Clinical trial NCT01813305
Conditions: Diabetic Foot Ulcer Diabetes Mellitus Wounds
Keywords: Diabetic Foot Ulcer Diabetes Mellitus Debridement Standard of Care wounds
Sponsor: Charsire Biotechnology Corp.
Start date: 2014-07-09; Completion date: 2020-01-07
Enrollment: 124 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objective of this study is to evaluate the efficacy and safety of CSTC1 in patient with diabetic foot ulcers.

Detailed description:
This study was designed as a randomized, double-blind, vehicle-controlled, multiple-center, and parallel trial to evaluate the efficacy and safety of CSTC1 in patients with diabetic foot ulcers (DFU). In each study site, eligible patients were randomized in a 4:1 ratio to receive either one of the topical applications of CSTC1 or CSTC1 matched vehicle, topical application on target diabetic foot ulcer (DFU), 2 times daily.

The treatment duration for each subject was 12 weeks or up to confirmed complete ulcer closure, whichever comes first. That was, subjects would receive treatment for at most 12 weeks, which consists of 8 visits located at weeks 1, 2, 3, 4, 6, 8, 10, and 12. Subjects who achieved confirmed complete ulcer closure during the treatment period would be arranged for a 12 week post-treatment follow-up. Subjects failed to achieve complete ulcer closure at week-12 visit would be arranged for 4 weeks of safety follow-up. If confirmation of complete ulcer closure was reached at a week-14 visit, the subject would continue the post-treatment follow-up visit until week-24 visit. Otherwise, the subject would be arranged for safety follow-up until week-16 visit.

Arms: arm 1: CSTC1 (vapor fraction from seeds of Glycine max (L.) Merr. and composition thereof), topical, two times daily arm 2: Matched vehicle, topical, two times daily
Interventions: intervention 1: CSTC1 intervention 2: CSTC1 Matched vehicle
Intervention descriptions: intervention 1: vapor fraction from seeds of Glycine max (L.) Merr. and composition thereof intervention 2: None

Protocol documents:
Prot_SAP_000.pdf: 
Charsire Biotechnology Corp 
Date: 23Nov2017 
 
 
Page 1 of 36
 
 
STUDY PROTOCOL 
 
 
A Randomized, Double-Blind, Vehicle-controlled, 
Parallel, Phase II Study to Evaluate Efficacy and 
Safety of CSTC1 in Patient with Diabetic Foot Ulcers 
 
 
 
 
Project Number: 
CSTC1-01 
Investigational Product: 
CSTC1 
Sponsor: 
Charsire Biotechnology Corp.  
 
 
Confidentiality Statement 
This protocol is provided for the purpose of conducting a clinical research study. The information contained in this 
document is confidential and, except to the extent necessary to obtain informed consent or IEC/IRB approval, 
cannot be disclosed unless required by governmental regulation. Persons to whom any portion of the contents of 
this document is disclosed must be informed that the information is confidential and may not be further disclosed 
by them. 
 
 


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Date: 23Nov2017 
 
 
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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
ANOVA 
Analysis of Variance 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
CHO-K1 
Chinese Hamster Ovary 
CABG 
Coronary Artery Bypass Graft 
CMH test 
Cochran-Mantel-Haenszel test 
CRA 
Clinical Research Associate 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CSTC1 
CHARSIRE Trauma Complex 
DFU 
Diabetic Foot Ulcer 
EC 
Ethics Committee  
EDC 
Electronic Data Capture 
GCP 
Good Clinical Practice 
A1 
Ethanol Extraction from Soybean 
C1 
Vapor Fraction from Soybean 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
ITT 
Intent-to-treat 
IQR 
Inter-quartile Range 
LDPE 
Low Density Polyethylene 
LEA 
Lower Extremity Amputations 
NOAEL 
No-observed-adverse-effect-level 
NZW 
New Zealand White 
PI 
Principle Investigator 
PII 
Primary Irritation Index 
PP 
Per-Protocol 
PTCA 
Percutaneous Transluminal Coronary Angioplasty 
RBC 
Red Blood Cell 
SAE 
Serious Adverse Event 
SOP 
Standard Operation Procedure 
STZ 
Streptozotocin 
WBC 
White Blood Cell 


Charsire Biotechnology Corp 
Date: 23Nov2017 
 
 
Page 3 of 36
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS .................................................................................................. 2 
TABLE OF CONTENTS ......................................................................................................... 3 
PROTOCOL SUMMARY ....................................................................................................... 5 
SCHEDULE OF ASSESSMENTS .......................................................................................... 9 
1. 
BACKGROUND AND RATIONALE ........................................................................ 11 
1.1 
GENERAL INTRODUCTION ...................................................................................................................... 11 
1.2 
RATIONALE AND JUSTIFICATION FOR THE STUDY .................................................................................. 13 
2. 
OBJECTIVES AND ENDPOINTS............................................................................. 14 
2.1 
OBJECTIVES ........................................................................................................................................... 14 
2.2 
ENDPOINTS ............................................................................................................................................ 14 
3. 
STUDY POPULATION ............................................................................................... 15 
3.1 
SUBJECTS ENROLLMENT ........................................................................................................................ 15 
3.2 
INCLUSION CRITERIA ............................................................................................................................. 15 
3.3 
EXCLUSION CRITERIA ............................................................................................................................ 15 
3.4 
WITHDRAWAL CRITERIA ........................................................................................................................ 16 
3.5 
SUBJECT REPLACEMENT ........................................................................................................................ 17 
3.6 
CONCOMITANT MEDICATIONS ............................................................................................................... 17 
4. 
STUDY DESIGN .......................................................................................................... 18 
4.1 
TREATMENT ASSIGNMENT ..................................................................................................................... 18 
4.2 
RANDOMIZATION AND BLINDING ........................................................................................................... 18 
5. 
TRIAL MATERIALS .................................................................................................. 19 
5.1 
TRIAL PRODUCT (S) ............................................................................................................................... 19 
5.2 
STORAGE AND DRUG ACCOUNTABILITY ................................................................................................ 19 
5.3 
DOSE REGIMEN ...................................................................................................................................... 19 
5.4 
TREATMENT COMPLIANCE ..................................................................................................................... 20 
6. 
STUDY MEASUREMENTS ....................................................................................... 21 
6.1 
ULCER ASSESSMENT .............................................................................................................................. 21 
6.2 
PHYSICAL EXAMINATION AND VITAL SIGNS .......................................................................................... 21 
6.3 
LABORATORY EXAMINATION ................................................................................................................ 21 
6.4 
STANDARD-OF-CARE PROCEDURES ....................................................................................................... 22 
7. 
STUDY SCHEDULE ................................................................................................... 23 
7.1 
SCREENING VISIT ................................................................................................................................... 23 
7.2 
INITIAL PHASE VISIT (ONLY FOR APPLICABLE SUBJECTS) ....................................................................... 24 
7.3 
RANDOMIZATION VISIT .......................................................................................................................... 24 
7.4 
EVALUATION VISITS DURING TREATMENT PERIOD IN VISITS 4 TO 11 ...................................................... 24 
7.5 
FOLLOW-UP VISITS ................................................................................................................................. 25 
7.6 
FINAL VISIT ............................................................................................................................................ 25 
8. 
ADVERSE EVENTS .................................................................................................... 26 
8.1 
DEFINITIONS .......................................................................................................................................... 26 
8.2 
AE/SAE INTENSITY AND RELATIONSHIP ASSIGNMENT ......................................................................... 26 
8.3 
COLLECTING, RECORDING AND REPORTING OF ADVERSE EVENTS ........................................................ 28 
9. 
DATA ANALYSIS ....................................................................................................... 29 
9.1 
DATA QUALITY ASSURANCE ................................................................................................................. 29 
9.2 
CLINICAL DATA MANAGEMENT ............................................................................................................. 29 


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10. 
SAMPLE SIZE AND STATISTICAL METHODS .................................................. 30 
10.1 
DETERMINATION OF SAMPLE SIZE ......................................................................................................... 30 
10.2 
STATISTICAL AND ANALYTICAL PLANS ................................................................................................. 30 
11. 
ETHICAL CONSIDERATIONS ................................................................................ 32 
11.1 
INFORMED CONSENT .............................................................................................................................. 32 
11.2 
IRB REVIEW ........................................................................................................................................... 32 
11.3 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS ............................................................................ 32 
12. 
PUBLICATIONS ......................................................................................................... 34 
13. 
RETENTION OF TRIAL DOCUMENTS ................................................................. 34 
14. 
REFERENCES ............................................................................................................. 35 
 


