Effect of Increasing Blood Flow by Botulinum Toxin Local Injection for Severe Peripheral Artery Occlusive Disease: Preliminary Report
Phase: PHASE4
Conditions: Chronic Ulcer of Lower Extremity, Wound, Botulinum Toxins, Type A, Blood Circulation
Sponsor: Yonsei University

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Clinical trial protocol 
 
Effect of increasing blood flow by botulinum toxin local injection for severe 
peripheral artery occlusive disease: preliminary report  
 
 
 
 
 
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CONFIDENTIAL 
 
 

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Contents 
1. Title .................................................................................................................................. 5 
2. Name and address of clinical trial institution ............................................................ 5 
3. Clinical trial person in charge ...................................................................................... 5 
3.1 Clinical trial person in charge ...................................................................................... 5 
3.2 Clinical trial staff, managing pharmacist, and co-researchers ..................................... 5 
4. Introduction ................................................................................................................... 6 
4.1 Background .................................................................................................................. 6 
4.2 Purpose ........................................................................................................................ 6 
5. Clinical investigational drugs ....................................................................................... 7 
6. Target Disease ............................................................................................................... 7 
7. Selection of test subjects ............................................................................................. 7 
7.1 Inclusion criteria ........................................................................................................... 7 
7.2 Exclusion criteria .......................................................................................................... 7 
7.3 Number of subjects ..................................................................................................... 8 
7.4 Determination of Sample Size ..................................................................................... 8 
8. Clinical trial period ........................................................................................................ 9 
9. Clinical test method ...................................................................................................... 9 
9.1 Preparation process for clinical investigational drugs ................................................. 9 
9.1.1 
Foreign body inspection ....................................................................................... 9 
9.1.2 
Dilution method .................................................................................................. 9 
9.2 Dosage and administration method of investigational drugs ................................... 10 
9.3 Storage, management and records of investigational drugs .................................... 10 
9.4 Concomitant drugs and treatment/Contraindicated drugs and 
treatment ................................................................................................................. 11 
9.4.1 
Concomitant drugs ............................................................................................ 11 
9.4.2 
Contraindicated drugs ....................................................................................... 11 

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10. Observation items, clinical examination items and observation test methods ..... 12 
10.1 Observation and clinical examination items by visit period ...................................... 12 
10.1.1 Visit 1 : Baseline (Day -7~0) .............................................................................. 12 
10.1.2 Visit 2: Day 0 ..................................................................................................... 13 
10.1.3 Visit 3 : Day 3 ± 2 ............................................................................................. 14 
10.1.4 Visit 4 : Day 7 ± 2 ............................................................................................. 14 
10.1.5 Visit 5 : Day 10 ± 2 ........................................................................................... 14 
10.1.6 Visit 6 : Day 14 ± 2 ........................................................................................... 15 
10.2 Dropout ..................................................................................................................... 15 
10.3 Unscheduled Visits ..................................................................................................... 15 
11. Criteria for plan violation, suspension and disqualification .................................... 16 
11.1 Criteria for plan violation ........................................................................................... 16 
11.2 Criteria for suspension and disqualification .............................................................. 16 
12. Effectiveness evaluation criteria, evaluation methods and interpretation methods
 ...................................................................................................................................... 17 
12.1 Primary endpoint ....................................................................................................... 17 
12.2 Secondary endpoint ................................................................................................... 17 
12.3 Effectiveness evaluation method ............................................................................... 17 
12.3.1 Local transcutaneous Oxygen Pressure Measurement, tcPO 2 .......................... 17 
12.3.2 Thermal imaging camera ................................................................................... 17 
12.3.3 Gross photo ....................................................................................................... 18 
13.3.3.1 photography equipment ....................................................................................... 18 
13.3.3.2 photography ........................................................................................................ 18 
13.3.3.3.2 photo conversion .............................................................................................. 19 
13.3.3.3.3 Photo data management .................................................................................. 19 
13.3.3.3.4 Photo recording and storage ............................................................................ 19 
13. Statistical analysis method ......................................................................................... 19 
13.1 Definition of analysis group ...................................................................................... 19 
13.1.1 FAS (Full analysis set) ........................................................................................ 19 
13.1.2 PPS (Per-Protocol set) ....................................................................................... 20 
13.1.3 Safety set ........................................................................................................... 20 
13.2 Principles of result analysis ........................................................................................ 20 
13.3 Demographic and basic information ......................................................................... 20 
13.4 Analysis of primary efficacy endpoints ...................................................................... 20 
13.5 Analysis of secondary efficacy endpoints .................................................................. 20 
13.6 Safety evaluation parameters .................................................................................... 21 

