by incorporation by the polymerase unlike most compounds in this class. Nu-3 is 
activated through an acidification step that results in an acidic or protonated compound that when 
dissolved in water has a pH between 2 and 2.5. The compound can be neutralized or unactivated 
by a strong base or buffer such as sodimn bicarbonate. In addition, the molecule was designed to 
minimize the potential for toxic side effects by utilizing a thymidine core molecule, natmal 
phosphate linkages, and even carbon butyl blocking groups, all of which are either found in the 
body and/or feed into natmal recycling pathways. 
A Sllllllllaty of the preclinical toxicology studies completed with Nu-3 is presented in Table 7. 
The data indicates that Nu-3 is safe and well-tolerated when adininistered systemically or 
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topically and no specific toxicities were identified that would be cause for concern for the 
proposed Phase I/IIa study in subjects with infected diabetic foot ulcers.  In general, the data 
indicates that Nu-3 functions through a membrane depolarization mechanism of action that is 
highly specific for bacterial cell membranes.   
Table 7. 
Summary of Nu-3 Preclinical Toxicology Data 
Study 
Test Article/Dose 
Species 
Lot # 
Result 
Genotoxicity 
Bacterial Reverse Mutation 
Assay (GLP) 
Nu-3 ACT 
In vitro 
LWA-01R-14 
Non-mutagenic 
Bacterial Reverse Mutation 
Assay (GLP) 
Nu-3 NEUT 
In vitro 
LWA-01R-14 
Non-mutagenic 
Systemic Toxicity Study 
IV Max Tolerated Dose 
(GLP) 
0, 250, 500, 
1000mg/kg 
Rats 
LWA-01-14 
NOEL and NOEAL at 250mg/kg 
LD50 ≥ 500mg/kg 
IV Max Tolerated Dose 
Study  (Non-GLP) 
0, 250, 500, 750, 
1000mg/kg 
Mice 
LWA-01-14 
LD50 ≥ 1000mg/kg 
Oral Max Tolerated Dose 
Study (Non-GLP) 
1000, 1256, 1580, 
1988, 2500mg/kg 
Mice 
PN 1127 
LD50 2001mg/kg according to 
the Karber method 
Acute Single IV Dose 
(Non-GLP) 
50, 200mg/kg 
Rat 
PN 1127 
No observable effect on blood 
chemistry (CBC, metabolic 
panel) 
Local Tolerance Studies 
Primary Dermal Irritation 
(GLP) 
10% Nu-3 
Rabbit 
LWA-01R-14 
Not a primary irritant 
MapTek EpiOcular™ 
Toxicity (GLP) 
1%, 2.5%, 5% 
Nu-3 
In vitro 
LWA-01-14 
1% - Non-irritating, Minimal; 
2.5%, 5% - Moderate irritants; 
No significant IL1α stimulation 
for all doses 
In vitro Sensitization 
(GLP) 
10%, 6.8%, 4.6%, 
3.2%, 2.2%, 
1.5%, 1%, 0.68% 
In vitro 
LWA-01-14 
4.6% Sensitizer, All other 
concentrations are Non-
sensitizers 
3 Day Ophthalmic 
Irritation Study (Non-GLP) 
1% 
Rat 
PN 1127 
No sign of irritation 
28 Day Repeat Dermal 
Toxicity (Non-GLP) 
10% 
Rat 
PN1127 
No pathological changes in skin, 
no clinical observations 
 


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1.2.3 Clinical Trials of Bisphosphocin Nu-3 
Bisphosphocin™ Nu-3 is a new chemical entity that has not been evaluated under a formal IND 
application in human clinical studies.  The compound was initial developed as a homeopathic 
treatment for a variety of topical infections as it was considered to fall under the HPUS DNA 
guidelines9.  As a homeopathic treatment, Nu-3 was used topically to treat a variety of infections 
including acne, urethritis, ear infections, diabetic foot infections, and certain fungal infections.  
The information collected from these homeopathic treatments revealed that Nu-3 applied 
topically in solution for up to 14 days was able to improve the severity of an infection based 
visual observation.   
1.3 
Rationale for Current Study 
The proposed clinical study is designed to demonstrate the safety and tolerability of increasing 
doses of topically applied bisphosphocin™ Nu-3 on a chronic diabetic foot infection.  In 
addition, the study is designed to collect data on the efficacy of Nu-3 as it relates to eradication 
of the infection and wound healing to enable planning of future trials.  The 3 dosing levels and 
length of treatment were selected based on in vitro, in vivo, and toxicology studies of Nu-3 and 
its unique mechanism of action.  While the current trial is considered a first-in-human trial, the 
design also has taken into consideration data collected from the development of the compound 
initially as a homeopathic treatment where the use as a topical solution was able to reduce the 
severity of several common topical infections.  Recent studies have shown that a high percentage 
of chronic wounds have biofilm associated which interferes with wound healing.  This trial is 
intended to evaluate the efficacy of an antimicrobial with known activity against biofilm encased 
bacteria to not only eliminate the infection but potentially improve wound healing.  For this 
reason, bisphosphocin Nu-3 is being formulated in a normal saline solution so that a clearer 
picture can be gained of its effect on the wound without the interference of an ointment base.  
Bisphosphocins have been shown to effectively reduce the severity of the infection in preclinical 
animal model testing, but these models have limits as they do not accurately mimic the complex 
environment of a chronic wound infection and in many cases rely on immunosuppression to 
maintain the infection.  This first in human study is designed to collect both safety and efficacy 
data so that the parameters such as dose, duration and frequency of administration can be refined 
for future more intensive studies.  Based on the data collected from preclinical studies, it is 
anticipated that a therapeutic effect may be observed in the middle (1% w/v, or 10 mg/ml) and 
high dose  (2% w/v, or 20 mg/ml) cohorts that could justify extending the treatment course an 
additional seven (7) days.  An extension of the treatment duration to fourteen (14) days would 
only be initiated after five subjects in Cohorts 2 and 3 completed the seven (7) day treatment and 
approval was given by the SMC.  The decision to extend treatment of individual subjects 
showing a clinical response would then be made by the principal investigator. 
Potential Risks 
This clinical trial is a first in human trial so there is a potential risk of an adverse reaction from 
the study drug.  Bisphosphocin Nu-3, however would be considered a nucleotide analog which is 
a class of drugs with a strong safety record so the likelihood of this particular risk is considered 


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minimal.  The major distinction between bisphosphocin Nu-3 and the nucleotide analog class are 
that both 3’ and 5’ sites are blocked and a novel acid activation step that is reversed when the 
compounds are exposed to buffers or base.  Therefore, it is anticipated that any reaction would be 
localized since the sodium bicarbonate in the blood would neutralize the compound which is 
supported by inhibitory effect of blood in MIC assays.  In addition to a reaction from the study 
drug, there is also the possibility that the drug is ineffective and the infection worsens which 
could necessitate more aggressive therapy or treatment. 
Potential Benefits 
The major benefit of the proposed study will be an improvement in the wound due to the 
elimination of the pathogenic bacteria that is inhibiting healing.  Bisphosphocin™ Nu-3 is a new 
class of broad spectrum antimicrobial so it is unlikely that the pathogenic bacteria will be outside 
its spectrum of activity or resistant, which is a major cause of treatment failure in this indication.  
The dose levels have been selected so that two cohorts are likely to be receiving efficacious dose 
levels of drug with a satisfactory safety margin so that some improvement in wound appearance 
is anticipated in some subjects in these cohorts.  In addition to directly eliminating the 
pathogenic bacteria associated with the chronic ulcer, it is anticipated that a second potential 
benefit would be the elimination of any biofilm encased bacteria that would then make the 
infection more susceptible to traditional therapy as a follow up therapy.  Any of these outcomes 
would benefit the subject as it would reduce the risk of limb loss due to a worsening infection 
and the pain. 
 


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2 
OBJECTIVES 
Objective/Purpose: The primary objective of this study is to assess the safety and tolerability of 
Nu-3 when applied topically to a chronically infected diabetic ulcer. 
A secondary objective of this study is to obtain preliminary data on the microbiological activity 
of Nu-3 as measured by clinical wound assessments and presence of pathogenic bacteria.  Based 
on preclinical data, it is anticipated that improvement in the clinical wound assessment may be 
observed in the middle and high dose (1% and 2%) cohorts that may justify extending the 
treatment for an additional 7 days. 
Statement of Hypothesis:  Nu-3 is a broad spectrum antimicrobial with a unique mechanism of 
action that involves cell membrane depolarization and rapid bacterial cell death.  As an 
antimicrobial it is particularly well suited for development as a topical for Diabetic Foot 
Infections for the following reasons: 
1. These infections tend to be polymicrobial in nature, involve biofilm, and require an 
antibiotic(s) with a broad spectrum of activity. 
2. Many of the bacteria in these infected wounds are resistant to one or more classes of 
antibiotics making treatment difficult 
3. The directly bactericidal mode of action of Nu-3 should translate into an immediate 
reduction in the severity of the infections and offers new options in dosing. 
4. Research has shown that many chronic infections are due to the formation of biofilm 
which is inherently resistant to treatment by traditional antibiotics but susceptible to 
treatment by bisphosphocins such as Nu-3 
 
The proposed clinical trial design is aimed at defining the initial parameters for safety and 
tolerability in an infected wound based on the currently available preclinical data on Nu-3. The 
trial is focused on DFIs of mild to moderate severity and an area of 0.5 to 6 cm2 as a population 
of subjects that will provide the data across a range of pathogens and ulcers of varying duration.  
In addition, the middle and high dose (1% and 2% Nu-3) cohorts were selected so that some 
clinical improvement in wound assessment be observed which will enable better planning of 
following clinical trials.   
Route of Administration: Study medication will be applied topically directly to the infected 
ulcer 
Dosage:  The study includes a sequential ascending dose protocol with Cohort 1 at 0.1% w/v or 
1mg/ml, Cohort 2 at 1% w/v or 10mg/ml, and Cohort 3 at 2% w/v or 20mg/ml.  In addition, two 
subjects in each cohort will be randomized to placebo.  The proposed doses and treatment 
duration were based on data from in vitro antimicrobial assays, preclinical animal efficacy 
models, and toxicology studies and were selected to demonstrate safety at a high dose that 
provides a significant therapeutic margin lethal to all tested bacteria and fungi to date while 
proven safe in animal toxicology studies.   


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Dosing Regime:  To establish the initial safety and tolerability of Nu-3, a twice daily application 
of the compound was selected for this clinical trial based on preclinical studies on efficacy and 
toxicology.  Subjects will be required to keep a daily log of when treatments are applied and 
contacted daily by a member of the study team. 
Ulcer Imaging and Area Calculations:  Photographs of the ulcer(s) will be taken at study visits 
using the Aranz Medical Silhouette™ system which allows capture of a color image and 
computer calculation of ulcer(s) area, depth, and volume with more consistent and greater 
accuracy than traditional methods. 
Randomization: Eligible subjects will be randomly assigned in a 4:1 ratio to Nu-3 (n=8) or 
matching placebo (n=2) in each cohort. 
Treatment Period:  Day 1 (in clinic): Day 2 (end of SAD/Beginning of MAD) through Day 8 or 
Day 15 (Cohort 2 and 3) 
Follow-Up Period: Day 15 or Day 22 (Cohort 2 and 3) 
Selection of Study Population:  Type I or II diabetes mellitus subjects with a chronic infected 
lower extremity ulcer defined as having a DUSS score of 0 to 3 and a DFI wound score 1 to 9 as 
this is the most appropriate population to assess the safety of a new drug aimed at treating 
infected wounds.  The proposed inclusion criteria have been selected to define a subject 
population that will have a diverse spectrum of pathogens and range of chronic ulcers to ensure a 
proper assessment of safety and tolerability as well as data to better define follow on clinical 
studies.  The DFI wound score range is intended to select subjects with mild to moderate 
infection which have the best potential to respond to the treatment.  A radiograph of the affected 
area will be performed during the screening period to rule out the possibility that the ulcer is due 
to an underlying osteomyelitis.  Preclinical studies have demonstrated the safety of Nu-3 on 
mammalian cells, intact and damaged skin, and when injected intravenously, therefore exposure 
of healthy subject to Nu-3 would provide little if any additional safety information. 
 