Charsire Biotechnology Corp 
Date: 23Nov2017 
 
 
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PROTOCOL SUMMARY 
Full Title 
 
A Randomized, Double-Blind, Vehicle-controlled, Parallel, Phase II 
Study to Evaluate Efficacy and Safety of CSTC1 in Patient with 
Diabetic Foot Ulcers  
Short Title 
CSTC1 for Diabetic Foot Ulcers Phase II Study 
Project No 
CSCT1-01 
Study Phase 
Phase II 
Sponsor 
Charsire Biotechnology Corp. 
Objectives 
 
To evaluate the efficacy and safety of CSTC1 in patient with 
diabetic foot ulcers  
Study Design 
Randomized, double-blind, vehicle-controlled, multiple center, 
parallel study 
Study Population 
Subject aged at least 20 years old in Taiwan diagnosed with diabetic 
ulcer (which is selected by investigator and defined as the target 
ulcer) on the foot that  not healing for at least 4 weeks 
Number of Subjects  
The sample size is determined to be 80 versus 20 subjects (4:1 ratio) 
for Treatment versus Control groups, 100 subjects in total. To 
ensure the completion of 100 evaluable subjects, around 125 
subjects will be recruited. 
Study Product, Dose, 
Route, Regimen 
CSTC1 (vapor fraction from seeds of Glycine max (L.) Merr. and 
composition thereof), topical application, 2 times daily 
Duration of 
Administration 
12 weeks of treatment or confirmed complete ulcer closure, 
whichever comes first, and up to 12 weeks of post-treatment follow-
up for subject with confirmed complete ulcer closure after at most 
12 weeks of treatment; Subjects failed to achieve complete ulcer 
closure will be arranged for 4 weeks of safety follow-up 
Subject Assignment 
Subjects who meet all eligible requirements for entry into the study 
will be randomized into one of the treatment group or vehicle 
control group in 4:1 ratio as shown below: 
Treatment Group: 
CSTC1  
Control Group: 
Matched vehicle  
Subjects Inclusion 
Criteria 
A subject is eligible for the study if all of the following apply:  
1. With either gender aged at least 20 years old 
2. With at least one diabetic foot ulcer (including ulcers on the 
lower legs) and not healing for at least 4 weeks. The largest 
diabetic foot ulcer will be selected as target ulcer. If two or more 
ulcers have the largest size, the one with worst grade will be 
selected. If two or more ulcers have the largest size and grade, 
the one with longest duration will be selected. 


Charsire Biotechnology Corp 
Date: 23Nov2017 
 
 
Page 6 of 36
3. The target ulcer is classified as grade 1 to 2 ulcer according to  
modified Wagner system and with ulcer size of 1cm2 to 50 cm2 
4. The target ulcer should be confirmed without active infection  
5. Subject should be free of any necrosis or infection in soft and 
bone tissue; 
6. Subject has signed the written informed consent form 
Subjects Exclusion 
Criteria 
Any subject meeting any of the exclusion criteria will be excluded 
from study participation. 
1. With active osteomyelitis 
2. With target ulcer size decreased by at least 30% after at least 2 
weeks of standard-of-care-only period or any other recorded 
regular therapy before Randomization visit 
3. With poor nutritional status (albumin < 2g/dl), poor diabetic 
control (HbA1c > 12%), anemia (hemoglobin < 8 g/dL), a 
leukocyte counts < 2,000/mm3, abnormal liver function (AST, 
ALT>3 x upper limit of normal range) 
4. Requiring treatment with systemic corticosteroids, 
immunosuppressive or chemotherapeutic agents within 28 days 
prior to Screening visit 
5. Presence of necrosis, purulence or sinus tracts that cannot be 
removed by debridement 
6. Receiving revascularization surgery or endovascular therapy  
performed <8 weeks before entry in the study  
7. With known or suspected hypersensitivity to any ingredients of 
study product and vehicle 
8. With coronary heart disease with myocardial infarction, 
coronary artery bypass graft (CABG), or percutaneous 
transluminal coronary angioplasty (PTCA) within 3 months 
prior to study 
9. Pregnant or lactating or premenopausal with childbearing 
potential but not taking reliable contraceptive method(s) during 
the study  period 
10. Enrollment in any investigational drug trial within 4 weeks 
before entering this study 
11. With any uncontrolled illness judged by the investigator that 
entering the trial may be detrimental to the subject 
Primary Endpoint 
Efficacy:  
The incidence of complete ulcer closure during up to 12 weeks of 
treatment period 
Complete ulcer closure is defined as 100% skin re-epithelialization 
without drainage or dressing requirements confirmed at two 
consecutive study visits 2 weeks apart. Subjects with complete ulcer 
closure at week-12 visit and confirmed at week-14 visit will be 
considered as success. 
 


Charsire Biotechnology Corp 
Date: 23Nov2017 
 
 
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Secondary Endpoints Efficacy: 
1. The ulcer closure time 
Defined as the time to complete ulcer closure 
2. The complete ulcer closure rate  
The proportion of subjects with complete ulcer closure at each 
post-treatment visit 
3. Percentage change in ulcer size for each post-treatment visit 
Calculated as (Ulcer size at post treatment visit - Ulcer size at 
baseline)/(Ulcer size at baseline) 
Safety: 
1. Adverse event incidence 
2. Change in physical examination results 
3. Net change from baseline in laboratory test results (include 
hemoglobin, hematocrit, RBC, platelet, WBC with differential 
counts, HbA1c, fasting glucose, AST, ALT, creatinine and 
BUN) 
4. Net change from baseline in vital signs 
Statistical Analysis 
Analysis population 
Three data sets will be introduced for statistical analysis.  
Intent-to-treat (ITT) population: 
– 
All randomized subjects who have received at least one dose 
study medication.  
Per-protocol (PP) population:  
– 
A subset of ITT population 
– 
Fulfill all inclusion and exclusion criteria 
– 
Dosed with at least 8 weeks study drug or with early confirmed 
complete ulcer closure AND with at least 70% treatment 
compliance 
– 
Did not receive any prohibited treatment during treatment 
period 
– 
With any post-treatment assessment relevant to efficacy 
endpoint up to end of dosing period 
Efficacy endpoints will be analyzed on ITT and PP population. 
Demographics, baseline characteristics, and safety endpoints will be 
analyzed on ITT population. Conclusion of efficacy will be made 
according to the result of ITT population analysis. 
Analysis 
Demographics and baseline characteristics will be summarized for 
each group by using descriptive statistics. 


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Date: 23Nov2017 
 
 
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The efficacy endpoints will compare the CSTC1 group with vehicle 
group. Incidence of ulcer closure and ulcer closure rate at endpoint 
will be analyzed with Cochran-Mantel-Haenszel test (CMH test). 
Ulcer closure time will be estimated by using Kaplan-Meier 
methods and compared between groups by using log-rank test. 
Percentage change in ulcer size will be analyzed by using Analysis 
of variance (ANOVA) model with treatment group and center as 
factors. Further efficacy analyses with ankle brachial index (ABI) 
(<0.7 or not) as stratification may be performed if necessary. 
For safety analyses, adverse events will be reported by treatment 
groups and by physiological systems as appropriate. Incidence of 
adverse events between treatments will be analyzed by CMH test. 
Changes in physical examinations will be displayed for each 
individual system. Net changes from pre-treatment laboratory test 
results and vital signs will be analyzed by descriptive statistics. 
Additionally, descriptive statistics will be provided for all of the 
endpoints. Frequency table will be provided for categorical data, 
while mean, standard deviation, maximum, minimum, median, inter-
quartile range (IQR), and 95% two-sided confidence interval will be 
calculated for continuous measurements. In addition, endpoints of 
time to event will be presented by using the life table and Kaplan-
Meier plot. The Kaplan-Meier estimates will also be provided for 
these time-to-event endpoints. 
All treatment group comparisons will be conducted with 
significance level of5. 
 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 9 of 36
SCHEDULE OF ASSESSMENTS 
 