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13.6.1 Adverse reactions .............................................................................................. 21 
13.6.2 Physical examination ......................................................................................... 21 
13.6.3 Laboratory tests ................................................................................................. 21 
13.7 Handling of missing values ....................................................................................... 21 
14. treatment standards for subjects after completion of clinical trial ........................ 21 
15. References ................................................................................................................... 22 
 
 
 

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1. TITLE 
Effect of increasing blood flow by botulinum toxin local injection for severe 
peripheral artery occlusive disease: preliminary report 
 
2. NAME AND ADDRESS OF CLINICAL TRIAL INSTITUTION 
Name 
address 
Yong in severance hospital 
Yongin Severance Hospital 363, 
Dongbaekjukjeon-daero, Giheung-gu, 
Yongin-si, Gyeonggi-do 16995, Republic 
of Korea 
 
3. CLINICAL TRIAL PERSON IN CHARGE 
3.1 
Clinical trial person in charge 
 
Hospital 
Major Field 
Title 
Name 
P.I. 
Yong in severance 
hospital 
Department of plastic and 
reconstructive surgery 
professor 
Hii sun, Jeog 
 
3.2 
Clinical trial staff, managing pharmacist, and co-researchers 
Hospital 
Major Field 
Co-researcher 
Name 
Yong in severance 
hospital 
Nephrology 
S.I. 
Jung eun, Lee 
Yong in severance 
hospital 
Cardiology 
S.I. 
O hyun, Lee 
Yong in severance 
hospital 
Endocrinology 
S.I. 
Gyung min, Kim 
Yong in severance 
hospital 
Plastic and 
reconstructive surgery 
S.I. 
Bok ki, Jung 
Yong in severance 
hospital 
Plastic and 
reconstructive surgery 
managing 
pharmacist 
Bok ki, Jung 
 
 

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4. INTRODUCTION 
4.1 Background 
Tissues below the calf are governed by blood flow from the anterior tibial artery, peroneal artery, 
and posterior tibial artery. When an ulcer occurs due to acute lower extremity ischemia, one of the 
three blood vessels is blocked by a blood clot, or when the blood vessel is narrowed due to 
perivascular inflammation, such as Buerger's disease, it occurs in a subacute or chronic form. In 
particular, in the case of elderly patients with ulcers or diabetic feet due to lower extremity ischemia, 
the symptoms are often worsened by chronic narrowing of blood vessels due to arteriosclerosis or 
blockage by blood clots. To treat this, prostaglandin or antithrombotic drugs are taken, and blood 
flow is resumed through stent surgery, but in the lower extremities, the recurrence rate is relatively 
high because the blood vessels are smaller than the iliac artery, femoral artery, and popliteal artery. 
Botulinum toxin is known to have a positive effect on wound healing and blood flow improvement. 
Botulinum toxin has been published several times in papers showing its spasmolytic effect by 
participating in the radiographic hip osteoarthritis Ras homolog A (RhoA)/Rock-assisted protein 
kinase (ROCK) pathway. In addition, botulinum toxin has a vasodilating effect related to the 
calcitonin gene-related peptide (CGRP) pathway, and is involved in increasing vascular endothelial 
growth factor (VEGF) from NO-mediated angiogenesis, inhibiting vasoconstriction, increasing blood 
flow rate, and promoting angiogenesis. 
In this study, in patients with chronic wounds on the feet, whose lower extremity blood vessels 
circulate mainly through collateral circulation, subcutaneous injection of Botulinum toxin in and 
around the wound improves blood flow around the wound and confirms the degree of wound 
healing. 
. 
4.2 Purpose 
This is a non-randomly assigned, open-label, prospective comparative clinical study to confirm the 
effectiveness and safety of the effect of improving blood flow around foot wounds by injecting 
botulinum toxin into patients with moderate to severe lower extremity ischemia. 
 