 


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3 
STUDY DESIGN 
Phase I/IIa, three cohort double-blind ascending dose with two dosing phases per cohort, study in 
diabetes mellitus subjects with a chronic infected diabetic ulcer defined as having a DUSS score 
between 0 and 3 and a DFI wound score between 1 and 9.  The study proposes to use the same 
subject(s) in SAD Phase 1 and MAD Phase 2 of each cohort 
The study is designed to conduct the cohorts sequentially with the completion of the first cohort 
before initiating the next dosing level.   Prior to initiating treatment of the ulcer, subjects will be 
administered a skin sensitivity test by applying a single drop of Nu-3 or placebo to the forearm 
and visually monitoring the area for 10-15 minutes for any acute reaction such as reddening, 
swelling, or irritation.  If the skin sensitivity test is negative the subject is cleared to begin Arm 1 
of the treatment. 
A subject who has cleared the skin sensitivity test will have a microbiological sample taken from 
the infected diabetic ulcer(s) prior to initiating treatment.  In Phase 1, eligible subjects will be 
treated with a single application of Nu-3 or placebo in a 4 to 1 ratio to judge the initial safety of 
Nu-3 over a brief 15-30 minute interval and 24 hour interval.  Bisphosphocin Nu-3 will be 
applied topically to the chronic infected ulcer (DUSS score: 1-3; DFI wound score: 1-9), and the 
area observed over a 15-30 minute time period and photographed.  If no visible reaction is 
observed, the ulcer is covered with a bandage.  The subject will be released with verbal 
instructions to leave the nonabrasive, hypoallergenic bandage on the wound, avoid getting the 
bandage wet, and return for a follow up visit within 24 ± 2h.  At the follow up visit, the bandage 
will be removed, the ulcer visually examined, photographed using the Aranz Medical 
Silhouette™ system, and the subject cleared for the MAD Phase 2 based on the 
recommendation/decision of the PI and on the absence of any drug related SAEs.   
In Phase 2, eligible subjects, which are those who have been approved by the PI after the Visit 2 
examination, will be instructed in the proper application of bisphosphocin Nu-3 or placebo.  In 
the clinic, the subjects will be observed applying the first dose to ensure compliance.  The 
subject will then be given a 7-day supply of study drug, non-abrasive bandages, a home diary, 
written instructions for care and hygiene, and sent home to continue treatment.  On Day 9 ± 2h or 
earlier in the case of any adverse events, subjects will return to the clinic for an examination, 
including visual evaluation of the ulcer, vital signs, adverse events, photo documentation using 
the Aranz Medical Silhouette™ system, collection of a sample for microbiological assessment, 
and concomitant medication use.  A final follow up visit will be scheduled for Day 15 for a 
complete examination as described above.  Based on the data collected from preclinical studies, 
it is anticipated that a therapeutic effect may be observed in the middle (1% w/v, or 10mg/ml) 
and high-dose (2% w/v, or 20mg/ml) cohorts that could justify extending the treatment course an 
additional seven (7) days.  An extension of the treatment duration to fourteen (14) days would 
only be initiated after five subjects in Cohorts 2 and 3 completed the seven (7) day treatment and 
approval was given by the SMC.  The decision to extend treatment of individual subjects 
showing a clinical response would then be made by the principal investigator. 
 


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SUBJECT POPULATION 
3.1 
Selection and Exclusion of Subjects 
Subjects, greater than 18 years of age, will be recruited from the area surrounding the Phase I/IIa 
Clinical Trial Centers as well as existing subjects of the center.  Strategies for recruiting subjects 
will include web based advertising and social media sites, or traditional advertising in local 
newspapers all using IRB-approved materials.  It is anticipated that 50-60 subject will be 
screened to identify the 30 subject needed for the proposed study.  Subjects will be allowed as 
much time as necessary to review and give informed signed prior to any study related procedures 
being performed.   
3.2 
Inclusion Criteria 
1. Non-hospitalized ambulatory subjects suffering from Diabetes mellitus, Type I or II 
2. Male or Female older than 18yrs 
3. Diabetic foot ulcer(s) with a DUSS Score of 0 to 3 
4. A radiograph, MRI, and/or CAT scan evaluation within the last seven (7) days to 
determine the ulcer is not caused by an osteomyelitic bone infection 
5. Infection as defined by the IDSA as ≥2 classic findings of inflammation or purulence 
6. Ulcerated area(s) of not more than two (2) ulcers between 0.5 to 6 cm2 as calculated by 
the Aranz Medical Silhouette™ system 
7. Infection must be localized to the area of the ulcer and defined as mild (superficial and 
limited in size and depth) with a DFI Wound Score between 1 and 9 
8. Subject must agree to be treated as an outpatient, follow all protocol procedures, return 
for all schedule visits, and provide informed consent. 
9. Any female of child bearing age must consent to use medically acceptable birth control 
for the duration of the study. 
 
3.3 
Exclusion Criteria 
1. A DUSS Score above 3 
2. Any ulcer caused by an underlying osteomyelitic bone infection 
3. Assessed with a Moderate to Severe Infection, including abscesses, extensive gangrene, 
symptoms of systemic infection, or a limb threatening infection.  DFI Wound Score 
above 9. 
4. An ulcer area(s) greater than 6 cm2 or more than two (2) ulcers. 
5. Any subject that has received systemic or topical antibiotics within the last seven (7) days 
6. Any subject on topical antimicrobial treatment for their infected diabetic foot ulcer whose 
ulcer is responding to treatment 
7. Any subject that would be unable to follow the protocol procedures, safely monitor the 
infection status at home, and return for schedule visits 
8. Positive pregnancy test at Screening or Visit 2 
 


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4 
STUDY DRUGS 
Original - 310ct2014 
4.1 
Randomization, Blinding, and Drug Product/Placebo Packaging and Labeling 
The drng product kits will be labeled as shown in Table 8 to ensme blinding of both the Principal 
Investigator and Subject. The kits will contain a 0.5 oz dropper bottle containing either 15 ml of 
Bisphosphocin™ Nu-3 or Placebo packaged in a cardboard a ca1ton with a foam inse1t for 
protection. Both the carton and dropper bottle will be labelled. 
The randomization will consist of two materials that have been allocated within 3 Cohorts. 
Material 1: Bisphosphocin ™ Nu-3 at 0.1 %, 1 %, or 2% w/v 
Material 2: Matching Placebo 
Within each Coho1t, a material blocking ratio of 4: 1 has been utilized with three (3) blocks per 
coho1t. All Coho1ts utilize a different bottle number range with ranges set at Coho1t 1 from 101 
to 115, Cohort 2 from 201-215, and Coh01t 3 from 301 to 315. All coh01t will have a different 
staiting Randomization Specification Seed value which will result in a different statistical 
outcome. 
Table 8. 
Product Label Specifications 
GC: X; LID: X; CARTON 
Protocol#: 
LAI2014-1 
Product: 
Bisphosphocin Nu-3 topical antibiotic or placebo 
Bottle Number: 
#### 
Lot Number*: 
14K### 
Directions for Use: 
As directed, apply twice daily to the affected area 
Storage: 
Room Temperature" 
Caution: 
New Drug-Limited by federal law to Investigational Use 
Keep out of reach of children 
Manufactmer: 
Lakewood Amedex Inc, Sarasota, FL (941)225-2515 
*Lot number represents the group of kits being assembled for each cohort. 
"Room temperatme is defined according to the USP as the temperature that is prevailing in a work area. 
4.2 
Description and Handling of Bisphosphocin Nu-3 
is a thymidine analog with both 
the 3' and 5' sites blocked by butyl phosphate which prevents it from causing eai·ly chain 
te1mination by inc01poration by the polymerase unlike most compounds in this class. Nu-3 is 
activated through an acidification step that results in an acidic or protonated compound that when 
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dissolved in water has a pH between 2 and 2.5.  The antibacterial activity can be neutralized by a 
strong base or buffer.  Prior non-human investigations indicate that all doses being used in this 
study are likely to be safe, medical personnel and caregivers should use caution when handling 
the drug to avoid exposure of sub-MIC levels through inadvertent application to the skin and 
normal skin flora that could potentially lead to resistant strains.   
4.3 
Dosage and Administration of Bisphosphocin Nu-3 or Placebo 
Bisphosphocin Nu-3 will be dosed at three different concentrations of 0.1% w/v (1mg/ml), 1% 
w/v (10mg/ml), and 2% w/v (20mg/ml) in 0.85% normal saline.  Placebo will be 0.85% normal 
saline solution.  The drug or placebo will be administered as a single dose and then twice daily 
for 7 days.  The drug or placebo will be administered to the infected diabetic foot ulcer and 
surrounding intact skin at a dose of 1-3 drops from a dropper bottle per 1 cm2 of ulcer area. 
4.4 
Prior and Concomitant Medications 
Subjects are allowed to continue on any concomitant prescribed medication for medical 
conditions not related to the treatment of the infected diabetic ulcer.  All systemic and topical 
antibiotics must be stopped seven (7) days prior to admission to the proposed study. 
 


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5 
STUDY PROCEDURES 
5.1 
Subject Enrollment and Treatment Assignment 
Screened subjects who sign the Informed Consent and meet all the inclusion criteria and none of 
the exclusion criteria will be considered eligible for study participation.  Eligible subjects will be 
assigned to Cohort 1 until a total of 10 subjects are enrolled, followed by Cohort 2, and Cohort 3.  
There will be a minimum of 7 days between the last subject completing the Day 9 assessment 
from each cohort and the first subject enrolled into the next cohort.  All subjects who are eligible 
for enrollment will be randomly assigned in a 4:1 ratio to NU-3 (n=8) or matching placebo (n=2) 
in each cohort.  The dose escalation will proceed following the Safety Monitoring Committee 
recommendation/approval.   
5.2 
Pre-treatment Assessments 
The schedule of evaluations and procedures that must be performed at specific time points is 
described in the following sections.  The Time and Event Schedule (Appendix B) summarizes 
the frequency and timing of various safety evaluations and clinical microbiological sampling.  As 
soon as a subject is considered to be a potential subject for this study and prior to any other study 
procedures, the nature of the study will be explained to him/her by the study investigator or 
designee and the potential subject will be asked to provide written informed consent.  Informed 
consent must be obtained prior to any study procedures being conducted. 
5.2.1 Screening Visits 
Screening Visit/Visit 1  
Subjects diagnosed with diabetes mellitus and suffering from a chronic infected diabetic foot 
ulcer will be evaluated for entry criteria during a screening period conducted within the 30 days 
prior to bisphosphocin Nu-3 or placebo treatment.  After informed consent has been obtained, the 
following procedures will be completed for each subject prior to inclusion in the study.   
• Medical History 
• Physical Examine with vital signs 
• Concomitant medications 
• Urine Pregnancy tests (HCG), if applicable 
• Drug and alcohol blood toxicity screen 
• Draw blood for CBC and CMP 
• Review of shoe gear worn 
• Radiograph, MRI, and/or CAT scan evaluations of the infected ulcer within the last seven 
(7) days to confirm the absence of osteomyelitis 
• Scoring of the diabetic ulcer using the DUSS Scoring system 


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• Rating of the infection using the DFI Wound Scoring  system 
• Examination of Chronic Ulcer(s)  
• Photo Documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ as described in Appendix C. 
 