Screening1 
Initial2 Phase 
Randomization3 
(Baseline) 
Dosing/Observation 
 
Follow-up 
Visit No. 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
1111 
12 
13 
14 
15 
Week relative to Baseline visit 
-3~-2 
-2~0 
0 
1 
2 
3 
4 
6 
8 
10 
12 
14 
16 
20 
24 
Visit Window (days) 
NA 
4 
NA 
4 
4 
4 
4 
4 
4 
4 
4 
4 
4 
4 
4 
Informed consent signed and given  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening number assignment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic data & medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion and exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test for applicable subjects 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Ulcer Evaluation4 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examinations 
X 
 
X  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Ankle Brachial Index5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory test: hemoglobin, hematocrit, 
RBC, platelet, WBC with differential 
counts, HbA1c, fasting glucose, AST, 
ALT, creatinine and BUN, albumin 
X6 
 
X7  
 
 
 
 
 
 
 
X8 
 
 
 
 
Record concomitant medication(s) 
X 
X 
X  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Standard of Care  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Record adverse events (AE)9 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Dispense trial medication 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Returned medication count 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Diary Dispensing 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Diary Return 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Complete exit form 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Dismiss subject10 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Footnote: 
1: Could be on the same day as the first day of Initial Phase or randomization visit if subject’s Initial Phase can be waived 
2: Can be waived if subject’s diabetic foot ulcer(s) has been treated for at least 2 weeks, and is still eligible to enter this study 
3: Could be on the same day as the last day of Initial Phase  
4,10: Subjects with confirmed complete ulcer closure up will be arranged for ulcer evaluation for follow-up period; Subjects not with complete ulcer closure will take 4 weeks 
of safety evaluation 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 10 of 36
5: The two blood pressures for calculating ABI, ankle and brachium, should be measured on the same side (left to left, right to right) and should be on the same side of target 
diabetic foot ulcer. However, the blood pressures of brachium may not be available for some patients. For such case, investigator is suggested trying to measure pressure of 
brachium of another side.  
6: Test results within 14 days before Screening visit are acceptable; HbA1c test result within 28 days can be accepted. Albumin test will only be done at Screening and 
Randomization visits 
7: Test results within 28 days before Randomization visit are acceptable 
8: Test results within time window are allowed 
9: AE should be recorded since informed consent is signed 
11: For subject who has been confirmed complete closure before week-12 visit should be arranged for visit 11 assessment. 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 11 of 36
1. 
BACKGROUND AND RATIONALE 
1.1 
General Introduction 
Diabetic foot ulcers (DFUs) are a common problem with clinically serious sequelae. It 
has been reported that annually, about 1% to 4% of those with diabetes, which is 
believed that more than 23 million people in the United States have, develop a foot 
ulcer; 10% to 15% of those with diabetes will have at least one foot ulcer during their 
lifetime. DFUs place patients at the risk of lower extremity amputations (LEAs), which 
is an extremely complicated. In the U.S., around 80,000 LEAs annually are conducted 
on diabetics.1,2 In 2005, the overall rate of hospital discharge for new LEA was about 
4.3 per 1,000 diabetic people.3 As a result, DFUs are wildly acknowledged a source of 
major distress and morbidity for diabetic patient and also an enormous drain on health-
care resources. It then becomes an important issue to have an effective treatment to 
alleviate the ulcer worsening and even to achieve complete ulcer closure for DFU 
patients. 
Wound healing process is a complex series of events that comprised of inflammatory, 
proliferative (including granulation, contraction, epithelialization) and remodeling 
phases. Excessive inflammatory stimuli may be deleterious and result in adverse 
consequence to wound healing. Antioxidants, on the other hand, significantly limit the 
delayed sequelae of tissue damage and stimulate wound healing process. As a result, 
many substances that intend to enhance the wound healing emphasis on their anti-
inflammatory and antioxidant effects.4,5 
Three main anthocyanins (cyanidin-3-glucoside, delphinidin-3-glucoside, and 
petunidin-3-glucoside) have been characterized in black soybean seed coats.6 
Anthocyanins have been reported with considerable interest in the health-promoting 
benefits, including antioxidant and anti-inflammatory effects, reduction in risk of 
coronary heart disease and prevention of ischemia and reperfusion heart injury.7-10 
Thus, the pharmacologic actions of anthocyanins, especially anti-inflammatory effect, 
resemble the wound healing enhancement process.  
Anthocyanins from black soybean seed coats were investigated to see whether it could 
stimulate wound healing while preventing excessive inflammation. The results 
indicated that treatment with anthocyanins from black soybean seed coats enhance 
wound healing process in vitro and in vivo through a cytoprotective effect, enhancement 
of angiogenesis, and an antiinflammatory effect.11,12 Therefore, it may be a potentially 
effective therapeutic strategy to promote and potentiate wound healing in chronic 
wounds and thus to attenuate and even prevent excessive inflammatory condition. 
Pharmacology of CSTC1 
Pharmacology studies about CSTC1have been performed. The dose effect of enhancing 
diabetic wound healing with different dose level of A1 (ethanol extraction from 
soybean) in combination with C1 (vapor fraction from soybean) on Streptozotocin 
(STZ, 65mg/kg) -induced rats with high blood sugar (over 300mg/DL) for 2 months 
was investigated. The most effective ratio for A1 and C1 was 0.3% of A1 combined 
with C1, i.e. 0.75g A1 and 213.9g C1 vapor fraction added to 34.4g cream base to make 
test ointment. 
Study was also performed to investigate the effect of CSTC1 (composed of 0.3% A1 
and C1) on diabetic wound closure in db/db mice. Results showed that the and very 
significantly better than group 1 (placebo).  Results showed that the wound closure 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 12 of 36
percentage in 1x (0.75g A1 and 213.9g C1 vapor fraction added to 34.4g cream base to 
make test ointment) and 2x (1.5g A1 and 213.9g concentrated vapor fraction from 400g 
C1 added to 34.4g cream base to make test ointment) strength of CSTC1-treated groups 
were higher than group of less strength of CSTC1 during early stage of wound healing 
period (day 4~day10) and very significantly higher than cream base 2 group (215.6g 
milli-Q water added to 34.4g cream base to make a 250g of test ointment), (p< 0.01) on 
day 10. 
The pharmacology of CSTC1 on enhancing diabetic wound healing is thus justified.  
Toxicology 
Several toxicology studies of CSTC1 were performed as follows: 
Bacterial Reverse Mutation Test 
CSTC1 did not present genotoxic effect at all concentrations on every testing strains 
used in this study, i.e. TA98, TA100, TA102, TA1535, and TA1537, in the condition of 
both presence of S9 metabolic mixture. In conclusion, CSTC1 was non-genotoxic in the 
testing system applied in this study.  
In Vitro Mammalian Chromosome Aberration Test 
CSTC1 would not induce significant chromosome aberration in all testing dosages 
compared with the concurrent negative control, indicating that CSTC1 would not be 
considered positive in causing structural chromosome aberration to Chinese hamster 
ovary (CHO-K1) cells. In conclusion, CSTC1 was not genotoxic to CHO-K1 cells under 
the testing condition employed. 
In Vivo Mammalian Erythrocyte Micronucleus Test 
There was no significant difference in micronucleus frequency between every testing 
group and concurrent negative controls at 48 and 72 hours. Moreover the polychromatic 
erythrocytes percentage of every testing group was greater than 80% of that in negative 
controls, which indicated that the test article would not inhibit erythropoiesis. 
Accordingly, CSTC1 would be considered negative in micronucleus test. In conclusion, 
CSTC1 has no genotoxicity and would not inhibit erythropoiesis in ICR mice under the 
test system employed in this study.  
Dermal Irritation Study 
All test animals were alive and appeared normal during the testing. No body weight loss 
was noted in test animals. The primary irritancy scores for three test animals were all 0. 
The primary irritation index (PII) for the test article was graded as 0 and the irritancy 
was classified as “Negligible”. Based on these results, the test article “CSTC1” 
administered to the New Zealand White (NZW) rabbits by dermal application did not 
cause irritation in vivo. 
Skin Sensitization Study 
All animals appeared clinical normal throughout the study period. The body weights of 
all animals at study ending were heavier than their weights at study beginning. 
Individual results of dermal scoring for the challenge phase were summarized in a 
tabular form. The sensitization rate in each group was calculated. All animals presented 
negative results (grading scale = 0). The sensitization rates were 0% in both control and 
treatment groups. It indicated that test article CSTC1 was considered not a sensitizer. 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 13 of 36
Acute Oral Toxicity Study in Rats 
Based on these results, the test article “CSTC1” administered to SD rats via a single oral 
administration did not cause animal deaths at the designated dose levels of 0, 1/4 diluted, 
1/2 diluted and original solutions.  
28-Day Repeated Dose Oral Toxicity Study in Rats 
No animal death and no test article related clinical signs were observed during the study 
period. No ocular lesions were noted in all animals before dosing and prior to necropsy. 
Results of mean body weights, mean body weight gains and food consumption showed 
that there were no significant toxicity evidences related to test article treatments in the 
male and female rats. 
In the clinical pathology, no test article related abnormalities were noted in hematology, 
serum chemistry and urinalysis. 
The gross necropsy showed that there were no observable abnormal findings in the 
study. There were no significant toxicity evidences in the organ weights between the 
vehicle control and test article treatment groups in male or female rats. The 
histopathological findings showed that no test article related changes were observed in 
the vehicle control and high-dose groups. 
Based on these study results, the NOAEL (no-observed-adverse-effect-level) of the 
testing article was 10mL original solution/kg b.w./day.  
1.2 
Rationale and Justification for the Study 
1.2.1 Rationale for the Study Purpose 
The purpose of the present phase II study is to explore the efficacy and safety of 
CSTC1 in subject with diabetic foot ulcers. This study is to be conducted via a 
randomized, double-blind, vehicle-controlled, multiple center and parallel trial 
fashion. Moreover, this phase II study is expected to provide some useful 
information for further phase III study in the future. 
1.2.2 Rationale for Doses Selected 
The dosing regimen and treatment duration were demonstrated to be as safe as 
vehicle based on the toxicology study results.  
1.2.3 Rationale for Study Population 
The study population designed to be enrolled is based on the Guidance for 
Industry: Chronic Cutaneous Ulcer and Burn Wounds - Developing Products for 
Treatment and should enroll patients with established diabetic foot ulcers. 
1.2.4 Rationale for Study Design 
A superiority design with vehicle control is used intending to explore the real 
efficacy of CSTC1 in treating diabetic foot ulcer. To avoid system bias, the study 
is designed as a double-blind, randomized study. 
 
