Clinical trial NCT04808622
Conditions: Healthy Subjects
Keywords: Transcutaneous oximetry
Sponsor: Diffusion Pharmaceuticals Inc
Start date: 2021-03-17; Completion date: 2021-03-25
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Randomized, double-blind, placebo-controlled, pharmacokinetic, pharmacodynamic study.

Subjects will be randomized to TSC or placebo to determine the effect of Trans Sodium Crocetinate (TSC) on Transcutaneous Oximetry Measurements (tcpO2) following a single administration of TSC in subjects breathing oxygen (O2).

Detailed description:
This is a randomized, double-blind, placebo-controlled, pharmacokinetic, pharmacodynamic study of Trans Sodium Crocetinate (TSC) in healthy volunteers. The primary objective is to determine the effect of TSC on transcutaneous oximetry measurements (tcpO2) following a single administration of TSC in subjects breathing oxygen (O2).

30 healthy volunteers will be randomized to one of 6 treatment groups, to include 5 TSC doses (0.5, 1.0, 1.5, 2.0, and 2.5 mg/kg) and placebo normal saline (7 mL). TcpO2 sensors will be applied to 4 standardized areas on the lower extremity. Subjects will be placed on O2 via simple face mask at 6 L/minute, and will remain on O2 for 70 minutes prior to study drug administration. The first 10 minutes will allow for equilibration of O2 levels, and the subsequent 60 minutes will serve as the baseline period. TcpO2 values and SpO2 will be recorded every 5 minutes during the above periods. At the end of the 70-minute equilibration/baseline period, subjects will receive a single IV bolus injection of TSC at a dose of 0.5, 1.0, 1.5, 2.0 or 2.5 mg/kg, or placebo. Study drug will be administered in a blinded fashion.

After study drug is administered, subjects will continue on O2 and be evaluated for an additional 60 minutes, with tcpO2 and SpO2 recorded at 1, 2, and 5 minutes post-dose, and every 5 minutes thereafter.

Prior to and following study drug administration, PK samples will be obtained pre-dose, and 1, 10, 30, and 90 minutes post-dose.

After the 60 minute post-treatment evaluation period, oxygen will be discontinued and the tcpO2 sensor electrodes removed. Subjects will remain in the procedure room for an additional 30 minutes to allow for collection of the 1.5-hour PK blood draw and repeat vital signs. Subjects will remain in the clinic overnight for observation and discharged the following morning.

Subjects will be contacted by telephone at 48 hours (+ 2 days) for a safety follow up to assess adverse events and new medications.

Arms: arm 1: TSC 0.5 mg/kg given as a one-time IV bolus injection arm 2: TSC 1.0 mg/kg given as a one-time IV bolus injection arm 3: TSC 1.5 mg/kg given as a one-time IV bolus injection arm 4: TSC 2.0 mg/kg given as a one-time IV bolus injection arm 5: TSC 2.5 mg/kg given as a one-time IV bolus injection arm 6: 7 mL normal saline given as a one-time IV bolus injection
Interventions: intervention 1: Trans-Sodium Crocetinate intervention 2: Placebo
Intervention descriptions: intervention 1: TSC given as a one-time IV bolus injection intervention 2: Placebo normal saline given as a one-time 7 mL one-time IV bolus injection

Protocol documents:
Prot_000.pdf: 
 
 
 
Title: 
Randomized, double-blind, placebo-controlled, pharmacokinetic, pharmacodynamic 
study of Trans Sodium Crocetinate utilizing Transcutaneous Oximetry Measurement in 
healthy volunteers 
 
NCT Number: 
NCT04808622 
 
Document Type: 
Protocol 
 
Document Date: 
09 March 2021 


Diffusion Pharmaceuticals Inc 
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Protocol 200-301 March 9, 2021 
 
Page 1 of 52 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confidential – Property of Diffusion Pharmaceuticals Inc. 
This document or its contents may not be used, divulged, published, or disclosed without consent of Diffusion 
Pharmaceuticals Inc. 
 