Diabetic Ulcer Wound Scoring System (DUSS) 
Parameter 
Score 
Palpable Pedal Pulses 
Presence = 0 
Absence = 1 
Probing to Bone 
No = 0 
Yes = 1 
Location of Ulcer 
Toe = 0 
Foot = 1 
Number of Ulcerations 
Single = 0 
Multiple = 1 
Score Range  
0 
4 
 
Diabetic Foot Ulcer Wound Infection Score 
 
Parameter 
Wound Infection Score 
0 
1 
2 
3 
Purulent discharge 
Absent 
 
 
Present 
Nonpurulant drainage (serious, 
sanguinous) 
Absent 
Mild 
 
 
Erythema 
None 
Mild pink, 
barely 
perceptible 
Moderate pale 
red, defined 
edges 
Severe red to 
dark red 
Induration 
None 
Mild 
Moderate 
Severe 
Tenderness (sign) 
None 
Mild 
Moderate 
Severe 
Pain (symptoms) 
None 
Mild 
Moderate 
Severe 
Local Warmth (relative to uninfected 
contralateral foot) 
Same 
Mildly increased 
Moderately 
increased 
Severely 
increased 
 
5.3 
Enrollment/Baseline in Single Ascending Dose Arm (SAD) 
Treatment Visit/Visit 2 
• A urine pregnancy test must be performed prior to starting Nu-3 treatment in all women 
of child-bearing potential and a negative result must be documented. 
• A urine drug and alcohol toxicology screen must be performed before starting Nu-3 
treatment for each subject and a negative result must be documented. 
• Draw blood for CBC and CMP 


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• Study subject will be assigned to the open cohort and treatment number per standard 
procedure.  The treatment number will correspond with the dropper bottle number that 
will be used for treatment. 
• A skin sensitivity test will be performed prior to initiating dosing by applying a single 
drop of Bisphosphocin Nu-3/Placebo to the subject’s forearm and observing the area over 
a 10-15 minute interval for any reddening, swelling, or irritation.  A negative result must 
be documented before initiating treatment of the ulcerated area. 
• Before the treatment begins, the following data/samples will be collected and procedures 
completed: 
o Concomitant medications 
o Vital signs 
o Debridement, if necessary 
o Collection of  sample from the ulcer for microbiological assessment 
• Study Treatment for SAD Arm will be applied topically to the chronic ulcer and the area 
observed over a fifteen minute period for any acute reactions prior to being covered by a 
non-abrasive hypoallergenic bandage.  The Nu-3 or placebo solution will be applied from 
a pre-packaged dropper bottle at a rate of 1-3 drops per 1 cm2 of ulcer making sure to 
cover the entire ulcer as well as up to 0.5 cm of surrounding tissue. 
• One hour after the treatment the following data will be collected: 
o Vital signs 
o Signs and symptoms of adverse events (e.g. redness of the ulcer, swelling, fever, 
chills) 
• One hour post-treatment the study subject will be allowed to leave the clinic and 
instructed to call or return to the office if any of the following occur: 
o Increased pain or swelling of the infected ulcer or surrounding area. 
o Any observed increase in redness or swelling of the ulcer. 
 
5.4 
Follow-up Single Ascending Dose Arm 
Post Treatment Visits/Visit Day 3 (24+/-2 h from Visit 2) 
Study subjects will return to the clinical site for a follow-up evaluation 24±2h post treatment 
prior to starting the multiple ascending dose arm (MAD).  At this follow-up visit, the subject will 
undergo the following procedures and evaluations to determine if they can be cleared for 
enrollment in the multiple ascending dose arm (MAD). 
• Vital sign measurements including, weight, temperature, resting blood pressure, heart 
rate, and respiratory rate. 
• Assessment of adverse events since the previous study visit 
• Visual examination of the ulcer  


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• Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ system 
 
5.5 
Enrollment/Baseline in Multiple Ascending Dose (MAD) Arm 
Treatment Visit/Visit 3  
• Subjects will be instructed on the proper procedure for applying study treatment for the 
MAD Arm and observed performing the first application to the chronic ulcer and 
applying the non-abrasive hypoallergenic bandage.  The Nu-3 or placebo solution will be 
applied from a pre-packaged dropper bottle at a rate of 1-3 drops per 1 cm2 of ulcer 
making sure to cover the entire ulcer as well as up to 0.5cm of surrounding tissue.  The 
subject will be provided the Subject Treatment Log in Appendix D. 
• The study subject will be discharged from Visit 3 with a seven-day supply of study 
medication and supplies.  The study subject will be instructed to continue treatment until 
the night before their next Visit. 
• The study subject will be allowed to leave the clinic and instructed to call or return to the 
clinic if any of the following occur: 
o Increased pain or swelling of the infected ulcer or surrounding area. 
o Any observed increase in redness or swelling of the ulcer. 
• Subjects will be provided a Subject Home Treatment Log (Appendix B) and instructed in 
the proper method of documenting the timing of treatments and recording of any 
observations 
.  
Follow-Up 7 Day Multiple Ascending Dose Arm Visit/Visit 4 (Day 9+1) 
The study subject will return for a follow-up evaluation to the clinical site on Day 8 (or the next 
day) following the final MAD treatment.  At this follow-up visit, the subject will undergo the 
following procedures and evaluations. 
• Vital sign measurements including weight, temperature, resting blood pressure, heart rate, 
and respiratory rate. 
• Radiograph, MRI, and/or CAT evaluations of the infected ulcer to rule out the 
development of osteomyelitis, if determined by the Principal Investigator to be necessary 
• Concomitant medication use 
• Draw blood for CBC and CMP. 
• Assessment of adverse events and interim history since the previous study visit 
• Visual examination of the ulcer  
• Scoring of the Ulcer(s) using both the DUSS and DFI Wound Scoring Systems 
• Photo documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ system 
• Collection of a sample from the ulcer for microbiological assessment 


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• Subjects will be given a 7 day supply of non-abrasive bandage and written instructions on 
the proper care and hygiene to include keeping the ulcer clean and bandage dry until the 
Day 14 follow up visit.  In addition, subjects will be told to call if there is any worsening 
of the ulcer with regard to pain, infection, or swelling.  
• In Cohorts 2 and 3, if after review of the safety data of the first 5 subjects, the SMC finds 
no safety concerns with the treatment, approval may be given by the PI to extend 
treatment an additional seven (7) days for those subjects whom are showing a clinical 
benefit in their judgment. Subject selected for continued treatment will be scheduled to 
return for Visit 5 on Day 16+1 for assessment. 
• Subjects not selected to continue treatment will be given a 7 day supply of non-abrasive 
bandage and given written instructions on proper care and hygiene which includes 
keeping the ulcer clean and bandaged dry until the Day 15 Final Study Visit.  In addition, 
subjects will be told to call if there is any worsening of the ulcer with regard to pain, 
infection, or swelling.  
 
Follow-Up 14 Day Multiple Ascending Dose Arm Visit/Visit 5 (Day 16+1) 
Study subject will return for a follow-up evaluation to the clinical site on Day 15 or the next day 
following the final MAD treatment.  At this follow-up visit, subject will undergo the following 
procedures and evaluations. 
• Vital sign measurements including weight, temperature, resting blood pressure, heart rate, 
and respiratory rate. 
• Radiograph, MRI, and/or CAT evaluations of the infected ulcer to rule out the 
development of osteomyelitis, if determined by the Principal Investigator to be necessary 
• Concomitant medication use 
• Draw blood for CBC and CMP. 
• Assessment of adverse events and interim history since the previous study visit 
• Visual examination of the ulcer  
• Scoring of the Ulcer(s) using both the DUSS and DFI Wound Scoring Systems 
• Photo documentation and Calculation of the Ulcer Area and Depth using the Aranz 
Medical Silhouette™ system 
• Collection of a sample from the ulcer for microbiological assessment 
• Subjects will be given a 7 day supply of non-abrasive bandage and given written 
instructions on proper care and hygiene which includes keeping the ulcer clean and 
bandaged dry until the Day 22 Final Study Visit.  In addition, subjects will be told to call 
if there is any worsening of the ulcer with regard to pain, infection, or swelling.  
 
5.6 
Final Study Visit – Visit 5 (7-Day MAD) or Visit 6 (14-Day MAD) 
Post Treatment Final Visit (Day 15 or 22 ± 1) 
All study subjects will have the following procedures performed at their final visit (Day 15 or 22 
± 2) 


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• Vital sign measurements including weight, temperature, resting blood pressure, heart rate 
and respiratory rate 
• Physical examinations 
• Visual examination of the chronic ulcer  
• Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ System 
• Assessment of adverse events and interim history since the previous study visit 
• Collection of a sample from the ulcer for microbiological evaluation 
 
5.7 
Early Termination Visit 
If a subject withdraws prior to completing the study, the reason for withdrawal will be 
documented on source documentation and in the CRF.  If a subject withdraws early due to an 
adverse event, he/she will be followed until resolution/stabilization of the adverse event. 
If a subject prematurely withdraws from the study they will be asked to complete the study 
procedures and evaluations performed in the final study visit at the time of withdrawal from the 
study (i.e. Study subject withdraws at the time of visit and consents to having 
procedures/evaluations done): 
• Vital sign measurements including weight, temperature, resting blood pressure, heart rate 
and respiratory rate 
• Physical examinations 
• Visual examination of the chronic ulcer   
• Photo documentation and calculation of ulcer area and depth using the Aranz Medical 
Silhouette™ system 
• Concomitant medication use 
• Assessment of adverse events and interim history since the previous study visit 
• Collection of a sample from the ulcer for microbiological evaluation 
 
Administrative withdrawals will be replaced to ensure that each cohort includes a total of 10 
subjects who have completed the entire treatment.  The study sponsor has designated the 
following person to be in charge of the shipping of study drug as they will be unblinded and able 
to match the proper replacement kit. 
 
 
 
  
 
 
 
  
 
 
 
  
 
 


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Subjects withdrawn from the study due to an AE/SAE related to the study drug will not be 
replaced. 
 
5.8 
Unscheduled Visit 
Unscheduled visits will be documented using the CRFs used for the Day 1 follow-up visit 
determined to be required by the PI, except in the case of photo documentation which must be 
performed.  If the unscheduled visit occurs as a result of an event, additional testing consistent 
with the event will be performed. 


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6 
ADVERSE EVENTS AND TOXICITY MANAGEMENT 
6.1 
Specification of Safety Parameters 
The primary endpoint safety variables will be the incidence of localized swelling surrounding the 
ulcer, development of a rash, joint pain, and any adverse changes in the appearance of the ulcer.   
The Safety Monitoring Committee (SMC) will receive safety reports outlining the number of 
subjects enrolled in each cohort to date, summary of the safety data Day 0 – 15 for the most 
recent cohort, adverse events, and serious adverse events listed by grade as well as all laboratory 
tests data.  These reports will present the data blinded and will be unblinded only if necessary to 
determine the cause of an AE or SAE at the request of the SMC.  In addition, safety data from 
completed cohorts will be summarized in the same manner and included in the report.  All 
information on adverse events will be provided in a table.   
6.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters 
6.2.1 Adverse Events 
Adverse Event: According to the ICH E6 guidelines, an AE is defined as any untoward medical 
occurrence in a subject/subjects in a clinical study regardless of its relationship to the 
administration of the study drug.  Therefore, an AE is any unintended, unfavorable, or 
unexpected sign, laboratory test, symptom, reaction, or disease temporarily associated with a 
clinical subject/subjects during the duration of the study.  The occurrence of an AE may come to 
the attention of study personnel at a study visit and during interviews of subjects seeking medical 
care, or upon reviews conducted by study monitors.  During all visits and interviews after 
administration of the study drug, clinical staff will inquire about any anticipated adverse events 
or symptoms using standardized questions.  The data will be recorded on the appropriate CRFs, 
All AEs whether local or systemic reactions not meeting the criteria for “serious adverse events” 
should be documented on the appropriate CRF.  The information to be recorded should include a 
description of the event, time of onset, clinician’s assessment of the severity, relationship to the 
study drug (assessment to be conducted only by those with the qualified training and authority to 
make a diagnosis and include the CMO, MD, PA, Nurse Practitioner, DO, DPM in podiatry and 
DDS), and time to resolution or stabilization. All AEs that occur during the study regardless of 
their association with the study drug must be documented appropriately and followed to adequate 
resolution. 
Any AE (i.e. a new event or an exacerbation of a pre-existing condition) with an onset date after 
the screening visit up to the last day on study (including follow-up), should be recorded as an AE 
on the appropriate CRF page(s). 
 