 


Study Protocol:  
 CSTC1 for Diabetic Foot Ulcers 
(Project Number: CSTC1-01) 
Confidential  
Version: 2.4 
Date: 23NOV2017 
 
 
Page 14 of 36
2. 
OBJECTIVES AND ENDPOINTS  
2.1 
Objectives 
2.1.1 Primary Objectives 
The primary objective of this study is to evaluate the efficacy of CSTC1 in 
patient with diabetic foot ulcers.  
2.1.2 Secondary Objectives 
The secondary objective of this study is to evaluate the safety of CSTC1 in 
patient with diabetic foot ulcers. 
The study purpose is for registration. The study results may be used for 
developing new drug application dossier.  
2.2 
Endpoints 
2.2.1 Primary endpoints 
Incidence of complete ulcer closure during up to 12 weeks of treatment period 
Complete ulcer closure is defined as 100% skin re-epithelialization without 
drainage or dressing requirements confirmed at two consecutive study visits 2 
weeks apart. 
2.2.2 Secondary efficacy endpoints 
1. The ulcer closure time 
Defined as the time to complete ulcer closure 
2. The complete ulcer closure rate  
The proportion of subjects with complete ulcer closure at each post-
treatment visit 
3. Percentage change in ulcer size for each post-treatment visit 
Calculated as (Ulcer size at post treatment visit - Ulcer size at 
baseline)/(Ulcer size at baseline) 
2.2.3 Safety endpoints 
1. Adverse event incidence 
2. Change in physical examinations 
3. Net change from baseline in laboratory test results (include hemoglobin, 
hematocrit, RBC, platelet, WBC with differential counts, HbA1c, fasting 
glucose, AST, ALT, creatinine and BUN) 
4. Net change from baseline in vital signs 
  
 


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3. 
STUDY POPULATION 
3.1 
Subjects Enrollment 
The sample size is determined to be 80 versus 20 subjects (4:1 ratio) for Treatment 
versus Control groups, 100 subjects in total. To ensure the completion of 100 evaluable 
(= per-protocol) subjects, around 125 subjects will be recruited. All of the subjects will 
be recruited in Taiwan. Any subject fulfills all of the recruitment criteria will be 
screening and/or randomized into the study.  
Re-screening for patient’s eligibility will be allowed if the patient is willing to 
participate in the study and one of the following conditions meets: 
1. If a patient consents to participate and meets the eligibility criteria but a delay occurs 
in starting to participate in due to certain change in situation such that some 
measurement relevant to eligibility became invalid owing to the delay 
2. If the cause of screen failure has now resolved or adequately treated such that 
patient’s medications have now stabilized 
In these situations, if randomization has not occurred; 
1. 
A new CRF will be used 
2. 
A new Screening number will be assigned to the person 
3. 
The person will be marked as having been re-screened on both the CRF and the 
site master list 
4. 
The patient needs to sign a new ICF as part of the screening procedure. 
It is not appropriate to re-screen a patient if he/she has previously failed to meet the 
eligibility criteria and no further changes or treatments have been able to indicate that 
the patient may be suitable. 
3.2 
Inclusion Criteria 
A subject is eligible for the study if all of the following apply: 
1. With either gender aged at least 20 years old 
2. With at least one diabetic foot ulcer (including ulcers on the lower legs) on the foot 
and not healing for at least 4 weeks. The largest diabetic foot ulcer will be selected 
as target ulcer. If two or more ulcers have the largest size, the one with worst grade 
will be selected. If two or more ulcers have the largest size and grade, the one with 
longest duration will be selected. 
3. The target ulcer is classified as grade 1 to 2 ulcer according to  modified Wagner 
system and with ulcer size of 1cm2 to 50 cm2 
4. The target ulcer should be confirmed without active infection. 
5. Subject should be free of any necrosis or infection in soft and bone tissue; 
6. Subject has signed the written informed consent form 
3.3 
Exclusion Criteria 
Any subject meeting any of the exclusion criteria will be excluded from study 
participation. 
1. With active osteomyelitis 


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2. With target ulcer size decreased by at least 30% after at least 2 weeks of standard-
of-care-only period or any other recorded regular therapy before Randomization 
visit 
3. With poor nutritional status (albumin < 2g/dl), poor diabetic control (HbA1c > 
12%), anemia (hemoglobin< 8 g/dL), a leukocyte counts < 2,000/mm3, abnormal 
liver function (AST, ALT>3 x upper limit of normal range) 
4. Requiring treatment with corticosteroids, immunosuppressive or chemotherapeutic 
agents within 28 days prior to Screening visit 
5. Presence of necrosis, purulence or sinus tracts that cannot be removed by 
debridement 
6. Receiving revascularization surgery or endovascular therapy performed <8 weeks 
before entry in the study 
7. With known or suspected hypersensitivity to any ingredients of study product and 
vehicle 
8. With coronary heart disease with myocardial infarction, coronary artery bypass 
graft (CABG), or percutaneous transluminal coronary angioplasty (PTCA) within 
3 months prior to study 
9. Pregnant or lactating or premenopausal with childbearing potential but not taking 
reliable contraceptive method(s) during the study period 
10. Enrollment in any investigational drug trial within 4 weeks before entering this 
study 
11. With any uncontrolled illness judged by the investigator that entering the trial may 
be detrimental to the subject 
3.4 
Withdrawal Criteria  
Subjects may be withdrawn from the trial due to any of the following conditions: 
1. Subjects decide to withdraw their consents 
2. Subjects found violating any enrollment criteria at eligibility confirmation (wrong 
entry) 
3. Investigators consider that there is of safety concerns for the subjects to remain in 
the trial 
4. Subjects are lost to follow-up 
5. Subjects become pregnant 
6. Termination of the study by sponsor, IRB or regulatory authority. 
7. If a subject’s ulcer becomes infected during treatment period, and topical 
antimicrobial treatment is necessary at investigator’s discretion 
8. Subjects ulcer became worse (e.g. grade III or above, ulcer increases in sizes needs 
deep debridement, develops evidence of infection requiring intravenous or local 
antibiotics, or osteomyelitis develops) 
9. Taking prohibited medication/treatment during treatment period 
The reasons for discontinuation must be recorded in the Case Report Form and in the 
subject’s medical records for all early terminated cases. All study participants 
withdrawn from the study before Week-12 will be requested to return for Week-12 visit 
assessments. If a subject is discontinued due to an AE, every effort will be made to 
follow the event until resolution. 