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5. CLINICAL INVESTIGATIONAL DRUGS 
1) Product name: Botulex®  
2) Product name: Botulinum toxin A  
3) Appearance: It is an injection containing white, freeze-dried powder in a colorless and 
transparent glass vial. When a solvent (physiological saline solution) is added, it should 
become a colorless and transparent liquid preparation..  
4) Amount of raw drug product: 100 unit/1 vial 
5) Storage conditions: Store in refrigerator (2~8℃). 
6) Period of use: 36 months from the date of manufacture 
7) Manufacturer: Hugel Co., Ltd. 
 
6. TARGET DISEASE 
Patients with chronic ulcer on foot among patients with moderate to severe lower extremity 
ischemia 
7. SELECTION OF TEST SUBJECTS 
7.1 
 Inclusion criteria 
1. Age ≥ 20 years old 
2. Patients who have a lower extremity wound among those who have been diagnosed with 
moderate or severe lower extremity ischemia 
3. Patients who are unable to perform additional procedures (angioplasty, etc.) by 
performing peripheral vascular examination and CT angiography 
4. Wound size ≥ 1x1cm2 to ≤ 3x3cm2 
5. Patients who can follow the clinical trial procedure well and abide by the visit schedule 
6. Written informed consent to participate in the study after having fully understood the 
contents of the protocol and restrictions. 
 
7.2 Exclusion criteria 
7. Patients with diseases that can affect neuromuscular function, such as myasthenia gravis, 
Eaton-Lamberton syndrome, amyotrophic lateral sclerosis, and motor neuropathy 

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8. Within 4 weeks before screening, aminoglycoside antibiotics, curare-like agents, or drugs 
that inhibit neuromuscular function (muscle relaxants, anticholinergics, benzodiazepines, 
benzamides, tetracyclines, Rinco Those who have taken mycin antibiotics, etc.) 
9. Those taking aspirin, NSAIDs or anticoagulants within 7 days before screening 
10. Those who have received botulinum toxin preparations within 3 months before screening 
11. Angiography or CT angiography If one or more of the three major blood vessels in the 
lower extremity are open 
12. Cases in which blood flow to the lower extremities can be preserved by performing 
balloon angioplasty even if all three major blood vessels in the lower extremity are 
blocked 
13. Those who are currently taking steroids or immunosuppressants that affect wounds, or 
those who have taken them within one month of screening 
14. Those who have applied injection drugs or wound coverings that help improve wounds 
within 1 week of screening 
15. Women who are pregnant, lactating, planning to become pregnant during the clinical 
period, or women of childbearing age who are not using available contraceptive methods 
(women of childbearing age must be negative in the pregnancy test prior to injection). 
16. Those who are allergic or sensitive to botulinum toxin 
17. Those who have participated in another clinical trial within 30 days before screening or 
those who have not passed the half-life of the investigational product of the clinical trial 
that they participated in, whichever is longer. 
18. Those who are not suitable for this clinical trial under the judgment of other investigators 
 
7.3 
 Number of subjects 
10 subjects 
 
7.4 Determination of Sample Size  
This study was planned as an investigator-led clinical trial. The purpose is to collect information 
on the initial safety and effectiveness of drugs, design follow-up confirmatory clinical trials, and 
provide evidence for evaluation items and evaluation methods, and is scheduled to be conducted 
on a small number of clinical trial subjects over a relatively short period of time. As this is an 
exploratory clinical trial using non-random allocation, open label, and clinical case studies, the 
number of subjects is not calculated statistically. 
It is expected that approximately one clinical trial subject will be registered per month. The wound 
healing treatment period is expected to be more than a month, and the clinical trial period for each 

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test subject is short, about two weeks, making it difficult for patients to drop out. Therefore, even 
if dropout is considered, if 10 clinical trial subjects who met the study period complete the clinical 
trial, the entire study will be terminated, and statistical results will be derived based on this. 
8. CLINICAL TRIAL PERIOD 
It is expected to take approximately 12 months from the date of IRB approval to completion of 
administration to the last test subject. 
 Test subject registration period: approximately 6 months 
 Clinical trial period for each test subject: approximately 2 weeks  
(follow-up for 2 weeks after one administration) 
 
9. CLINICAL TEST METHOD 
9.1 
Preparation process for clinical investigational drugs 
9.1.1 
Foreign body inspection 
Carefully inspect the presence of foreign substances and discoloration in the vial before and after 
dilution.. 
 