 


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An AE does not include: 
• Medical or surgical procedures) e.g. surgery, endoscopy, tooth extraction, transfusion); 
the condition that leads to the procedure is an adverse event. 
• Pre-existing diseases or conditions or laboratory abnormalities present or detected prior 
to the screening visit, that do not worsen. 
• Situations where an untoward medical occurrence has not occurred (e.g. hospitalization 
for elective surgery, social and/or convenience admissions). 
• Overdose of either study drug or concomitant medication without any signs or symptoms 
unless the subject is hospitalized for observation. 
• Events related to the underlying condition under study should not be reported as adverse 
events unless they are considered to be study related. 
Severity of Event: All AEs will be assessed by the investigator and recorded on the appropriate 
CRF page, including the date of onset and resolution, severity, relationship to study drug or study 
procedures, outcome and action taken with study medication.  The clinician will assess all AEs 
using a protocol defined grading system as follows. 
Scale 
Definition 
Grade 1 
No intervention required because the event is mild or asymptomatic.  A clinical or diagnostic 
observation. 
Grade 2 
Minimal to moderate intervention and/or localized event that requires non-invasive treatment 
Grade 3 
A debilitating or incapacitating condition, severe or medically significant that is not life threatening 
that requires hospitalization or prolonged treatment 
Grade 4 
Requiring urgent intervention because the event is life threatening 
Grade 5 
Death of a subject due to an adverse event. 
 
Relationship to Study Products: 
6.2.2 Reactogenicity 
Adverse events associated with the study product may include vesicle (blistering) development, 
burning sensation at the ulcer site (may be masked by diabetic neuropathy), increase in local 
temperature, and expansion of necrotic tissue surrounding the ulcer. 
 


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6.2.3 Serious Adverse Events 
A serious adverse event (SAE) is defined as follows: 
• Any adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
• Death; 
• Life-threatening situation (subject is at immediate risk of death); 
• In-patient hospitalization or prolongation of existing hospitalization (excluding those for 
study therapy or placement of an indwelling catheter, unless associated with other serious 
events); 
• Persistent or significant disability/incapacity; 
• Congenital anomaly/birth defect in the offspring of a subject who received study drug; 
• Other: medically significant events that may not result in death, be immediately life-
threatening, or require hospitalization, may be considered an SAE when, based upon 
appropriate medical judgment, they jeopardize the Subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
Examples of such events are: 
• Intensive treatment in an emergency room or at home for allergic bronchospasm 
• Blood dyscrasias or convulsions that do not result in hospitalization 
• Development of drug dependency or drug abuse  
 
Clarification of Serious Adverse Events 
• Death is an outcome of an adverse event, and not an adverse event in itself.  In reports of 
death due to “Disease Progression”, where no other information is provided, the death 
will be assumed to have resulted from progression of the disease being treated with the 
study drug(s). 
• All deaths, regardless of cause or relationship, must be reported for subjects on study and 
for deaths occurring within 30 days of last study drug dose or within 30 days of last study 
evaluation, whichever is longer. 
• “Occurring at any dose” does not imply that the subject is receiving study drug at the 
time of the event.  Dosing may have been given as treatment cycles or interrupted 
temporarily prior to the onset of the SAE, but may have contributed to the event. 
• “Life-threatening” means that the subject was at immediate risk of death from the event 
as it occurred.  This does not include an event that might have led to death, if it had 
occurred with greater severity. 
• Complications that occur during hospitalizations are AEs.  If a complication prolongs 
hospitalization, it is an SAE. 


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• “In-patient hospitalization” means that the subject has been formally admitted to a 
hospital for medical reasons, for any length of time.  This may or may not be overnight.  
It does not include presentation and care within an emergency department. 
• The investigator should attempt to establish a diagnosis of the event based on signs, 
symptoms and/or other clinical information.  In such cases the diagnosis should be 
documented as the AE and/or SAE and not the individual signs/symptoms.    
 
A distinction should be drawn between serious and severe AEs.  An AE that is assessed as Grade 
4 (potentially life-threatening) should not be confused with a SAE.  Severity is a category 
utilized for rating the intensity of an event; and both AEs and SAEs can be assessed as Grade 4.  
An event is defined as “serious” when it meets one of the pre-defined outcomes as described 
above in Section 6.2.3.  
6.2.4 Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
Laboratory abnormalities are usually not recorded as adverse events or serious adverse events 
unless they are associated with clinical signs and/or symptoms. However, laboratory 
abnormalities (e.g. clinical chemistry, hematology, urinalysis, etc.) independent from the 
underlying medical condition, that require medical or surgical intervention, or lead to study drug 
interruption or discontinuation must be recorded as an AE, as well as an SAE, if applicable.  In 
addition, laboratory or other abnormal assessments (e.g. electrocardiogram, X-rays, vital signs) 
that are associated with sign and/or symptoms must be recorded as an AE or SAE if they meet 
the definition of an adverse event (or serious adverse event) as described in the protocol.  If the 
laboratory abnormality is part of a syndrome, record the syndrome or diagnosis.   
Severity should be recorded and graded according to Common Terminology Criteria for Adverse 
Events v4.0 (CTCAE).  For adverse events associated with laboratory abnormalities, the event 
should be graded based on the clinical severity in the context of the underlying conditions, which 
may or may not be in agreement with the grading of the laboratory abnormality. 
6.3 
Serious Advent Event Reporting Procedures 
The study sponsor is required to expedite to worldwide regulatory authorities reports of Serious 
Adverse Drug Reactions or Suspected Unexpected Serious Adverse Reactions (SUSARs); 
therefore, the study sponsor or its delegate must be notified immediately regarding the 
occurrence of any SAE that occurs after the subject consents to participate in the study, including 
SAEs resulting from protocol-associated procedures performed from screening onwards. The 
procedures for reporting all SAEs, regardless of causal relationship, are as follows: 
• Record the SAE on the AE CRF and complete the “Serious Adverse Event Report” form. 
• E-mail or fax the SAE form to the attention of the Drug Safety Manager within 24 hours 
of the investigator’s knowledge of the event. Contact information is as follows: 


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• 
For fatal or life-threatening events, also e-mail or fax copies of hospital case repo1is, autopsy 
repo1is, and other documents when requested and applicable. Transmission of such 
documents should occur with Personal Subject Details de-identified, without losing the 
traceability of a document to the Subject Identifiers. 
• 
The study sponsor or its delegate may request additional info1mation from the investigator to 
ensure the timely completion of accurate safety reports. 
The investigator must take all therapeutic measures necessaiy for resolution of the SAE. Any 
medications necessa1y for treatment of the SAE must be recorded onto the concomitant 
medication section of the subject's CRF and the event description section of the SAE fo1m. 
Follow-up of adverse events will continue through the last day on study (including the follow-up 
off-study medication period of the study) and/or until the investigator and/or study sponsor 
dete1mine that the subject's condition is stable. The study sponsor or its delegate may request 
that ce1iain adverse events be followed until resolution. 
6.3.1 
Reporting Pregnancy 
The risks of treatment with bisphosphocin during pregnancy has not been evaluated. 
The subject must be instmcted to discontinue all study diugs and info1m the investigator 
immediately if she becomes pregnant during the study. 
The investigator should repo1i all pregnancies to Lakewood-Amedex within 24 hours of 
becoming aware of the pregnancy. The investigator should counsel the subject regarding the 
possible effects of prior study diug exposure on the fetus and the need to info1m the study site of 
the outcome of the pregnancy. 
Any pregnancy complication or elective termination of a pregnancy for medical reasons will be 
recorded as an AE or an SAE. 
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A spontaneous abortion is always considered to be a SAE and will be reported as described in 
the adverse and Serious Adverse Events section.  Furthermore, any SAE occurring as an adverse 
pregnancy outcome post-study must be reported to Lakewood-Amedex. 
Additionally, all pregnancies that occur during the study should be reported using the Pregnancy 
Report CRF page.  Monitoring of the subject should continue until the conclusion of the 
pregnancy.  The outcome should be reported to Lakewood-Amedex using the Pregnancy 
Outcome CRF page(s).  If the end of the pregnancy occurs after the study has been complete the 
outcome should be reported directly to Lakewood-Amedex.  Pregnancies that occur after the 
subject has discontinued study drugs do not require monitoring.  
6.4 
Safety Oversight 
6.4.1 Independent Safety Monitor Committee 
Clinical Response Evaluations: 
The clinical outcome will be based on the Principle Investigator’s judgment as to the 
improvement of the wound status from Visit 1 to Visit 4 (7-Day MAD) and Visits 5 and 6.  The 
PI will score the ulcer(s) using both the DUSS and DFI Wound Scoring Systems. 
Diabetic Ulcer Wound Scoring System (DUSS) 
Parameter 
Score 
Palpable Pedal Pulses 
Presence = 0 
Absence = 1 
Probing to Bone 
No = 0 
Yes = 1 
Location of Ulcer 
Toe = 0 
Foot = 1 
Number of Ulcerations 
Single = 0 
Multiple = 1 
Score Range  
0 
4 
 


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Diabetic Foot Ulcer Wound Infection Score 
Parameter 
Wound Infection Score 
0 
1 
2 
3 
Purulent discharge 
Absent 
 
 
Present 
Nonpurulant drainage (serious, 
sanguinous) 
Absent 
Mild 
 
 
Erythema 
None 
Mild pink, 
barely 
perceptible 
Moderate pale 
red, defined 
edges 
Severe red to 
dark red 
Induration 
None 
Mild 
Moderate 
Severe 
Tenderness (sign) 
None 
Mild 
Moderate 
Severe 
Pain (symptoms) 
None 
Mild 
Moderate 
Severe 
Local Warmth (relative to uninfected 
contralateral foot) 
Same 
Mildly 
increased 
Moderately 
increased 
Severely 
increased 
 
An improvement in the DFI Wound Score will be used to indicate a clinical improvement of the 
wound and a change in the DUSS Score wound be used to indicate the healing of a small 
ulceration. 
Microbiological Response Evaluations: 
This assessment will be conducted on samples collected by the investigator on Visit 2, Visit 4, 
Visit 5, and Visit 6.of the study which will be cultured to determine: microbial load and identity 
of the infected pathogens or wound flora.  The microbiological response will be determined by a 
comparison of the results pre- and post-treatment. 
SMC Safety Reports: 
Safety reports will be written by the SMC three times during the course of the study.  The first 
will follow the first interim analysis (once the first SAD/MAD cohort 1 reaches Day 9), the 
second report will follow the second interim analysis (once the second SAD/MAD cohort 2 
reaches Day 9), and the third report will follow the third interim analysis (once the third 
SAD/MAD cohort 3 reaches Day 9).  The study reports will be presented to the Principal 
Investigator at each clinical site and Lakewood-Amedex Inc.    
6.4.2 Study Termination 
This study may be terminated at any time by Lakewood-Amedex Inc.  The PI and SMC may 
provide Lakewood-Amedex with recommendations regarding termination of the trial, however, 
Lakewood-Amedex ultimately makes the final decision.  Reasons for terminating the study 
include: 


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• If a serious or unexpected adverse event occurs and the event is judged to be probably or 
definitively related to exposure to Nu-3, the study will be immediately suspended by the 
Principal Investigator pending review of all appropriate safety data.  The event will be 
reported to the SMC, IRB, and Sponsor Medical Monitor within 24 hours or notification 
of its occurrence.  No additional subjects will receive Nu-3 until a joint decision is 
reached between the Sponsor, PI, and SMC as to whether further doses can be given or 
the trial is terminated.  Subjects currently in a MAD Arm will be contacted to ascertain 
treatment status and if any AEs have occurred.  Absent any findings, subjects in the 
ongoing MAD arms will be allowed to complete treatment. 
• Safety Monitoring Committee terminates the study based on interim safety review 
• In the event of a clinical hold being placed on the study following a report of an AE to the 
FDA 
• At the discretion of Lakewood-Amedex. 
 