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3.5 
Subject Replacement 
All subjects who drop out from the study will not be replaced. New subjects will be 
enrolled with a newly assigned subjects number until the target evaluable subjects is 
reached for this study. 
3.6 
Concomitant Medications 
All medications and supplements taken by the study participant beginning four weeks 
prior to the screening visit and throughout the study will be recorded on the appropriate 
page of the Case Report Form (CRF). This record will include the name of the 
medication, frequency, unit dose, dates of the drug was started and stopped, and the 
indication for the use of the drug. 
Subjects are allowed to receive routinely used medications or treatments for other 
indications which is judged by the investigator as not affecting the efficacy and safety 
assessments of this study.  
- 
Prohibited Medications/Treatment during Treatment Period 
(1) Treatment 
with 
systemic 
corticosteroids, 
immunosuppressive 
or 
chemotherapeutic agents 
(2) Revascularization surgery or endovascular therapy on lower extremity that is 
of the same side as target ulcer 
(3) Any treatment/therapy applied to foot ulcer area that may affect the evaluation 
of study medication at investigator’s discretion 
 
 


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4. 
STUDY DESIGN 
This study is designed as a randomized, double-blind, vehicle-controlled, multiple 
center and parallel trial to evaluate the efficacy and safety of CSTC1 in patients with 
diabetic foot ulcer.  
The potential subjects with diabetic foot ulcer at Screening visit will be arranged to enter 
an initial phase for 2 weeks in which subjects receive only standard of care, unless 
subject has receive any other regular therapy for at least 2 weeks. Subjects whose study 
ulcer size decreases by at least 30% during this initial standard-of-care phase or previous 
regular therapy period will be excluded from the study at investigator’s discretion. The 
remaining eligible subjects will be randomly assigned to receive either one of topical 
application of CSTC1 dose or CSTC1 matched vehicle, 2 times daily.  
The treatment duration for each subject is 12 weeks or up to confirmed complete ulcer 
closure, whichever comes first. That is, subjects will receive treatment for at most 12 
weeks, which consists of 8 visits located at weeks 1, 2, 3, 4, 6, 8, 10, and 12. Subjects 
confirmed complete ulcer closure during treatment period will be arranged for a 12 week 
post-treatment follow-up. Subjects failed to achieve complete ulcer closure at week-12 
visit will be arranged for 4 weeks of safety follow-up. Subjects happening to attain 
complete ulcer closure at week-12 visit will not receive further treatment but needs to 
be confirmed at week-14 visit. If confirmation of complete ulcer closure is reached at 
week-14 visit, the subject will continue the post-treatment follow-up visit until week-24 
visit. Otherwise, the subject will be arranged for safety follow-up until week-16 visit.  
After the treatment period, the post-treatment follow-up period for subjects confirmed 
complete ulcer closure will consist of 4 visits at weeks 2, 4, 8, 12 after confirmation of 
complete ulcer closure. The safety follow-up period for subjects failed to achieve 
complete ulcer closure at week-12 visit will consist of 2 visits at weeks 14 and 16.  
4.1 
Treatment Assignment 
Eligible subjects will be assigned randomization number in sequential order and each 
of the randomization will determine the allocation of one of the two treatment groups 
in 4:1 ratio as shown below: 
1. CSTC1 two times daily 
2. CSTC1 matched vehicle, two times daily 
Treatments should be continued for a total of 12 weeks or until confirmation of complete 
ulcer closure, whichever comes first. After treatment period, additional 12 weeks of 
follow-up period is arranged for subjects confirmed complete ulcer closure to help 
distinguish actual ulcer healing from transient ulcer coverage, determine if the 
investigational product affects the strength of ulcer closure relative to standard care, and 
monitor for adverse effects on surrounding tissue 
4.2 
Randomization and Blinding 
Permuted block randomization method will be applied to generate randomization codes. 
Each randomization number will be assigned to individual subject according to the time-
sequence for screened subject becoming eligible. 
Both CSTC1 and its matched vehicle will be prepared to be identical in all aspects to 
achieve the double-blind purposes. All subjects will take proper amount of CSTC1 or 
CSTC1 matched vehicle per application to ulcer area. 
 


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5. 
TRIAL MATERIALS 
5.1 
Trial Product (s) 
Topical formulation of CSTC1 is a vapor fraction from seeds of Glycine max (L.) Merr. 
and composition thereof. The marker ingredients (impurity) of CSTC1 are Raffinose 
pentahydrate, Stachyose hydrate, Daidzin, and Genistin.  The comparator used in this 
study is CSTC1 matched vehicle. Both investigational products will be applied daily 
twice a day. 
Soybean was extracted using two methods, vapor fraction (C1) and ethanol extraction 
followed by concentration and drying (A1). 0.3% of A1, shown to reach the most active 
in wound healing in animal model when combined with the water extracted composition 
to form the drug substance. The final drug product, CSTC1, contains 0.3% of A1 and 
85.95% water extracted composition in a cream base with a total of 30g per tube shown 
as below: 
Code Name 
CSTC1 
Component 
2148.75g C1 and 7.5g A1; cream base 343.75 g 
Amount per tube 
30g/tube 
*Cream base: Stearic acid, Stearyl alcohol, Tween 80, Borneol, Potassium hydroxide, Benzyl alcohol. 
The placebo was made with a cream base and coloring agents with the ingredients and 
amount as follows: 
The active and vehicle cream is manufactured and packed with 30 g in weight in a LDPE 
tube by Charsire Biotechnology Co., Ltd.   
5.2 
Storage and Drug Accountability 
Adequate supplies of either CSTC1 or CSTC1 matched vehicle will be packaged for 
one visit dispense. The total number of tubes of investigational product planned for each 
subject will be 100 and each subject is expected to use at most 1 tube per day. Each 
subject will be dispensed for up to 8 visits at visits 3 to 10. At each visit of visits 3 to 9, 
investigational product should be supplied to the subject for use based on the number of 
tubes of unused investigational product the subject possesses and to the extent of date 
that the subject may return with remaining eligibly for next visit. Therefore, the number 
of tubes supplied at each visit will be calculated as (date of subject expected to return 
for next visit – date of current visit + 4 days of time window - number of tubes the 
subject possesses).  
The trial medication for both treatment groups will be packaged in an identical way. 
Drug accountability will be recorded for the weight of test drug given to the subjects 
and returned to site by the subjects. Subject will be dispensed diary cards to record the 
times in each day they apply the study medications to the treated area. 
All the products should be stored at room temperature (under 30o C) and be managed 
by the hospital pharmacies.  
5.3 
Dose Regimen 
The investigational product, CSTC1 and CSTC1 matched vehicle, will be applied two 
times daily until the end of 12 weeks treatment or up to confirmation of complete ulcer 
closure. The study medication will be applied in such an amount that the ulcer area is 
fully covered with thin and even layer. The maximum amount must not exceed 2 
millimeter in thickness, 15g per administration, 30g per day. The investigator/study 


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nurse will give a demonstration and training of how to apply the medication at the 
Randomization visit. 
5.4 
Treatment Compliance 
Subject’s treatment compliance will be assessed by the following formula: 
# of times of IP actually administered 
*100% 
((Last day – first day +1)) * (2 times of administration/day) 
 
In the formula above, last day and first day indicates the last and first days the subject 
administers IP. Therefore “last day – first day +1” means the number of days the subject 
is exposed to IP. 
 