9.1.2 
Dilution method 
To dissolve the freeze-dried investigational drug, use preservative-free sterile saline solution (0.9% 
sodium chloride injection). If the investigational drug bubbles or undergoes similar vigorous 
agitation, it will denature, so gradually add the diluent to the vial. If the diluent does not enter the 
vial under vacuum, discard the vial. Record the date and time of dissolution on the label and 
administer within 24 hours after dissolution. During this period, the dissolved investigational drug 
is stored in the refrigerator (2~8℃). The dissolved clinical trial drug should be colorless and 
transparent and no unusual substances should be visible. As the formulation and diluent do not 
contain preservatives, it is not recommended to use one vial for more than one patient. In this 
clinical trial, 1 vial of Botulex 100 Units is prepared by diluting it in 4 ml of 0.9% physiological saline 
solution. Before administration, foreign matter inspection, dilution, and syringe filling are performed 
by a designated dilution manager, and then administration is performed by the researcher in charge 
of medication administration. 

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9.2 
Dosage and administration method of investigational drugs 
1) Botulex® (Main ingredient: Clostridium botulinum toxin type A, Strain: Clostridium 
Botulinum CBFC26) 100 units(1 vial) is prepared by dissolving in 4 cc normal saline for test 
subjects who meet the selection criteria as a result of the screening test. 
2) Treatment method 
① Dissolve 100 units (1 vial) of Botulex in 4 cc of normal saline. 
② Prepare 4 syringes by filling 4 cc of Botulex solution into each 1 cc syringe. 
③ Attach a 30G, 1/2’’ needle to a 1cc syringe filled with 1cc of Botulex solution, and prepare 
alcohol cotton, gauze, and surgical gloves. 
④ Inject the Botulex solution into the subcutaneous fat layer at a dose of 5 units per 1x1 
cm2 into and around the wound of a patient with moderate to severe lower extremity 
ischemia. 
⑤ After administration, disinfect the administration site and dress the administration site 
and wound area. 
⑥ The maximum dosage at one time should not exceed 100 units.  
 
9.3 
Storage, management and records of investigational drugs 
1) Responsibility for the management of investigational drugs used in clinical trials lies with the 
principal investigator and the investigational drug manager (hereinafter referred to as manager) of 
the relevant clinical trial institution. 
2) The provider of investigational drugs must distribute the investigational drugs used in clinical 
trials to the manager, and obtain and preserve a receipt of receipt. 
3) The manager checks whether the price of the investigational drug used in the clinical trial matches 
the packaging list, whether it is in good condition, and whether the quantity matches the quantity 
on the receipt. 
4) Managers must store and manage investigational drugs used in clinical trials to ensure that they 
are not used for purposes other than clinical trials. 
5) During the conduct of clinical trials, the manager prepares receipt/disbursement records 
regarding the receipt/release, inventory, and use status of investigational drugs and the return or 
disposal of unused investigational drugs. The quantity and storage conditions of investigational 
drugs used in clinical trials must be confirmed, and measures must be taken to ensure that clinical 
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6) The clinical trial director or manager confirms that the quantity of the receipt and payment record 
of the investigational drug used in the clinical trial matches the contents of the case record. If there 
is a discrepancy, investigate the cause, record the results, and take necessary action. 
7) Drugs for clinical trials are stored in a storage container with a locking device in the drug 
refrigerator in the plastic surgery outpatient treatment room on the 2nd floor of Yongin Severance 
Hospital under the supervision of the clinical research director or manager. 
8) Once an investigational drug is assigned, it cannot be reassigned to another subject. 
9) After injecting the clinical trial drug, the syringe and packaging used must be managed so as not 
to be reused. 
10) The provider of investigational drugs shall recall unused investigational drugs when the clinical 
trial is suspended or terminated or the clinical trial manager does not conduct the test according 
to the protocol. At this time, after consultation with the clinical trial director, the manager must 
return unused clinical trial drugs to the company and keep the return certificate. 
11) The provider of investigational drugs must confirm the quality of the investigational drugs by 
conducting quality control tests by batch number before the start of clinical trials. 
 