6.4.3 Removal of Subjects from the Trial or Study Drug 
Subjects will be free to withdraw at any time for any reason, or they may be withdrawn if 
necessary, to protect their health and safety or the integrity of the study data.  A subject will be 
removed from participation in the study if any of the following occur: 
• Any clinical adverse event (AE), laboratory abnormality, intercurrent illness, or other 
medical condition or situation arises such that in the judgment of the PI continued 
participation in the study would not be in the best interest of the subject 
• A protocol violation/deviation occurs that might compromise the integrity of the data, 
compliance, or subject safety 
• Informed consent is withdrawn 
• Subject is lost to follow-up 
 
6.4.4 Handling Withdrawals 
If a subject is withdrawn prematurely from the study after receiving Nu-3, the reason for 
withdrawal is to be documented in the source document and in the CRF. If the withdrawal of a 
subject is due to an AE, the subject will be followed until resolution/stabilization of the adverse 
event. 
In the case of a subject prematurely withdrawing from the study, the following procedures and/or 
evaluations will be performed if possible at the time of withdrawal or as soon thereafter as 
possible (i.e. study subject withdraws at time of visit and consents to the procedures or consents 
to come in for a visit and the procedures) 
• Photo documentation of ulcer site using the Aranz Medical Silhouette™ system. 
• Collection of a sample for microbiological assessment 


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• Assessment of adverse events 
In the event a subject is lost to follow-up, the site staff must make reasonable attempts to contact 
the subject. A minimum of two documented phone calls followed by a certified mailed letter is 
considered reasonable. The measures taken to follow up must be documented. 
Administrative withdrawals will be replaced in order to achieve the objective of 10 subjects per 
cohort who receive a complete treatment regime.  Subjects withdrawn from the study due to an 
AE/SAE related to the study drug will not be replaced. 


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7 
STATISTICAL CONSIDERATIONS 
7.1 
Study Hypothesis 
Study Hypothesis – Bisphosphocin Nu-3 solution applied topically to a chronic infected diabetic 
ulcer will be safe and well tolerated in subjects suffering from diabetes mellitus.  In addition, the 
Nu-3 MAD Arm subjects may show improvement in the ulcer appearance, reduction in size, and 
improvement in both the DUSS score and DFI Wound Score. 
The study hypothesis will be judged by the following two study objectives: 
• Primary objective: To assess the safety and tolerability of escalating single and multiple 
doses of Nu-3, a novel antimicrobial of the bisphosphocin class, topically to an open 
wound/diabetic ulcer 
• Secondary objectives: To assess clinical response of subjects of the ascending multiple 
dose arm of Nu-3 applied topically to an open wound/diabetic ulcer to determine clinical 
and microbiological response.  
 
7.2 
Sample Size Considerations 
The number of treated subjects is based on the desire to gain preliminary safety, tolerability and 
efficacy information to support future work, while exposing a minimal number of subjects to the 
study procedures and medication.   Inferential statistical testing is not the primary intent of the 
study, therefore, no formal sample size calculation was considered.  Ten subjects per Cohort (1, 
2, and 3), for a total not to exceed 30, is considered sufficient to show clinical significance for 
safety and preliminary efficacy analysis.  The sample sizes for this study were set based on the 
trial design for dose escalation and safety evaluation requirements and not for statistical analysis. 
7.3 
Planned Interim Analysis 
There will be an interim analysis to review safety data on each cohort of subjects when all 
members of a cohort complete Visit 4 (7-Day MAD Follow Up).  In addition, an interim analysis 
will be conducted after the first 5 subjects of Cohort 2 and 3 complete Visit 4 (7-Day MAD 
Follow Up).  The interim analysis will be performed by the Safety Monitoring Committee 
(SMC) which will be composed of three physicians and may include one infectious disease 
specialist, one diabetologist, and one orthopedic surgeon.  The SMC will communicate by email 
or teleconference on at least 3 occasions during the study and more frequently if necessary. 
No formal statistical interim analyses are planned for this Phase I/IIa study since it is primarily 
focused on evaluating safety and tolerability.  Interim safety data on each cohort will be provided 
to the SMC when all members of a cohort have reached Day 3.  The SMC will decide if the 
study can proceed to the next dose cohort (majority vote).  The SMC will also determine if the 
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(majority vote).  The Chairman of the SMC will share their findings and recommendations with 
Lakewood Amedex and Principal Investigators.  
7.3.1 Safety Review 
Subjects will be monitored closely for the occurrence of study related adverse events during their 
24-hr admission in the SAD Arm and for up to 7 days following their last Nu-3 treatment in the 
study.  If any grade 3 or 4 adverse events occur and the event is judged to be probably or 
definitely related to having received the Nu-3, the study will be immediately suspended by the 
Principal Investigator pending review of all safety data.  Adverse events determined to be related 
to the study drug will be reported to the SMC, Lakewood-Amedex, and second PI within 
24 hours of notification of its occurrence.  No additional subjects will receive Nu-3 depending on 
the joint decision of the SMC, Principal Investigator, and Lakewood Amedex Project 
Coordinator and CMO as to whether further doses can be given or the entire study should be 
terminated.  In addition, the SMC will review all interim safety data prior to the next cohort 
beginning treatment.  At each review, the SMC will determine whether the study may proceed to 
the next dose cohort (majority vote). 
7.4 
Statistical Analysis Overview 
This study is designed to evaluate the safety, tolerability, and preliminary efficacy of a minimum 
of three dose levels of Nu-3 relative to placebo.  Data summaries will be presented in order to 
assess the safety of Nu-3 and each Nu-3 dose group will be compared with placebo to determine 
the presence or absence of a treatment effect.  Evaluation across all dose levels of Nu-3 will be 
conducted to determine if there is evidence of a dose response to any treatment effect noted.   All 
subjects treated with placebo from all cohorts will be combined for any analysis.  Evaluations 
will also be conducted within each subject over time to assess the rate of change (if any) in study 
outcome measures over the full course of the study treatment and follow-up periods.  Inferential 
statistical testing is not the primary interest of this study. 
All subjects receiving any amount of study treatment will be evaluated for safety, tolerability, 
and efficacy.  An “intent to treat”  approach will be followed in all data summaries, using all 
available data in the summaries and analysis for an assessment of safety and tolerability while an 
“as treated” approach will be used for microbiological and clinical wound assessment data 
summaries and reports.  
All descriptive summaries and analyses will be presented in tabular and/or graphical form from 
all subjects as appropriate.  These summaries and analysis will be described in detail in the 
Statistical Analysis Plan (SAP) which will be produced prior to breaking the study blind.  All 
statistical summaries and analysis will be conducted using SAS®. 
 
 


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8 
RESPONSIBILITIES 
8.1 
Investigator Responsibilities 
8.1.1 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of the 
“Declaration of Helsinki” (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonization (ICH) guidelines, or with the laws and 
regulations of the country in which the research is conducted, whichever affords the greater 
protection to the study subject. For studies conducted under a United States IND, the investigator 
will ensure that the basic principles of “Good Clinical Practice,” as outlined in 21 CFR 312, 
subpart D, “Responsibilities of Sponsors and Investigators,” 21 CFR, part 50, 1998, and 21 CFR, 
part 56, 1998, are adhered to. 
Since this is a “covered” clinical trial, the investigator will ensure that 21 CFR, Part 54, 1998, is 
adhered to; a “covered” clinical trial is any “study of a drug or device in humans submitted in a 
marketing application or reclassification petition subject to this part that the applicant or FDA 
relies on to establish that the product is effective (including studies that show equivalence to an 
effective product) or that make a significant contribution to the demonstration of safety.” This 
requires that investigators and all sub-investigators must provide documentation of their financial 
interest or arrangements with Lakewood-Amedex or proprietary interests in the drug being 
studied. This documentation must be provided before participation of the investigator and any 
sub-investigator. The investigator and sub-investigator agree to notify Lakewood-Amedex of any 
change reportable interests during the study and for one year following completion of the study. 
Study completion is defined as the date that the last subject has completed the protocol defined 
activities. 
This study is also subject to and will be conducted in accordance with 21 CFR, part 320, 1993, 
“Retention of Bioavailability and Bioequivalence Testing Samples.” 
8.1.2 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) 
Approval 
This protocol and any accompanying material to be provided to the subject (such as 
advertisements, subject information sheets, or descriptions of the study used to obtain informed 
consent) will be submitted by the investigator to an IRB in compliance with FDA regulations for 
IRBs (21CFR Part 56). Approval from the IRB must be obtained before starting the study and 
must be documented in a letter to the investigator specifying the protocol number, protocol 
version, protocol date, documents reviewed, and date on which the committee met and granted 
the approval. 
Any modifications made to the protocol after receipt of IRB approval must also be submitted to 
the IRB for approval before implementation. 


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8.1.3 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must utilize an IRB approved consent form for documenting written informed 
consent. Each informed consent will be appropriately signed and dated by the subject or the 
subject’s legally authorized representative and the person obtaining consent.  The information in 
the informed consent must comply with 21CFR Part 50, Subpart B.  The elements of the 
informed consent must be consistent with the requirements of 21CFR Part 50.25. 
8.1.4 
Confidentiality 
The investigator must assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, and an 
identification code (i.e., not names) should be recorded on any form or biological sample 
submitted to the Sponsor, IRB, or laboratory. The investigator must keep a screening log 
showing codes, names, and addresses for all subjects screened and for all subjects enrolled in the 
trial. 
The investigator agrees that all information received from  Lakewood-Amedex, including but not 
limited to the Investigator Brochure, this protocol, CRFs, the investigational new drug, and any 
other study information, remain the sole and exclusive property of  Lakewood-Amedex during 
the conduct of the study and thereafter. This information is not to be disclosed to any third party 
(except employees or agents directly involved in the conduct of the study or as required by law) 
without prior written consent from Lakewood-Amedex. The investigator further agrees to take 
all reasonable precautions to prevent the disclosure by any employee or agent of the study site to 
any third party or otherwise into the public domain. 
8.1.5 
Study Files and Retention of Records 
The investigator must maintain adequate and accurate records to enable the conduct of the study 
to be fully documented and the study data to be subsequently verified. These documents should 
be classified into at least the following two categories: (1) investigator’s study file, and 
(2) subject clinical source documents. 
The investigator’s study file will contain the protocol/amendments, CRF and query forms, IRB 
and governmental approval with correspondence, informed consent, drug records, staff 
curriculum vitae and authorization forms, and other appropriate documents and correspondence. 
The required source data are listed in the Source Data verification Plan, and should include 
sequential notes containing at least the following information for each subject: 
• subject identification (name, date of birth, gender); 
• documentation that subject meets eligibility criteria, i.e., history, physical examination, 
and confirmation of diagnosis (to support inclusion and exclusion criteria); 


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Page 50 
 
• participation in trial (including trial number); 
• trial discussed and date of informed consent; 
• dates of all visits; 
• documentation that protocol specific procedures were performed; 
• results of efficacy parameters, as required by the protocol; 
• start and end date (including dose regimen) of trial medication (preferably drug 
dispensing and return should be documented as well); 
• record of all adverse events and other safety parameters (start and end date, and 
preferably including causality and intensity); 
• concomitant medication (including start and end date, dose if relevant; dose changes 
should be motivated); 
• date of trial completion and reason for early discontinuation, if applicable. 
 
All clinical study documents must be retained by the investigator until at least 2 years after the 
last approval of a marketing application in an ICH region (i.e., United States, Europe, or Japan) 
and until there are no pending or contemplated marketing applications in an ICH region; or, if no 
application is filed or if the application is not approved for such indication, until 2 years after the 
investigation is discontinued and regulatory authorities have been notified. Investigators may be 
required to retain documents longer if required by applicable regulatory requirements, by local 
regulations, or by an agreement with Lakewood-Amedex. The investigator must notify 
Lakewood-Amedex before destroying any clinical study records. 
Should the investigator wish to assign the subject records to another party or move them to 
another location, Lakewood-Amedex should be notified in advance. 
If the investigator cannot guarantee this archiving requirement at the study site for any or all of 
the documents, special arrangements must be made between the investigator and  Lakewood-
Amedex to store these in sealed containers outside of the site so that they can be returned sealed 
to the investigator in case of a regulatory audit. When source documents are required for the 
continued care of the subject, appropriate copies should be made for storage outside of the site. 
8.1.6 
Case Report Forms 
For each subject enrolled, a CRF must be completed and signed by the principal investigator or 
sub-investigator (as appropriate) within a reasonable time period after data collection. This also 
applies to records for those subjects who fail to complete the study (even during a pre-
randomization screening period if a CRF was initiated). If a subject withdraws from the study, 
the reason must be noted on the CRF. If a subject is withdrawn from the study because of a 
treatment-limiting adverse event, thorough efforts should be made to clearly document the 
outcome. 