 


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6. 
STUDY MEASUREMENTS 
6.1 
Ulcer assessment 
The surface area of the ulcer will be estimated at each evaluation in the manner as 
follows. After ulcer debridement, the ulcer along with an ulcer measuring ruler will be 
photographed. The images will be processed and analyzed using the ImageJ software 
package, 
downloaded 
from 
the 
Internet 
(National 
Institutes 
of 
Health, 
http://rsb.info.nih.gov/ij/download.html). The ImageJ software read the computed 
tomographic data and calibrated the images automatically. 
The grading according to the Wagner system is described below: 
 
Grade 0: no open ulceration 
 
Grade 1: full thickness ulceration, but depth does not go beyond loss of skin 
 
Grade 2: deeper tendon or joint capsule may be exposed 
 
Grade 3: open to bone, osteomyelitis may be present 
 
Grade 4: wet or dry gangrene, plus or minus cellulitis 
 
Grade 5: extensive gangrene, indication higher amputation 
(Source: Wagner FW. The dysvascular foot: A system for diagnosis and treatment. Foot 
Ankle. 1981; 2:64-122)23 
The eligible subjects should have at least one diabetic ulcer on the foot and not healing 
for at least 4 weeks. All ulcers should be documented appropriately of size, grade, 
location and duration. The largest diabetic foot ulcer will be selected as target ulcer. If 
two or more ulcers have the largest size, the one with worst grade will be selected. If 
two or more ulcers have the largest size and grade, the one with longest duration will be 
selected. 
Increment in wound size or worsening of its status should be reported as an adverse 
event irrespective of causality to the investigational products. The reporting procedures 
of adverse events can refer to Chapter 8: Adverse Event. 
6.2 
Physical Examination and Vital Signs 
Physical examination conducted in this study will include the following items: general 
appearance, skin, eyes, ears, nose, throat, head and neck, heart, joints, chest and lungs, 
abdomen, lymph nodes, musculoskeletal, nervous system, and others 
Vital signs measurement will consist of blood pressures, pulse rate, respiratory rate, and 
body temperature.  Respiratory rate, heart rate and blood pressure (systolic/diastolic) 
will be obtained after the subject has been at rest for at least 5 minutes in a sitting 
position. 
Physical examination and vital signs will be performed at all visits throughout this study 
except initial phase visit. 
6.3 
Laboratory Examination 
Laboratory examination to be measured in this study will consist of  
 
Hematology: hemoglobin, hematocrit, RBC, platelet, WBC with differential 
counts 


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 
Biochemistry:  
AST, ALT, fasting glucose, HbA1c, serum creatinine, blood urea 
nitrogen (BUN), albumin 
Laboratory examination will be conducted at Screening visit, Randomization (baseline) 
visit, and week 12 visit. Albumin will only be test at Screening and Randomization visits 
for judging subject eligibility. Laboratory test results within 14 days before Screening 
visit and 28 days before Randomization visit are acceptable; HbA1c test result within 
28 days can be accepted.  
6.4 
Standard-of-Care Procedures 
Standard care procedures for chronic cutaneous ulcer trials should be conducted in the 
initial phase and treatment period. The procedures will include the following: 
 
Debridement of ulcer 
 
Wound cleansing and disinfection using normal saline and betadine respectively. 
Disinfection using topical anti-septic treatment can be applied in the initial phase. 
If using topical anti-septic treatment is deemed necessary at investigator’s 
discretion in the treatment period, the subject should be discontinued from the 
study. 
 
Maintenance of a moist wound environment 
 
Off-loading procedure to lower down the pressure over subject’s ulcer  
Standard care procedure, including debridement and off-loading procedures, will be 
documented in CRF including timing, frequency, and amount of tissue removed. 
 
 


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7. 
STUDY SCHEDULE 
This study is designed as a randomized, double-blind, parallel, vehicle-controlled trial. 
The potential subjects with diabetic foot ulcer may be arranged to enter an initial phase 
for 2 weeks in which subjects receive only standard of care before randomization. The 
subjects confirmed eligible at the randomization visit will be randomly assigned to 
receive either one of CSTC1 or its matched vehicle treatment, two times daily. The 
subjects in either treatment group will be given 12 weeks of treatment and arranged a 4 
week safety follow-up (if not achieving complete ulcer closure at week 12 visit) or be 
given up to confirmed complete ulcer closure and arranged 12-week post-treatment 
follow-up (if achieving confirmed complete ulcer closure before or at week-12 visit).  
The schedule of assessment is tabulated in front of the protocol main text. The 
assessments or procedures to be performed at each study visit are listed below. The 
results of protocol specific assessments and procedures will be recorded in the source 
documents for all study subjects and on the appropriate page of the CRF.  
7.1 
Screening visit  
(-3 to -2 weeks relative to Randomization (baseline) visit or within 1week 
randomization visit if initial phase is not required) 
1. 
Explain the nature of the study and have subject to read and sign an Informed 
Consent Form 
2. 
Assign subject screening number 
3. 
Obtain Demographic, including: 
- 
Gender 
- 
Date of Birth 
- 
Height 
- 
Weight 
4. 
Screen subject for inclusion/exclusion criteria 
5. 
Medical history, including: 
- 
History of diabetic mellitus 
 
Date of onset of diabetic mellitus 
 
Date of onset of first and current episode of diabetic ulcer foot  
 
Therapy for current episode of diabetic ulcer foot 
- 
General medical history: All medical histories within one year before 
screening visit are to be recorded, using the body system categories outlined 
below. For each history, the specific medical terminology for the 
disease/disorder/condition, the date of diagnosis, and the history status 
(resolved or ongoing) will be documented. 
6. 
Perform pregnancy test for female with childbearing potential (Note: not with 
childbearing potential means post-menopausal (defined as at least one year 
without any menstruation) or documented status of post hysterectomy) 
7. 
ABI measurement 
8. 
Vital signs 
9. 
Perform physical examinations 


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10. Obtain blood samples for hematologic and biochemical tests (Test results within 
14 days before screening visit are acceptable; HbA1c test results can be within 28 
days.): 
11. Ulcer evaluation 
12. Record concomitant medication(s) 
13. Record adverse event(s) 
7.2 
Initial Phase visit (only for applicable subjects) 
(-2 to 0 weeks relative to Randomization (baseline) visit) 
1. Dispense instruction and material for Standard-of-Care 
2. Record of Adverse Events 
3. Record changes on concomitant medication(s) 
7.3 
 Randomization visit  
(Day 0) 
1. Re-confirm eligibility of subject 
2. Ulcer evaluation 
3. Vital signs 
4. Perform physical examinations 
5. Obtain blood samples for hematologic and biochemical tests (The tests can be 
waived if the tests have been performed within past 28 days) 
6. Assign randomization number to eligible subject 
7. Record changes on concomitant medication(s) 
8. Dispense material of standard-of-care 
9. Record adverse event(s) 
10. Dispense trial medication 
11. Dispense diary to patient 
7.4 
Evaluation visits during treatment period in visits 4 to 11  
(Weeks 1, 2, 3, 4, 6. 8, 10, 12 with time window 4 days) 
Subjects confirmed complete ulcer closure before week-12 visit will be arranged for 
post-treatment follow-up. 
1. Ulcer evaluation 
2. Vital signs 
3. Perform physical examinations 
4. Obtain blood samples for hematologic and biochemical tests (will only be performed 
at week 12 visit or when subject complete the treatment period) 
5. Record changes on concomitant medication(s) 
6. Dispense material of standard-of-care 


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7. Record adverse event(s) 
8. Collect unused treatment medication(s) (which is dispensed at prior visit) 
9. Dispense trial medication (excluding week 12 visit) 
10. Collect and check diary (which recorded from previous visit) 
11. At week-12 visit only, perform pregnancy test for female with childbearing 
potential  
7.5 
Follow-up visits 
The follow-up visits will be performed at 2, 4, and 8 weeks after for subjects achieving 
confirmed complete ulcer closure during treatment period and at 2 weeks after treatment 
period (week 14 or visit 12) only for those not. 
(The visit day will be week 14 for visit 12, week 16 for visit 13, and week 20 for visit 
14, each with time window4.) 
1. Ulcer evaluation 
2. Vital signs 
3. Perform physical examinations 
4. Record changes on concomitant medication(s) 
5. Record adverse event(s) 
7.6 
Final visit 
The final visit will be performed at 12 weeks  4 days after confirmation of complete 
ulcer closure for subjects achieved and at visit 13 (week 16  4 days) for those not. 
1. 
Ulcer evaluation 
2. 
Vital signs 
3. 
Perform physical examinations 
4. 
Record changes on concomitant medication(s) 
5. 
Record adverse event(s) 
6. 
Complete an exit form in the case report form and dismiss the subject 