9.4 
Concomitant drugs and treatment/Contraindicated drugs and treatment 
9.4.1 
Concomitant drugs 
Concomitant drugs and combination therapies that are not defined as treatment contraindications 
in this clinical trial protocol are permitted. For all drugs taken during the clinical trial period, the 
drug name, dosage, dosage, and period of use must be recorded. If non-drug treatment such as 
rehabilitation therapy was received, the treatment method and purpose must be recorded in the 
case record. 
9.4.2 
Contraindicated drugs 
The following drugs that may affect muscle tone are permitted if taken steadily within 4 weeks prior 
to participating in the clinical trial. However, there should be no change in dosage during the clinical 
trial period. 
1) Muscle relaxants 
classification 
drug details 
peripheral  
muscle relaxant 
Alcuronium, Tubocurarine, Dimethyltubocurarine, Pancuronium, 
Gallamine, 
Vecuronium, 
Atracurium, 
Hexafluorenium, 
Pipecuronium bromide etc. 
central muscle relaxant Phenprobamate, Carisoprodol, Styramate, Baclofen, Tizanidine, 

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Pridinol, Tolperisone etc. 
muscle relaxant 
Dantrolene 
 
2) Benzodiazepines: Diazepam, Chlordiazepoxide, Medazepam, Oxazepam, Potassium 
clorazepate, Lorazepam etc. 
 
The following drugs are prohibited from co-administration during the clinical trial period.  
1) Aminoglycoside antibiotics: Streptomycins, Tobramycin, gentamicin, Kanamycin, Neomycin, 
Amikacin, Netilmicin, Sisomicin, Dibekacin etc. 
2) antibiotics: Spectinomycin, Polymyxin, Tetracycline, lincomycine etc. 
3) Anticholinergics: 
Trihexyphenidyl, 
Biperiden, 
Metixene, 
Procyclidine, 
Profenamine, 
Dexetimide, Phenglutarimide, Mazaticol, Benzatrophin etc. 
4) Benzamide-based drugs: tiapride hydrochloride, sulpiride etc. 
In addition to the administration of clinical trial drugs, concomitant drugs, including general drugs, 
may be permitted if necessary if they are not contraindicated drugs. During the clinical trial, 
concomitant medications must be recorded in the case record including drug name, dosage, 
dosage, and period of use. 
 
In the case of drugs that are used at the discretion of the investigator for reasons such as safety to 
manage adverse reactions, the reasons may be documented and the clinical trial may be continued. 
 
10. OBSERVATION ITEMS, CLINICAL EXAMINATION ITEMS AND 
OBSERVATION TEST METHODS 
10.1 Observation and clinical examination items by visit period 
10.1.1 Visit 1 : Baseline (Day -7~0) 
Researchers obtain written consent from test subjects prior to participation in clinical trials. On the 
day of administration, collect the following data and conduct clinical trials. 
 
The following process occurs at the screening visit (Visit 1). 
1)  Written consent 
2)  Demographic information: Check the test subject’s information such as initials, gender, 
and age. 

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3)  Confirmation of prior medications and treatments: Investigate medications and 
treatments administered or being administered within 4 weeks prior to the baseline 
visit. 
4)  Investigate the history of botulinum toxin injections. 
5)  Check medical history and surgical history: Check medical history and surgical history 
within 6 months before the baseline visit. 
6)  Physical examination 
- Wound size, wound depth, condition of the wound bed, degree of discharge of the 
wound, and whether the wound is inflamed. 
7)  Vital signs: Measure blood pressure (systolic/diastolic) and pulse. 
8)  Pregnancy test: Serum β hCG pregnancy test or urine hCG test is performed only on 
women who have started menarche and are capable of becoming pregnant. 
9)  Laboratory tests: The following laboratory tests are performed. (However, if results are 
available within 2 weeks prior to the screening visit, those results can be used instead.) 
- Hematology test: WBC, RBC, Hemoglobin, Hematocrit, Platelet count, WBC differential 
count (Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil) 
- Blood biochemical test: Na, K, Cl, Ca, P, BUN, Glucose, Total bilirubin, Total cholesterol, 
Uric acid, Total protein, Albumin, Alkaine phosphatase (ALP), SGOT. SGPT, Creatinine 
- Urine test: pH, Specific Gravity, Glucose, Protein, Color 
10)  Evaluation of selection/exclusion criteria 
11)  Dressing 
 