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8.1.7 
Drug Accountability 
The investigator or designee (i.e., pharmacist) is responsible for ensuring adequate accountability 
of all used and unused investigational medicinal product, placebos, and comparators. This 
includes acknowledgment of receipt of each shipment of study product (quantity and condition), 
subject dispensing records, and returned or destroyed study product. Dispensing records will 
document quantities received from  the study sponsor and quantities dispensed to subjects, 
including kit number, date dispensed, subject identifier number, subject initials, and the initials 
of the person dispensing the medication. 
At study initiation, the monitor will evaluate the site’s standard operating procedure for 
investigational medicinal product disposal/destruction in order to ensure that it complies with 
Lakewood-Amedex requirements. Drug may be returned on an ongoing basis during the study, if 
appropriate. At the end of the study, following final drug inventory reconciliation by the monitor, 
the study site will ship any remaining investigational product (to include empty containers) to 
Xcelience Inc. for disposal.   
All drug supplies and associated documentation will be periodically reviewed and verified by the 
study monitor over the course of the study. 
8.1.8 
Inspections 
The investigator understands that source documents for this trial must be made available to 
appropriately qualified personnel from Lakewood-Amedex or its representatives, to IRBs or to 
regulatory authority or health authority inspectors. 
8.1.9 
Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements.  The noncompliance maybe either on the part of 
the subject, the investigator, or the study site staff.  As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly. 
These practices are consistent with ICH E6: 
• Compliance with Protocol sections 4.5.1, 4.5.2, and 4.5.3 
• Quality Assurance and Quality Control, section 5.1.1 
• Noncompliance sections 5.20.1, and 5.20.2 
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations 
within 5 working days of identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity.  All significant deviations must be promptly reported to 


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Page 52 
 
Lakewood Amedex Inc. to the attention of 
 via fax.  Reports can be sent to 
Lakewood -Amedex as specified below copying the CRA assigned to your site: 
• Via the web-based PD forms submission 
• FAX submission (1
): utilizing the Protocol Deviation form and the 
email/fax transmittal form, or 
• E-mail submission (
) utilizing the Protocol Deviation 
form and the email/fax transmittal form. 
 
All deviations from the protocol must be addressed in the study subject source documents.  A 
completed copy of the LAI Protocol Deviation form must be maintained in the regulatory file, as 
well as in the subject’s source document.  Significant protocol deviations must be sent to the 
local IRB/EC per their guidelines.  The site PI/study staff is responsible for knowing and 
adhering to their IRB/EC requirements. 
8.2 
Sponsor Responsibilities 
8.2.1 
Protocol Modifications 
Protocol modifications, except those intended to reduce immediate risk to study subjects, may be 
made only by Lakewood-Amedex. All protocol modifications must be submitted to the IRB in 
accordance with local requirements. Approval must be obtained before changes can be 
implemented. 
8.2.2 
Study Report and Publications 
A clinical study report will be prepared by Protocol Coordinator and provided to the regulatory 
agency(ies).  Lakewood-Amedex will ensure that the report meets the standards set out in the 
ICH Guideline for Structure and Content of Clinical Study Reports (ICH E3). Note that an 
abbreviated report may be prepared in certain cases. 
Lakewood-Amedex Inc. will have the sole decision on whether or not to publish the results of 
this research.  The Company encourages its scientists and collaborators to published the results 
of any research sponsored by the Company but only after a review of the data by the Company to 
ensure that any discoveries or trade secrets are properly protected by issued, pending, or newly 
filed patents or patents applications.   
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials registration policy as a condition for publication.  This policy requires that all clinical 
trials except Phase I studies be registered in a public trials registry such as ClinicalTrials.gov, 
which is sponsored by the National Library of Medicine.  Other biomedical journals are 
considering adopting similar policies.  Lakewood-Amedex, Inc. will be responsible for 
registering the clinical trial with an acceptable registry.  Any clinical trial starting enrollment 
after 01 July 2005 must be registered on or before subject enrollment. 


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The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subject to intervention or comparison groups to study the cause-and-effect relationship between 
medical intervention and a health outcome.  Studies designed for other purposes, such as to study 
pharmacokinetics or major toxicity (e.g. Phase I trials) would be exempt from this policy. 
8.3 
Joint Investigator/Sponsor Responsibilities 
8.3.1 
Access to Information for Monitoring 
In accordance with ICH Good Clinical Practice (ICH GCP) guidelines, the study monitor must 
have direct access to the investigator’s source documentation in order to verify the data recorded 
in the CRFs for consistency. 
The monitor is responsible for routine review of the CRFs at regular intervals throughout the 
study to verify adherence to the protocol and the completeness, consistency, and accuracy of the 
data being entered on them. The monitor should have access to any subject records needed to 
verify the entries on the CRFs. The investigator agrees to cooperate with the monitor to ensure 
that any problems detected in the course of these monitoring visits are resolved. 
8.3.2 
Access to Information for Auditing or Inspections 
Representatives of regulatory authorities or of Lakewood-Amedex may conduct inspections or 
audits of the clinical study. If the investigator is notified of an inspection by a regulatory 
authority the investigator agrees to notify the Lakewood-Amedex medical monitor immediately. 
The investigator agrees to provide to representatives of a regulatory agency or Lakewood-
Amedex access to records, facilities, and personnel for the effective conduct of any inspection or 
audit. 
8.3.3 
Study Discontinuation 
Both the sponsor and the investigator reserve the right to terminate the study at any time. Should 
this be necessary, both parties will arrange discontinuation procedures and notify the appropriate 
regulatory authority(ies), and IRB/IECs. In terminating the study, Lakewood-Amedex and the 
investigator will assure that adequate consideration is given to the protection of the subjects’ 
interests. 
 


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9 
REFERENCES 
1. 
National intelligence estimate: the global infectious disease threat and its 
implications for the United States. Environ Change Secur Proj Rep. 2000 
Summer;(6):33-65. Publication NIE 99-17D. 
2. 
Institute of Medicine, America’s Vital Interest in Global Health: Protecting Our 
People, Enhancing Our Economy, and Advancing our International Interests 
(National 
Academy 
Press, 
Washington, 
DC, 
1997); 
http//www.nap.edu/readingroom/books/avi/. 
3. 
World Health Organization 2007 Tuberculosis Facts. and 2014 TB Facts 
http://www.who.int/features/factfiles/tuberculosis/en/. 
4. 
Lewis, K.. Platforms for antibiotic discovery.  Nature Reviews Drug Discovery 
2013 12:p371-387 
5. 
U.S. Department of Health and Human Services, Centers for Disease Control and 
Prevention. Antibiotic resistant threats in the United States, 2013. 
6. 
Dale, R.M.K., Schnell, G., Zhang, R.J.D., and Wong, J.P..  Therapeutic efficacy of 
aerosolized liposome-encapsulated nubiotic against pulmonary Pseudomonas 
aeruginosa infection. Therapy (2007) vol 4 (4) p441-9.   
7. 
Dale, R.M.K., Schnell, G., and Wong. J.P..  Therapeutic Efficacy of “Nubiotics” 
against Burn Wound Infections by Pseudomonas aeruginosa. Antimicrobial 
Agents and Chemotherapy  Aug (2004) vol 48 (8), p2918-2923.   
8. 
Cao, S., Sun, L.Q., and Wang, M... Antimicrobial activity and mechanism of 
action of Nu-3, a protonated modified nucleotide. Annals of Clinical Microbiology 
And Antimicrobials 2011 10(1) p1-9. 
9. 
Roderic M.K. Dale, United States Patent No. 7,868,162 B1 “ Antimicrobial and 
Antiviral Compounds and Methods of Their Use”  Jan 11, 2011 
10. 
Lipsky, B., Berendt, A.,Cornia, P., Pile, J., Peters, E., Armstrong, D.,Deery, H., 
Embil, J., Joseph, W., Karchimer, Pinzor, M., and Sennerville, E.. 2012 Infectious 
Diseases Society of America Clinical Practice Guideline for the Diagnosis and 
Treatment of Diabetic Foot Infections. IDSA Guidelines for Diabetic Foot 
Infections. CID 2:254p 1679-1684 
11. 
Chen., L. and Wen, Y. 2011 The role of bacterial biofilm in persistent infections 
and control strategies. Int. J. Oral Sci. 3:p66-73. 


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12. 
Akiyoshi, D., Dilo, J., and DiTullio, P., Novel Bisphosphocin Nu-3 Demonstrates 
Rapid Killing of Bacteria Encased in Biofilm In vitro.  Abstract 2013, 53rd ICAAC 
Sept. 10-13, Denver, CO. 
 
 
 
 
 


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10 
 APPENDICES 
 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood Amedex 
Appendix A. Project Timeline and Flow Chart 
Obtain Informed Consent 
Screen Subject 
Assign to Open Cohort 
SAD Ann 1: Cohort 1 - 0.1% or 
l mg/ml 
(n=lO) 
(2 Placebo; 8 Nu-3) 
Perform Informed Consent and 
Docwnent 
Pregnancy and Drug Screen 
<24h Before Treatment 
Perform Skin Sensitization Test, 
Apply Treatment 
Follow up Visit 3 
PI Review 
r+ SAD Ann 1 :Cohort 2 - 1 % or 1 Omg/ml 
at 24hr Examination 
fp=IO) 
No Safety Issues. Proceed to MAD Arm -
Cohort 1 
(2 Placebo; 8 Nu-3) 
Perform Informed Consent and 
Docmnent 
Instruct on Treatment 
Pregnancy and Dmg Screen 
Observe Subject Apply 1st Treatment 
<24h Before Treatment 
Follow up Visit 5 
Sensitization Test, Apply Treatment 
Follow up Visit 3 
SMC Review After All Subject 
PI Review at 24hr Examination 
Have Completed Cohort 1 
No Safety Issues. Proceed to MAD Arm 
No Safety Issues. Proceed to Next Cohort 
Cohort2 
-
Instruct on Treatment 
Observe Subject Apply 1
51 Treatment 
Follow up Visit 5 
SMC Review after first 5 subjects 
completed 
If approval given by SMC extension 
of treatment to fourteen (14) days 
MAD at PI discretion 
Follow-up Visit 6 
SMC Review After All Subject 
Have Completed Cohort 2 
Page 57 
Original - 310 ct2014 
.. SAD Ann 1: Cohort 3 - 2% or 20mg/ml 
(n=lO) 
(2 Placebo; 8 Nu-3) 
Perform Informed Consent and 
Document 
Pregnancy and Drug Screen 
<24h Before Treatment 
Review Infonned Consent & 
Document 
Perform Skin Sensitization Test, 
Apply Treatment 
Follow up Day 2 
PI Review at 24hr Examination 
1-
No Safety Issues. Proceed to MAD 


Bisphosphocin NU-3 
Protocol #LAI2014-1 
Lakewood Amedex 
No Safety Issues. Proceed to Next 
Cohort 
Page 58 
Original - 310 ct2014 
Ann 2 Cohort 3 
Instruct on Treatment 
Obseive Subject Apply 1
51 Treatment 
Follow up Visit 5 
SMC Review after first 5 subjects 
completed 
If approval given by SMC extension 
of treatment to fourteen (14) days 
MAD at PI discretion 
Follow-up Visit6 


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Appendix B:  Schedule of Events 
Key 
a Visit window 24 hours ± 2 hours post Visit 2 
b Visit window 8 days + 1 day post Visit 2 
c Visit window 14 days ± 1 day post Visit 2 for 7-Day MAD subjects or 15 days +1 day post Visit 2 for 14-Day MAD subjects 
d Visit window 22 days ± 1 day post Visit 2  (Visit 6 only necessary for 14-day MAD subjects) 
1 Height and weight (screening, Day 1, follow-up visit and ET), temperature, resting blood pressure (BP), heart and respiration rate 
2 Resting BP, heart and respiration rate to be obtained prior to and 1 hour post SAD Treatment 
3 Urine pregnancy test for all female subjects of childbearing potential 
4  Radiography, MRI and/or CT evaluation required within 2 weeks of Visit 1 (Screening Visit), at all other visits only necessary if determined by PI  
5 Upon SMC review and safety approval of 1st five (5) subjects in Cohorts 2 and 3, Principal Investigator (PI) may elect to extend treatment to fourteen (14) days if in the opinion 
of the PI the subject is showing improvement at Visit 4. 
6 Signs and symptoms of adverse events to be obtained 1 hour post SAD Treatment 
Event 
Visit 1 
Screening 
Visit  
Visit 2 
SAD 
Treatment  
Visit 
3a 
 