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8. 
ADVERSE EVENTS 
8.1 
Definitions 
- 
Adverse Event (AE): 
An AE is any untoward medical occurrence in a patient or clinical investigation 
participant administered a study medication and that does not necessarily have a 
causal relationship with this treatment. An AE can therefore be any unfavorable and 
unintended sign, symptom, or disease temporally associated with the use of a study 
medication, whether or not related to the study medication. Laboratory 
abnormalities should not be recorded as AEs unless determined to be clinically 
significant by the Investigator. 
- 
Expected AE: 
Expected AE are defined as any event, the specificity or severity of which is 
consistent with the current investigator brochure or other technique documents.  
- 
Unexpected AE: 
Unexpected AE is defined as any event, the specificity or severity of which is not 
consistent with the current investigator brochure or other technique documents. 
"Unexpected", as used in this definition, refers to an adverse drug experience that 
has not been previously observed (e.g., included in the investigator brochure) rather 
than from the perspective of such experience not being anticipated from the 
pharmacological properties of the pharmaceutical product. 
- 
Serious Adverse Event (SAE): 
Serious Adverse Event (SAE): A Serious Adverse Event is defined as an AE meeting 
one of the following conditions: 
 
Death during the period of protocol defined surveillance 
 
Life Threatening Event (defined as a participant at immediate risk of death at 
the time of the event) 
 
An event requiring inpatient hospitalization or prolongation of existing 
hospitalization during the period of protocol defined surveillance  
 
Results in congenital anomaly or birth defect 
 
Results in a persistent or significant disability/incapacity 
 
Based upon appropriate medical judgment, the event may jeopardize the 
participant and may require medical or surgical intervention to prevent one of 
the outcomes listed above. 
8.2 
AE/SAE Intensity and Relationship Assignment 
- 
AE/SAE Intensity 
The investigator must rate the intensity for all AEs that occur during the study using 
the grades provided below: 
 Grade 1 (Mild): events require minimal or no treatment and do not interfere with 
the subject’s daily activities. 


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 
Grade 2 (Moderate): events result in a low level of inconvenience or concern 
with the therapeutic measures.  Moderate events may cause some interference 
with functioning. 
 
Grade 3 (Severe): events interrupt a subject’s usual daily activity and may 
require systemic drug therapy or other treatment.  Severe events are usually 
incapacitating. 
 
Grade 4 (Life threatening):  Any adverse drug experience that places the subject 
or participant, in the view of the investigator, at immediate risk of death from 
the reaction as it occurred, i.e., it does not include a reaction that had it occurred 
in a more severe form, might have caused death. 
 
Grade 5 (Death) 
 
- 
AE/SAE Relationship Assessment 
The investigator will be asked to assess all AEs with respect to their causal 
relationship to the study drug according to the following classification:  
 
Definitely Related: There is clear evidence to suggest a causal relationship, and 
other possible contributing factors can be ruled out. The clinical event, including 
an abnormal laboratory test result, occurs in a plausible time relationship to 
study agent/intervention administration and cannot be explained by concurrent 
disease or other drugs or chemicals. The response to withdrawal of the study 
agent/intervention (dechallenge) should be clinically plausible. The event must 
be pharmacologically or phenomenologically definitive, with use of a 
satisfactory rechallenge procedure if necessary. 
 
Probably Related: There is evidence to suggest a causal relationship, and the 
influence of other factors is unlikely. The clinical event, including an abnormal 
laboratory test result, occurs within a reasonable time sequence to administration 
of the study agent/intervention, is unlikely to be attributed to concurrent disease 
or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this 
definition.  
 
Possibly Related: There is some evidence to suggest a causal relationship (e.g., 
the event occurred within a reasonable time after administration of the trial 
medication).  However, the influence of other factors may have contributed to 
the event (e.g., the subject’s clinical condition, other concomitant events). 
Although an adverse drug event may rate only as “possible” soon after discovery, 
it can be flagged as requiring more information and later be upgraded to probable 
or certain as appropriate. 
 
Unlikely: A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study agent/intervention administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time 
after administration of the trial medication) and in which other drugs or 
chemicals or underlying disease provides plausible explanations (e.g., the 
subject’s clinical condition, other concomitant treatments).  
 
Not related: The AE is completely independent of study agent/intervention 
administration, and/or evidence exists that the event is definitely related to 


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another etiology. There must be an alternative, definitive etiology documented 
by the clinician Expected Events Related to Disease Process: Provide explicit 
definitions of the type(s), grade(s), and duration(s) of adverse event(s) that will 
be considered disease related. 
8.3 
Collecting, Recording and Reporting of Adverse Events 
8.3.1 Collecting and Recording of AE 
AEs may be volunteered spontaneously by the study subject, discovered as a 
result of general questioning by the study staff, or determined by physical 
examination. All AEs starting from signing ICF will be recorded on the CRF. 
For all AEs, the investigator must pursue and obtain adequate information both 
to determine the outcome of the AE and to assess whether it meets the criteria 
for classification as a serious AE requiring immediate notification. Follow-up of 
the AE, even after the date of study drug discontinuation, is required if the AE 
persists. The study subject will be followed until the event resolves or stabilizes 
at a level acceptable to the investigator. For patients receiving hemodialysis, 
taking regular arteriovenous fistula will not be considered as an AE. 
8.3.2 Reporting of AE 
Serious, alarming and/or unusual adverse events must be reported to the 
Sponsor/CRO contact within 24 hours of the investigator's knowledge of the 
event. Any event requiring hospitalization (or prolongation of hospitalization) 
that occurs during the course of a subject’s participation in a clinical study must 
be reported as a serious adverse event, except hospitalizations for the following: 
• Social reasons in absence of an adverse event 
• Surgery or procedure planned before entry into the study (must be 
documented in the CRF 
The investigator is responsible to communicate details of medical emergencies 
in trial subjects to the Ethics Committee. Sponsor is responsible to inform the 
events to the regulatory authorities.  
Fatal or life-threatening, unexpected ADRs should be notified to Taiwan 
National ADR Reporting Center by sponsor/CRO as soon as possible, but no 
later than 7 calendar days, after first acknowledged by the investigator, and a 
complete report should be followed 8 additional calendar days. This report must 
include an assessment of the importance and implication of the findings and/or 
previous experience on the same or similar medical products. Serious, 
unexpected ADRs that are not fatal or life-threatening must be filed as soon as 
possible but no later than 15 calendar days after first acknowledged by the 
investigator that a case qualifies. 
 


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9. 
DATA ANALYSIS 
9.1 
Data Quality Assurance 
The sponsor will provide paper CRFs or electronic CRFs (via Electronic Data Capture 
(EDC) system) for the recording and collection of subject data and for the collection of 
data. All CRFs will be completed as soon as possible after the subject’s visit. 
Corrections to data on the CRFs will be documented or traced. The investigator will 
review CRFs to indicate that, to his/her knowledge, they are complete and accurate. If 
further changes are made after this, the investigator will need to again sign the 
investigator signature page or approve electronically. Designated source documents will 
be signed and dated by the appropriate study personnel. 
9.2 
Clinical Data Management 
The investigator must agree to complete and maintain source documents and CRFs for 
each subject participating in the study. 
The sponsor/CRO will be responsible for the processing and quality control of the data. 
Data management will be carried out as described in the sponsor/CRO’s standard 
operating procedures (SOPs) for clinical studies. 
The handling of data, including data quality control, will comply with regulatory 
guidelines and the sponsor/CRO’s SOPs as well as provisions of the study-specific Data 
Management Plan. 
 