10.1.2 Visit 2: Day 0  
The following data are collected on the day of administration of the clinical trial drug. 
1) Evaluation of exclusion criteria 
2) Administration of clinical trial drugs 
3) Dressing 
4) tcPO2 measurement 
5) Thermal imaging camera measurement 
6) Photography 
7) Concomitant medications 
8) Adverse reactions: Check whether the test subject has any signs or symptoms of an infusion 
reaction within 30 minutes after administration. Anaphylaxis, severe allergic reaction, and severe 

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infusion reaction are defined as Grade 3 or higher in each of the National Cancer Institute-Common 
Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0.  
 
10.1.3 Visit 3 : Day 3 ± 2  
The following data are collected 3 days after administration of the clinical trial drug. 
1) Physical examination 
2) Vital signs 
3) Dressing 
4) Concomitant medications 
5) Adverse reactions 
 
10.1.4 Visit 4 : Day 7 ± 2 
The following data are collected 7 days after administration of the investigational drug. 
1) Physical examination 
2) Vital signs 
3) Dressing 
4) tcPO2 measurement 
5) Thermal imaging camera measurement 
6) Photography 
7) Concomitant medications 
8) Adverse reactions 
 
10.1.5 Visit 5 : Day 10 ± 2 
The following data are collected 10 days after administration of the investigational drug. 
1) Physical examination 

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2) Vital signs 
3) Dressing 
4) Concomitant medications 
5) Adverse reactions 
 
10.1.6 Visit 6 : Day 14 ± 2 
The following data are collected 14 days after administration of the investigational drug. 
1) Physical examination 
2) Vital signs 
3) Pregnancy test 
4) Laboratory tests 
5) Dressing 
6) tcPO2 measurement 
7) Thermal imaging camera measurement 
8) Photography 
9) Concomitant medications 
10) Adverse reactions 
 
10.2  Dropout 
 Subjects who drop out must measure the items at the last visit (Visit 6) of the clinical trial. 
 
10.3 Unscheduled Visits 
If the test subject visits on a day other than the scheduled date, vital signs, physical examination, 
laboratory tests, concomitant drug/non-drug treatment investigation, and confirmation of adverse 
reactions are performed. Unscheduled visits should not change the schedule of the study plan. 
 

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11. CRITERIA FOR PLAN VIOLATION, SUSPENSION AND DISQUALIFICATION 
11.1 Criteria for plan violation 
If it is known that the protocol has been violated during the clinical trial, the investigator must 
decide whether the subject should continue or discontinue the clinical trial. If a student drops out 
due to violation of the plan, the details must be recorded in the case report form. 
11.2 Criteria for suspension and disqualification 
Subjects who have received the investigational drug but are unable to participate during the entire 
clinical trial period for any reason are classified as ‘withdrawals.’ If the test subject requests it or the 
investigator determines that the test subject should be eliminated, the test subject may be 
eliminated at any time. The reasons for elimination are as follows. 
1)  Voluntary termination of test by subject (withdrawal of consent) 
2)  Subjects who participated in violation of selection/exclusion criteria 
3)  When the subject does not visit (failure to follow-up) 
4)  When a concomitant contraindicated drug was administered or when it was deemed 
necessary to administer a concomitant contraindicated drug 
5)  When a female test subject is pregnant 
6)  When it is difficult to perform the test due to adverse reactions 
7)  If a serious violation of the clinical trial protocol occurs 
8)  If a systemic disease that was not detected in the pre-procedure examination is discovered 
9)  During the clinical trial period, if medication or procedures that could affect the test results 
were administered without the doctor's instructions. 
10)  In cases where the clinical trial can no longer proceed due to other investigators' judgment 
 
In order to evalute the fact that an adverse reaction has occurred that the tester is not aware of, 
the test subject who refuses to visit must be contacted directly using all methods such as phone, 
letter, or in-person visit. The reason for exclusion must be recorded in the case report form. 
Evaluation at the last visit should also be conducted for subjects who drop out. Subjects who fail 
are unable to re-participate. 