 Visit 
4b   
 
Visit 
5c  
 
Visit 
6d 
 
Early 
Termination 
(ET) Visit 
Informed Consent Process 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
Physical Exam 
X 
 
 
 
X 
X 
X 
Vital Signs1  
X 
X2 
X 
X 
X 
X 
X 
CBC, CMP Blood Draw 
X 
X 
 
X 
X 
X 
X 
Collect ulcer tissue sample for microbiological evaluation 
X 
X 
 
X 
X 
X 
X 
Urine Pregnancy Test (HCG)3 
X 
X 
 
 
 
Drug and ETOH toxicity screen 
X 
X 
 
 
 
 
 
Visual Examination of Chronic Ulcer 
X 
X 
X 
X 
X 
X 
X 
Radiograph, MRI and/or CT evaluation of the infected ulcer4 
X 
 
 
X 
 
 
 
Photo Documentation and Calculation of target ulcer area and depth – 
ARANZ Silhouette™ Medical System 
X 
X 
X 
X 
X 
X 
X 
Score target ulcer using DUSS Scoring System 
X 
X 
 
X 
X 
X 
 
Rating of the infected ulcer using DFI Wound Scoring system 
X 
 
 
X 
X 
X 
 
Skin Sensitivity Test 
 
X 
 
 
 
 
 
Investigational Product 
 
X 
X 
X5 
 
 
 
Concomitant Medication 
X 
X 
X 
X 
X 
X 
X 
Adverse Event 
X 
X6 
X 
X 
X 
X 
X 


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Page 60 
 
Appendix C:  Use of the Aranz Medical Silhouette™ System for Photo documentation of 
the DFU and Calculation of Area and Depth, and Volume. 
 
Capturing a Wound Image 
 
1. Log in to Silhouette Connect and Select the Patient to be imaged. 
2. Review the patient details and select Capture Images Button.  The laser lines should 
come on from the Silhouette Star Camera. 
3. To capture the wound image position the Silhouette Star Camera directly above the 
wound and move the camera up or down so that the laser lines form a star.  The center of 
the laser line star should be over the deepest part of the wound. 
4. Capture an image of the wound by pressing the bottom on the camera.   
5. Repeat the process to capture multiple images. 
6. All images will be uploaded to the SilhouetteConnect Database on the tablet. 
 
Calculating the Ulcer Area, Volume, and Depth 
 
1. Select the Ulcer image to be used for assessment on the tablet. 
2. Enlarge the image so the perimeter of the ulcer is easily visible 
3. Outline the perimeter of the ulcer by either clicking around the perimeter so that the 
entire ulcer is surrounded by dots or tracing the perimeter of the ulcer with a pointer or 
finger.   
4. Clicking on the circular target to initiate calculation of the area, volume, and depth of the 
ulcer by the SilhouetteConnect software. 
5. Click on the Notes(?) button to enter comments on the ulcer margins, appearance, and 
surrounding skin 
6. Click Generate pdf report and Save to Store it to the database. 
7. Click Send to export to the ClinicalInk Database. 
 
 


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Appendix D: Subject Home Treatment Log:  
 
Subject ID No.: 
 
 
 
 
 
 
 
 
 
Study Drug Number: 
 
 
 
 
 
 
 
Issue Date: 
 
 
 
 
 
Return Date:  
 
 
 
 
Instructions:  Subjects/caregivers should log both the date and approximate time of each 
treatment and the person administering the study drug should initial in the corresponding box.  A 
space is also provided for the subject/caregiver to write any comments concerning the 
administration or observations of the ulcer. 
 
Day 
Treatment 
1 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Treatment 
2 
Date/Time 
Initials 
Therapeutic 
Footwear  
Worn 
Length of 
Time 
Footwear 
Worn 
Comments 
1 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
2 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
3 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
4 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
5 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
6 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
7 
 
 
☐   Yes        
☐   No       
☐1-4 hrs.      
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
☐   Yes        
☐   No        
☐1-4 hrs.       
☐ 5-8 hrs.      
☐ 9-12 hrs.    
 
 
 

SAP_001.pdf: 
 
 
August 25, 2017 
Page 1 of 53 
 
 
 
 
Statistical Analysis Plan 
 
 
Lakewood Amedex 
LAI2014-1 
 
A Phase I/IIa, Randomized Double Blind, Placebo-Controlled, Dose Escalating 
Study to Evaluate the Safety and Tolerability of Topically Applied 
Bisphosphocin™ Nu-3 on Infected Diabetic Ulcers of Subjects  
With Type I or II Diabetes Mellitus 
 
Protocol Version: 3.1 
15 September 2016 
 
 
 
 
Sponsor: 
 
Lakewood Amedex Inc. 
3030 University Pkwy 
Sarasota, FL 34243 
 
 
Prepared By: 
 
 
 
 
 
 
 
Version: 
 
Final 
 
Date of Creation: August 31, 2017 
 
 
 


Lakewood Amedex 
 
Statistical Analysis Plan 
LAI2014-1 
 
August 31, 2017 
Page 2 of 53 
 
 
Approvals: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
      
 
 
 


Lakewood Amedex 
 
Statistical Analysis Plan 
LAI2014-1 
 
August 31, 2017 
Page 3 of 53 
 
Table of Contents 
LIST OF ABBREVIATIONS ......................................................................................................... 4 
DEFINITIONS ................................................................................................................................ 5 
1. 
INTRODUCTION ................................................................................................................... 6 
2. 
OBJECTIVES .......................................................................................................................... 6 
3. 
STUDY OVERVIEW.............................................................................................................. 6 
4. 
GENERAL ANALYSIS CONSIDERATIONS ...................................................................... 7 
5. 
ANALYSIS POPULATIONS ................................................................................................. 7 
5.1 
Intent-to-Treat (ITT) Population ...................................................................................... 7 
5.2 
Safety Population ............................................................................................................. 7 
5.3 
Per-Protocol (PP) Population ........................................................................................... 8 
6. 
SUBJECT DISPOSITION ....................................................................................................... 8 
7. 
DEMOGRAPHIC AND BASELINE CHARACTERISTICS ................................................ 8 
8. 
EFFICACY ANALYSIS ......................................................................................................... 8 
8.1 
Definition of Efficacy Endpoints ..................................................................................... 8 
8.2 
Analysis of Efficacy Endpoints ........................................................................................ 9 
9. 
SAFETY ANALYSES ............................................................................................................ 9 
9.1 
Adverse Events ................................................................................................................. 9 
9.2 
Vital Signs ...................................................................................................................... 10 
9.3 
Physical Examinations ................................................................................................... 11 
9.4 
Prior and Concomitant Medications ............................................................................... 11 
10. 
SAMPLE SIZE AND POWER .......................................................................................... 11 
11. 
APPENDICES ................................................................................................................... 12 
APPENDIX A: LIST OF TABLES AND LISTINGS .................................................................. 12 
List of Tables ............................................................................................................................. 12 
List of Data Listings .................................................................................................................. 13 
APPENDIX B: TABLES .............................................................................................................. 14 
APPENDIX C: LISTINGS ........................................................................................................... 31 
 
 
 


Lakewood Amedex 
 
Statistical Analysis Plan 
LAI2014-1 
 
August 31, 2017 
Page 4 of 53 
 
LIST OF ABBREVIATIONS 
 
AE  
 
Adverse Event 
ATC   
Anatomical/Therapeutic/Chemical 
BMI   
Body Mass Index 
CRF   
Case Report Form 
CSR   
Clinical Study Report 
DFI                  Diabetic Foot Ulcer Wound Infection Score 
DUSS              Diabetic Ulcer Severity Score 
ITT 
 
Intent-to-Treat 
MedDRA  
Medical Dictionary for Regulatory Activities 
PK  
 
Pharmacokinetic 
PP 
 
Per Protocol 
SAE   
Serious Adverse Event 
SD  
 
Standard Deviation 
TEAE   
Treatment-Emergent Adverse Event 
WHO   
World Health Organization 
 
 


Lakewood Amedex 
 
Statistical Analysis Plan 
LAI2014-1 
 
August 31, 2017 
Page 5 of 53 
 
DEFINITIONS 
 
Intent-to-Treat (ITT) Population 
Includes all randomized subjects. 
 
Safety Population  
Includes all subjects administered any amount of the study 
drug. 
 
Per-Protocol (PP) Population  
Includes all randomized subjects who received all doses  
of the of investigational product as required by the protocol 
and had no major protocol violations.  
 
Treatment-emergent   
 
AEs with an onset time after dosing and pre-existing  
Adverse Events (TEAE) 
 
AEs that worsen during the study. 
 
 
 


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1. INTRODUCTION 
 
This document outlines the statistical methods to be implemented during the analyses of clinical 
trial data collected within the scope of Lakewood Amedex protocol LAI2014-1 [A Phase I/IIa, 
Randomized Double Blind, Placebo-Controlled, Dose Escalating Study to Evaluate the Safety 
and Tolerability of Topically Applied Bisphosphocin™ Nu-3 on Infected Diabetic Ulcers of 
Subjects with Type I or II Diabetes Mellitus]. The purpose of this plan is to provide specific 
guidelines from which the analysis performed by 
 will proceed. 
Pharmacokinetics (PK) data was not collected for this study. Any deviations from these 
guidelines will be documented in the clinical study report (CSR). 
2. OBJECTIVES 
 
The primary objective of the study is as follows: 
• To assess the safety and tolerability of a twice daily, 7-day repeat topical doses of Nu-3 
in adult male and female subjects with chronically infected diabetic ulcer. 
 
The secondary objectives of the study are as follows: 
• To assess the clinical and microbiological response to Bisphosphocin™ Nu-3. 
• To support determination of the appropriate dose range of Nu-3 to be employed in future 
clinical studies. 
3. STUDY OVERVIEW 
 
This is a phase 1/2a prospective, multi-center, blinded, randomized, dose escalation study. This 
study will be conducted at four investigational sites. Subjects with diabetic chronic foot ulcer(s) 
and a Diabetic Ulcer Severity Score (DUSS) of 0 to 3, ulcerated area(s) of not more than two 
ulcers between 0.5 and 6 cm2, and no underlying DUSS Probing to Bone=’Yes’ are eligible to be 
enrolled into the study. 
 
Doses of Nu-3 will be escalated in three planned sequential cohorts with 10 subjects in each 
cohort. Randomization will be 4:1 active to placebo in each cohort. The dose levels in each 
cohort are: 
 
• Cohort 1 – 0.1% solution of Nu-3 in 0.9% saline with vehicle 
• Cohort 2 – 1% solution of Nu-3 in 0.9% saline with vehicle 
• Cohort 3 – 2% solution of Nu-3 in 0.9% saline with vehicle 
 
Each cohort has placebo defined as 0.9% saline with vehicle. Dosing will be twice daily for 7 
consecutive days. 
 
Subjects will have scheduled study visits as follows: 
• Screening (Day -7 to -4 hours) 
• Single dose visit (Day 1) 


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• Safety review and multiple dose visit (Day 2) 
• Safety review visit (Day 9 ±1 day) 
• Final Visit (Day 15 ±2 days) 
 
At the conclusion of each cohort, a Safety Review Committee will review the safety data and 
determined if it is safe to proceed to the next cohort. The committee includes, but is not limited 
to the Sponsor’s designated Medical Monitor, the Medical Director of the CRO, and the Medical 
Director of Lakewood Amedex. Additional members can be added as the sponsor deemed 
appropriate.  
4. GENERAL ANALYSIS CONSIDERATIONS 
 
The statistical analyses will be reported using summary tables and data listings. For all efficacy 
parameters, a comparison will be made of each active treatment group versus placebo using the 
appropriate statistical test. Unless otherwise indicated, all statistical tests will be two-sided and 
differences will be considered statistically significant if the associated p-value is less than 0.05. 
Continuous variables will be summarized with means, standard deviations (SD), medians, 
minimums, and maximums. Categorical variables will be summarized by counts and by 
percentage of subjects in corresponding categories. All data will be summarized by dose group 
(cohort), with all placebo subjects combined into one placebo arm. 
 