 


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10. 
SAMPLE SIZE AND STATISTICAL METHODS 
10.1 
Determination of Sample Size  
Based on the previous animal studies, we expect the investigational product, CSTC1, 
has the effect of reducing the ulcer size and even of achieving complete ulcer closure. 
The primary endpoint of this study is incidence of complete ulcer closure during 12 
weeks of treatment period. The sample size is arbitrarily determined to be 80 versus 20 
subjects (4:1) for Treatment versus Control groups.  
To ensure the completion of 100 evaluable subjects, around 125 subjects will be 
recruited. Evaluable subjects will be per-protocol population subjects with primary 
efficacy endpoint measurement. 
10.2 
Statistical and Analytical Plans 
10.2.1 Analysis Population 
Three data sets will be introduced for statistical analysis.  
Intent-to-treat (ITT) population: 
– All randomized subjects who have received at least one dose study 
medication.  
Per-protocol (PP) population: 
– A subset of ITT population 
– Fulfill all inclusion and exclusion criteria 
– Dosed with at least 8 weeks study drug or with early confirmed complete 
ulcer closure AND with at least 70% treatment compliance 
– Did not receive any prohibited treatment during treatment period 
– With any post-treatment assessment relevant to efficacy endpoint up to end 
of dosing period 
Efficacy endpoints will be analyzed on ITT and PP population. Demographics, 
baseline characteristics, and safety endpoints will be analyzed on ITT 
population. The conclusion of efficacy will be made according to the results of 
ITT population analysis. 
10.2.2 Efficacy Analyses 
- 
Primary efficacy endpoint 
The primary endpoint of this study is incidence of complete ulcer closure 
during up to 12 weeks of treatment period. Subjects with complete ulcer 
closure at week-12 visit and confirmed at week-14 visit will be considered 
as success. 
Let pt and pv represent the incidence of test and vehicle groups, respectively. 
We wish to test for equal proportions vs. the one-tailed alternative, since we 
are looking for improvement in one direction.  
The primary hypothesis will be: 
H0: pt = pv 
H1: pt ≠ pv 


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The test will be performed by Cochran-Mantel-Haenszel test (CMH test). 
Test treatment group will be declared superiority if the null hypothesis is 
rejected and incidence of complete ulcer closure of test product group is 
greater than that of matched vehicle group. 
- 
Secondary efficacy endpoints 
Ulcer closure rate at endpoint will be analyzed with CMH test. Ulcer closure 
time will be estimated by using Kaplan-Meier methods and compared 
between groups by using log-rank test. Percentage change in ulcer size will 
be analyzed by using Analysis of variance (ANOVA) model with treatment 
group and center as factor. Further efficacy analyses with ankle brachial 
index (ABI) (<0.7 or not) as stratification may be performed if necessary.  
All treatment group comparisons will be conducted with significance level 
of 0.05, using 2-tailed tests. 
10.2.3 Safety Analyses 
- 
Adverse Events (AEs) 
- 
Change in physical examinations results 
- 
Net change from baseline in laboratory test results 
- 
Net change from baseline in vital signs 
For safety analyses, adverse events will be reported by treatment groups and by 
physiological systems as appropriate. Incidence of adverse events between 
treatments will be analyzed by CMH test. Changes in physical examinations will 
be displayed for each individual system. Net changes from pre-treatment 
laboratory test results and vital signs will be analyzed by descriptive statistics. 
All treatment group comparisons will be conducted with significance level of 
0.05, using 2-tailed tests. 
10.2.4 Demography and baseline characteristics 
Demographics and baseline characteristics will be summarized for each group 
by using descriptive statistics. 
Additionally, descriptive statistics will be provided for all of the endpoints. 
Frequency table will be provided for categorical data, while mean, standard 
deviation, maximum, minimum, median, inter-quartile range (IQR), and 95% 
two-sided confidence interval will be calculated for continuous measurements. 
In addition, endpoints of time to event will be presented by using the life table 
and Kaplan-Meier plot. The Kaplan-Meier estimates will also be provided for 
these time-to-event endpoints. 
10.2.5 Interim Analyses 
There is no planned interim analysis for this study. 
 
 


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11. 
ETHICAL CONSIDERATIONS 
11.1 
Informed Consent 
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continuing throughout the individual’s study participation.  
Extensive discussion of risks and possible benefits of this therapy will be provided to 
the participants and their families.  
Consent forms will be IRB approved and the participant will be asked to read and review 
the document. Upon reviewing the document, the investigator will explain the research 
study to the participant and answer any questions that may arise.  The participants will 
sign the informed consent document prior to any procedures being done specifically for 
the study.  The participants should have sufficient opportunity to discuss the study and 
process the information in the consent process prior to agreeing to participate.  The 
participants may withdraw consent at any time throughout the course of the trial.  A 
copy of the informed consent document will be given to the participants for their 
records.  The rights and welfare of the participants will be protected by emphasizing to 
them that the quality of their medical care will not be adversely affected if they decline 
to participate in this study. 
11.2 
IRB review 
This protocol and the associated informed consent documents must be submitted to the 
IRB for review and approval. The study will not be initiated until the IRB provides 
written approval of the aforementioned documents and until approval documents have 
been obtained by the Principal Investigator and sponsor or CRO.  
No changes from the final approved protocol will be initiated without the IRB/EC’s 
prior written approval or favorable opinion of a written amendment, except when 
necessary to eliminate immediate hazards to the study participants or when the change 
involves only logistics or administration. Any significant deviation from the protocol 
when no approved amendment exists will be regarded as a protocol violation, and will 
be addressed as such during the reporting of the study. 
11.3 
Confidentiality of Data and Patient Records 
Include procedures for maintaining participant confidentiality, any special data security 
requirements, and record retention per the sponsor’s requirements. 
Participant confidentiality is strictly held in trust by the participating investigators, their 
staff, and the sponsor(s) and their agents.  This confidentiality is extended to cover 
testing of biological samples and genetic tests in addition to the clinical information 
relating to participating subjects. 
The study protocol, documentation, data and all other information generated will be held 
in strict confidence.  No information concerning the study or the data will be released 
to any unauthorized third party, without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all 
documents and records required to be maintained by the Investigator, including but not 
limited to, medical records (office, clinic or hospital) and pharmacy records for the 
participants in this study.  The clinical study site will permit access to such records. 


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The information provided by sponsor in this protocol and the associated Clinical 
Investigator's Brochure and the data generated by this clinical study are to be considered 
as confidential property of sponsor. 
 


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12. 
PUBLICATIONS 
The data and information associated with this study may be used by sponsor now and 
in the future for the purposes of presentation, publication at discretion of sponsor or for 
submission to regulatory agencies. In addition, relative to the release of any proprietary 
information, sponsor reserves the right of prior review of any publication or presentation 
of data from this study. 
In signing this protocol, the investigator agrees to the release of the data from this study 
and acknowledges the above publication policy. 
 
 
13. 
RETENTION OF TRIAL DOCUMENTS 
Records for all participants, including CRFs, all source documentation (containing 
evidence to study eligibility, history and physical findings, laboratory data, results of 
consultations, etc.) as well as IRB records and other regulatory documentation should 
be retained by the PI in a secure storage facility. The records should be accessible for 
inspection and copying by authorized authorities.  
All study documentation at the clinical site and sponsor records will be archived in 
accordance with ICH GCP, applicable regulations, the sponsor’s quality standards and 
SOPs. Study records should not be destroyed without prior written agreement between 
sponsor or CRO and the study investigator. 
 
 
 


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14. 
REFERENCES 
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Points #1. Data Points Publication Series [Internet]. Rockville (MD): Agency for 
Healthcare Research and Quality (US); 2011-.2011 Feb 17. 
2. Margolis DJ, Malay DS, Hoffstad OJ, Leonard CE, MaCurdy T, de Nava KL, Tan 
Y, Molina T, Siegel KL. Incidence of Diabetic Foot Ulcer and Lower Extremity 
Amputation Among Medicare Beneficiaries, 2006 to 2008: Data Points #2. Data 
Points Publication Series [Internet]. Rockville (MD): Agency for Healthcare 
Research and Quality (US); 2011-.2011 Feb 17.  
3. U.S. Department of Health and Human Services. Guidance for industry, Chronic 
Cutaneous Ulcer and Burn Wounds — Developing Products for Treatment. June 
2006.  
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5. Thang PT, Patrick S, Teik LS, Yung CS. Anti-oxidant effects of the extracts from 
the leaves of Chromolaena odorata on human dermal fibroblasts and epidermal 
keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase 
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6. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, Kang KH. Isolation 
and determination of anthocyanins in seed coats of black soybean (Glycine max 
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Matthews D, MacLean MR, Lean ME, Duthie GG, Crozier A. Relationship among 
antioxidant activity, vasodilation capacity, and phenolic content of red wines. J 
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soybean seed coat inhibit the expression of TNF-alpha-induced genes associated 
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SC, Chung JI, Kang D, Zhang D. Anthocyanins From Black Soybean Seed Coat 
Enhance Wound Healing. Ann Plast Surg. 2013 Feb 12. 


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13. Effects of A1 and C1 on wound closures in STZ-induced diabetic rats. Development 
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