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12. EFFECTIVENESS EVALUATION CRITERIA, EVALUATION METHODS AND 
INTERPRETATION METHODS 
12.1 Primary endpoint 
1) Change in tissue oxygen tension in tcPO2 (mmHg) in the wound 14 days after administration 
compared to baseline  
 
12.2 Secondary endpoint 
1) Change in the range of colors in the wound areas of both feet on the thermal imaging camera 
14 days after administration compared to baseline. 
2)  Changes in wound area in photography 14 days after administration compared to baseline  
 
12.3 Effectiveness evaluation method 
12.3.1 Local transcutaneous Oxygen Pressure Measurement, tcPO 2 
Transcutaneous oxygen partial pressure measurement is a technology that attaches a sensor, a 
measuring device, around the wound, and non-invasively measures the oxygen value diffusing in 
the subcutaneous blood vessels using an electrical signal. To determine the transcutaneous oxygen 
partial pressure, the transcutaneous oxygen partial pressure is measured using the PF5040 TcpO2 
unit (PeriFlux System5000; Perimed AB, Stockholm, Sweden) and dedicated software (PeriSoft for 
Windows 2.50; Perimed AB). 
The probe of the transdermal oxygen partial pressure meter is attached to the location of the 
wound or further distal to it with the patient lying horizontally to accurately reflect the amount of 
oxygen coming from the capillaries. Measurements are taken when the temperature of the skin 
surface reaches 43.0 C after an equilibrium period of 10 to 15 minutes. The measured room 
temperature was maintained at 25 to 26 C during the study period. For control comparison, normal 
areas of the same area are also measured and compared at each measurement. 
 
12.3.2 Thermal imaging camera 
After removing the patient's dressing from the wound area, the results of the thermal imaging 
camera are immediately measured to obtain static thermal imaging camera results. 
After applying isopropyl alcohol to the affected area, apply a breeze with a fan for 60 seconds to 
bring the skin temperature to 23-25 C. Then, use a thermal imaging camera to record the pattern 
of reheating the skin temperature(dynamic thermal imaging). 

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They appear as hot spots where perforating blood vessels exist, and they appear as cold spots 
where there is no subdermal blood vessel layer. Changes in blood flow in the skin near the wound 
are visually observed using a thermal imaging camera. 
 
12.3.3 Gross photo 
12.3.3.1. 
photography equipment 
① canon EOS 80D with Macro ring lite MR-14EX II (EF-S 35mm lens) 
② Camera tripo with adjustable height 
③ Green Background screen in PS OPD clinic 
④ Using the lighting installed in the photography room(PS OPD clinic) and the flash (Macro 
ring lite MR-14EX II) attached to the camera, take pictures under appropriate lighting to 
best express the wound area. 
 
12.3.3.2. 
 photography 
A total of 4 photos are taken: 2 photos each of detailed photos including the wound and 
the entire lower extremity with the wound. 
 
① Detailed photo: Take a close-up photo centered on the wound so that the ‘test subject 
identification sticker’ and ‘ruler for measuring the area of the wound area’ are visible. 
② Full photo: When taking a full photo, use the wound as a reference to include one joint 
above and below to confirm the location, and shoot to include the entire leg on the left 
and right. Photograph the entire area, including both the ‘test subject identification sticker’ 
and the ‘ruler for measuring the area of the wound area.’ 
(examples) 
 
< Full photo > 
 

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< Detailed photo > 
 
12.3.3.3. 
photo conversion 
Photos taken with a camera are used to measure the area of the wound using ImageJ 1.45s 
software (National Institutes of Health, Rockville, MD; http:// https://imagej.nih.gov/ij/). 
 