Individual subject data obtained from the electronic data capture system, study lab, and any 
derived data will be presented by cohort for each subject in data listings. 
 
The analyses described in this plan are considered a priori, in that they have been defined prior 
to database lock for individual subjects. Any analyses performed subsequent to database lock 
will be considered post-hoc and exploratory. Post-hoc analyses will be identified in the CSR. 
 
All analyses and tabulations will be performed using SAS® Version 9.4 or higher on a Windows 
platform. Tables and listings will be presented in PDF format. Upon completion, all SAS® 
programs will be validated by an independent programmer. In addition, all program output will 
undergo a senior level statistical review. The validation process will be used to confirm that 
statistically valid methods have been implemented and that all data manipulations and 
calculations are accurate. Checks will be made to ensure accuracy, consistency with this plan, 
consistency within tables, and consistency between tables and corresponding data listings. Upon 
completion of validation and quality review procedures, all documentation will be collected and 
filed by the project statistician or designee. 
5. ANALYSIS POPULATIONS 
5.1 Intent-to-Treat (ITT) Population 
All randomized subjects will be included in the ITT Population.  
5.2 Safety Population 


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All subjects administered any amount of investigational product will be included in the Safety 
Population. 
5.3 Per-Protocol (PP) Population 
The PP Population will include all randomized subjects who receive all doses of investigational 
product as required by the protocol and have no major protocol violations. Major protocol 
violations will be reviewed and the subjects in the PP population will be identified prior to 
unblinding the study. 
6. SUBJECT DISPOSITION 
 
Subject disposition information will be summarized for all subjects by dose group. Summaries 
will include: the number of screened and screen failure subjects, the number of subjects in the 
ITT, Safety and PP Populations, the number of subjects completing the study, and the primary 
reason for discontinuing the study.  
7. DEMOGRAPHIC AND BASELINE CHARACTERISTICS 
 
Demographic information (age, sex, race, and ethnicity) and baseline characteristics (height, 
weight and BMI) will be summarized by dose group. These demographic and baseline 
characteristics summaries will be analyzed for the Safety Population. Inferential testing is not 
planned on any demographic and baseline characteristic data. 
8. EFFICACY ANALYSIS 
8.1 Definition of Efficacy Endpoints 
All efficacy endpoints will be summarized by visit and evaluated statistically at the end of the 
MAD portion of the study. Efficacy endpoints include: 
• The proportion of subjects with a normal culture 
• The change from baseline in the DUSS score 
• The change from baseline in the DFI score 
• The absence of each of the following as reported by the investigator on the CRF: 
o Purulent discharge 
o Nonpurulant drainage 
o Erythema 
o Induration 
o Tenderness 
o PainLocal warmth (no change) 
• Data captured on the total wound size for each subject using the Aranz Medical 
Silhouette™ System: 
o Wound area (cm2) 


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o Percentage of area reduction 
o Perimeter (mm) 
o Maximum depth (mm) 
o Mean depth (mm) 
o Volume (cm3) 
o Length (mm) 
o Width (mm)  
8.2 Analysis of Efficacy Endpoints 
For each dose group, the proportion of subjects with a normal culture at the end of the MAD 
portion of the study will be compared to the proportion of subjects with a normal culture at the 
end of the MAD portion of the study for the combined placebo group using a Fisher’s exact test.  
A subject with a normal culture at this time will be considered a success. Any subject with a 
missing value at the end of the MAD portion of the study will not be included in the analysis. A 
p-value less than 0.05 will be considered statistically significant and a p-value less than 0.0167, 
employing a Bonferroni correction for multiple comparisons, will be considered highly 
conclusive.  
For continuous efficacy endpoints, data will be summarized descriptively at and a comparison 
using the Wilcoxon Rank Sum test between each of dose groups and the combined placebo 
group will be performed at each visit. For these endpoints, no imputation for missing values will 
be performed.  
For categorical endpoints, a Fisher’s exact test will be performed in a similar manner. Subjects 
missing a value for a secondary endpoint will be considered a failure (counted in the 
denominator) for that endpoint.  
For all tests, a p-value less than 0.05 will be considered statistically significant and a p-value less 
than 0.0167, employing a Bonferroni correction for multiple comparisons, will be considered 
highly conclusive. Analysis for each secondary endpoint will be performed on the ITT 
population and the PP population. 
Ulcer measurements and percentage of area reduction will be summarized descriptively by visit 
and statistical testing using a Wilcoxon Rank Sum test between each dose group and the 
combined placebo group. For all tests, a p-value less than 0.05 will be considered statistically 
significant. Analysis for each additional endpoint will be performed on the ITT population and 
the PP population. 
9. SAFETY ANALYSES 
 
For all safety analyses, the safety population will be used.  
9.1 Adverse Events 
 
All AE summaries will be restricted to Treatment-Emergent Adverse Events (TEAE), which are 


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defined as those AEs that occurred after dosing and those pre-existing AEs that worsened during 
the study. Verbatim terms on case report forms will be mapped to preferred terms and system 
organ classes using the MedDRA dictionary (version 14.1). 
 
Each AE summary will be displayed by dose group. Summaries that are displayed by system 
organ class and preferred terms will be ordered by descending order of incidence of system 
organ class and preferred term within each system organ class. Summaries of the following types 
will be presented: 
 
• Subject incidence of TEAEs and total number of unique TEAEs by MedDRA system 
organ class and preferred term. 
• Subject incidence of TEAEs by MedDRA system organ class, preferred term, and 
highest severity. At each level of subject summarization, a subject is classified 
according to the highest severity if the subject reported one or more events. An event 
with missing severity will not be imputed.  
• Subject incidence of TEAEs by MedDRA system organ class, preferred term, and 
closest relationship to study drug (Not Related, Unlikely, Possibly, Probable, 
Definite). At each level of subject summarization, a subject is classified according to 
the closest relationship if the subject reported one or more events. AEs with a missing 
relationship will be considered definite related for this summary. 
• Subject incidence of serious TEAEs and total number of unique serious TEAEs by 
MedDRA system organ class and preferred term. 
 
9.2 Vital Signs 
 
Vital sign assessments will be analyzed by dose group at baseline and each scheduled post-
baseline visit. Mean observed measurements and mean changes from baseline will be 
summarized. Baseline is defined as the last non-missing value obtained prior to receiving study 
drug. 
 
An outlier analysis will be performed to provide counts and percentage of subjects above or 
below certain cutoffs for specific vital sign parameters. The following cutoffs will be used in the 
outlier analysis for the vital signs data: 
 Heart Rate 
• Any post-baseline value > 120 bpm or > 130 bpm 
• Any increase from baseline of ≥ 20 bpm or ≥ 30 bpm 
 
Systolic Blood Pressure 
• Any post-baseline value > 150 mmHg or < 90 mmHg 
• Any increase from baseline of ≥ 20 mmHg or ≥ 30 mmHg 
• Any decrease from baseline of ≥ 20 mmHg or ≥ 30 mmHg 
 


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Diastolic Blood Pressure 
• Any post-baseline value > 100 mmHg or < 50 mmHg 
• Any increase from baseline of ≥ 20 mmHg or ≥ 30 mmHg 
• Any decrease from baseline of ≥ 20 mmHg or ≥ 30 mmHg 
 
The above outlier analysis will be conducted for post-baseline vital sign assessments collected 
throughout the entire study period.  
9.3 Physical Examinations 
 
Physical examination results will be provided in a listing. 
9.4 Prior and Concomitant Medications 
 
Verbatim terms on case report forms will be mapped to Anatomical/Therapeutic/Chemical 
(ATC) Level 4 categories and Drug Reference Names using the World Health Organization 
(WHO) dictionary (WHODDE B2 format, March 1, 2011 release). 
 
Prior medications are those medications taken within the last 14 days prior to the dose of study 
drug, and will be provided in a by-subject listing. Ongoing prior medications taken during the 
study will be considered concomitant medications. 
 
Concomitant medications are those medications taken after the dose of study drug. Concomitant 
medications will be summarized by WHO ATC class and medication name for each dose group. 
The summary will present the number and percentage of subjects using each medication. 
Subjects may have more than one medication per ATC category and medication. At each level of 
subject summarization, a subject is counted once if s/he reported one or more medications at that 
level. Each summary will be ordered by descending order of incidence of ATC class and generic 
drug name within each ATC class. 
10.    SAMPLE SIZE AND POWER 
 
This is a pilot dose-ranging study. No sample size calculations were conducted. 
 
 


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11. 
APPENDICES 
APPENDIX A: LIST OF TABLES AND LISTINGS 
 
List of Tables 
Table Number 
Table Description 
14.1.1 
Subject Disposition (All Subjects) 
14.1.2 
Demographic and Baseline Characteristics (Safety Population) 
14.2.1.1 
Summary of Microbiology Findings (ITT Population) 
14.2.1.2 
Summary of Microbiology Findings (PP Population) 
14.2.2.1 
Summary of DUSS Scores (ITT Population) 
14.2.2.2 
Summary of DUSS Scores (PP Population) 
14.2.3.1 
Summary of DFI Scores (ITT Population) 
14.2.3.2 
Summary of DFI Scores (PP Population) 
14.2.3.3 
Summary of DFI Components (ITT Population) 
14.2.3.4 
Summary of DFI Components (PP Population) 
14.2.4.1 
Summary of Ulcer Measurements (ITT Population) 
14.2.4.2 
Summary of Ulcer Measurements (PP Population) 
14.3.1.1 
Treatment-Emergent Adverse Events by System Organ Class and 
Preferred Term (Safety Population) 
14.3.1.2 
Treatment-Emergent Adverse Events by System Organ Class, 
Preferred Term and Severity (Safety Population) 
14.3.1.3 
Treatment-Emergent Adverse Events by System Organ Class, 
Preferred Term and Relationship to Study Drug (Safety Population) 
14.3.1.4 
Treatment-Emergent Serious Adverse Events by System Organ 
Class and Preferred Term (Safety Population) 
14.3.2.1 
Vital Signs (Safety Population) 
14.3.2.2 
Vital Signs – Outlier Analysis (Safety Population) 
14.3.3 
Concomitant Medications (Safety Population)  
 
 


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List of Data Listings 
Listing Number 
Listing Description 
16.2.1 
Subject Disposition  
16.2.2 
Subject Demographics and Characteristics 
16.2.3 
Inclusion/Exclusion Criteria 
16.2.4 
Protocol Deviations 
16.2.5 
Medical History 
16.2.6.1 
In-Clinic Study Drug Administration (Visit 2 SAD Treatment) 
16.2.6.2 
In-Clinic Study Drug Administration (Visit 3 MAD Treatment) 
16.2.6.3 
Study Drug Return 
16.2.7 
Concomitant Medications 
16.2.8 
Microbiology Results 
16.2.9 
DUSS Scores 
16.2.10 
DFI Scores 
16.2.11 
Ulcer Measurements 
16.2.12.1 
Adverse Events 
16.2.12.2 
Serious Adverse Events  
16.2.12.3 
Adverse Events Leading to Drug Withdrawn 
16.2.13.1 
Vital Signs 
16.2.13.2 
Vital Signs – Subjects with Outlying Heart Rate at Post-Dose 
Time Points 
16.2.13.3 
Vital Signs – Subjects with Outlying Systolic Blood Pressure at 
Post-Baseline Time Points 
16.2.13.4 
Vital Signs – Subjects with Outlying Diastolic Blood Pressure at 
Post-Baseline Time Points 
16.2.14 
Physical Examination 
16.2.15 
Visual Examination of Chronic Ulcer