12.3.3.4. 
Photo data management 
The photographs taken are important data for evaluating the effectiveness of this clinical trial, and 
the photographic materials can only be accessed by the principal investigator and those authorized 
by the principal investigator, and can be accessed by regulatory authorities as necessary. Photo 
data management personnel must pay special attention to the security of collected data. 
 
12.3.3.5. 
Photo recording and storage 
Photos taken for research purposes are prohibited from being used for purposes other than this 
clinical trial. In addition, stored photos must not be provided to anyone other than the research 
staff of the conducting institution and the person in charge of the study. 
 
 
13. STATISTICAL ANALYSIS METHOD 
 
13.1 Definition of analysis group 
This clinical trial is a superiority clinical trial, and the efficacy evaluation analysis is primarily 
conducted on FAS, and the analysis on PPS is performed as a supplement. Additionally, 
safety evaluation analysis is performed on the safety set.  
13.1.1 FAS (Full analysis set) 
The subjects are those who received the investigational drug and measured the primary efficacy 
endpoint at least once. 

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13.1.2 PPS (Per-Protocol set) 
Among the subjects included in FAS, the subjects are those who completed the clinical trial without 
any serious violation of the clinical trial protocol. The reasons for PPS exclusion are as follows. 
1) Violation of selection/exclusion criteria 
2) Administration of drugs prohibited from concomitant use 
3) Dropout 
4) Other serious violations of the plan 
 
13.1.3 Safety set 
The subjects are those who have received the clinical trial drug at least once. 
 
13.2 Principles of result analysis 
Demographic and baseline information is summarized for FAS. When evaluating effectiveness, FAS 
is used as the main analysis group, and statistical analysis is additionally performed on PPS. When 
evaluating safety, the safety set is the target. 
 
For continuous variables, descriptive statistics (mean, standard deviation, median, minimum, 
maximum, etc.) are calculated and presented, and for categorical variables, frequencies and ratios 
are presented. 
13.3 Demographic and basic information 
Regarding basic survey data such as demographic surveys, medical history, and prior medications, 
the average and standard deviation are calculated for continuous data, and frequencies and ratios 
are calculated and analyzed for categorical data. 
 
13.4 Analysis of primary efficacy endpoints 
1) The change in local transcutaneous oxygen partial pressure measured on the 7th and 14th days 
after the final administration compared to the baseline value is calculated and compared through 
the Mann-Whitney test, a non-parametric statistic. 
13.5 Analysis of secondary efficacy endpoints 
1) The changes in blood flow near the wound using a thermal camera imaging measured 7 and 14 
days after the final administration compared to the baseline value. 

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2) The change in wound area is calculated through photographs taken on the 7th and 14th days of 
final administration compared to the baseline value, and confirmed and compared using the Mann-
Whitney test, a non-parametric statistic. 
13.6 Safety evaluation parameters 
13.6.1 Adverse reactions 
Determine the frequency and proportion of test subjects who experienced adverse reactions (TEAEs) 
after administration of the investigational drug and adverse reactions related to the investigational 
drug. 
 
13.6.2  Physical examination 
The frequency and ratio of subjects are presented for each physical examination item, and the 
frequency and percentage of normal/abnormal changes are presented by comparing the subjects' 
results on the 14th day of final administration compared to the baseline value. . 
 
13.6.3 Laboratory tests 
Descriptive statistics (mean, standard deviation, median, minimum, and maximum) for laboratory 
test results performed at each visit are presented. In addition, the results of subjects on the 14th 
day of final administration compared to the baseline are compared to present the frequency and 
percentage of normal/abnormal changes. 
 
13.7 Handling of missing values 
Analysis is performed using raw data without data correction. If missing values occur for reasons 
such as a subject dropping out before the end of a clinical trial or missing a test at a specific time, 
the data are not replaced and analyzed and treated as missing unless specifically specified 
(Observed Case). 
 
 
14. TREATMENT STANDARDS FOR SUBJECTS AFTER COMPLETION OF 
CLINICAL TRIAL 
Subsequent treatment for subjects who terminated the clinical trial early and for subjects who 
completed the clinical trial shall follow standard treatment methods. 
 

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