 
 
 
 
 
 
 
DOCUMENT TYPE: 
Clinical protocol  
DOCUMENT NUMBER: 
200-301 
COMPOUND: 
Trans Sodium Crocetinate 
STUDY TITLE: 
Randomized, double-blind, placebo-controlled, 
pharmacokinetic, pharmacodynamic study of Trans 
Sodium Crocetinate utilizing Transcutaneous Oximetry 
Measurement in healthy volunteers 
CLINICAL PHASE: 
1 
INDICATION: 
Healthy volunteers 
IND NUMBER: 
68579 
SPONSOR: 
Diffusion Pharmaceuticals Inc. 
1317 Carlton Avenue, Suite 200 
Charlottesville, VA 22902 
DOCUMENT 
VERSION/STATUS: 
 
 Final 
DOCUMENT 
RELEASE/AMENDMENT 
DATE: 
Amendment 2: March 9, 2021 
Amendment 1: February 12, 2021 
January 14, 2021 




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Table of Contents 
PROTOCOL SYNOPSIS ............................................................................................................... 8 
1 
INTRODUCTION ................................................................................................................ 14 
1.1 
Study Rationale .............................................................................................................. 14 
1.1.1 
TSC Background ..................................................................................................... 16 
1.2 
TSC as a Therapeutic ..................................................................................................... 16 
1.3 
Preclinical Experience .................................................................................................... 17 
1.3.1 
Animal Pharmacokinetics ....................................................................................... 17 
1.3.2 
Safety Pharmacology .............................................................................................. 18 
1.3.3 
Animal Toxicology ................................................................................................. 18 
1.3.4 
Hemocompatibility ................................................................................................. 19 
1.3.5 
Intravenous Irritation .............................................................................................. 19 
1.3.6 
Genotoxicity ............................................................................................................ 19 
1.3.7 
Reproductive Toxicology........................................................................................ 20 
1.3.8 
Absorption, Distribution, Metabolism, and Excretion ............................................ 20 
1.4 
TSC in Model of Hemorrhagic Shock ........................................................................... 23 
1.5 
Experience in Humans ................................................................................................... 26 
2 
STUDY METHODS ............................................................................................................. 30 
2.1 
Study Overview .............................................................................................................. 30 
2.2 
Study Objectives ............................................................................................................ 30 
2.2.1 
Primary Endpoint .................................................................................................... 30 
2.2.2 
Safety Endpoints ..................................................................................................... 30 
2.3 
Study Population ............................................................................................................ 30 
2.3.1 
Inclusion Criteria .................................................................................................... 30 
2.3.2 
Exclusion Criteria ................................................................................................... 31 
2.4 
Enrollment and Consent ................................................................................................. 31 
2.5 
Withdrawal of Subjects .................................................................................................. 31 
2.5.1 
Criteria for Early Withdrawal ................................................................................. 31 
2.5.2 
Screening Failures ................................................................................................... 32 
2.5.3 
Subjects Lost to Follow Up..................................................................................... 32 
2.5.4 
Replacement of Subjects ......................................................................................... 32 
2.5.5 
Study Stopping Rules .............................................................................................. 32 
2.6 
Study Costs ..................................................................................................................... 32 
2.7 
Study Drug ..................................................................................................................... 32 


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2.7.1 
TSC Packaging........................................................................................................ 32 
2.7.2 
Reconstitution of TSC............................................................................................. 33 
2.7.3 
Placebo .................................................................................................................... 33 
2.7.4 
Randomization ........................................................................................................ 33 
2.7.5 
Blinding Protocol .................................................................................................... 33 
2.7.6 
Dosing Regimen...................................................................................................... 34 
2.7.7 
Drug Accountability................................................................................................ 34 
2.8 
Concomitant Medications .............................................................................................. 34 
2.9 
Schedule of Events ......................................................................................................... 35 
2.9.1 
Screening Evaluation (Day -21 to Day 0) ............................................................... 35 
2.9.2 
Treatment Visit (Day 0) .......................................................................................... 35 
2.9.3 
48 Hour Follow Up Telephone Visit ...................................................................... 37 
2.10 
Laboratory and PK Assessment Parameters ............................................................... 37 
2.10.1 Laboratory ............................................................................................................... 37 
2.10.2 PK Procedures ......................................................................................................... 37 
3 
ADVERSE EVENT REPORTING....................................................................................... 38 
3.1 
Definitions ...................................................................................................................... 38 
3.1.1 
Adverse Event ......................................................................................................... 38 
3.1.2 
Suspected Adverse Reaction ................................................................................... 39 
3.1.3 
Serious Adverse Event ............................................................................................ 39 
3.1.4 
Unexpected ............................................................................................................. 39 
3.1.5 
Unanticipated Problem (UP) ................................................................................... 39 
3.2 
Adverse Events Previously Reported with TSC ............................................................ 40 
3.3 
Adverse Event Reporting Requirements ........................................................................ 40 
3.3.1 
Expedited Reporting Requirements for Unanticipated Problem SAEs .................. 40 
3.4 
Research Related Injuries ............................................................................................... 41 
3.5 
Contraception and Pregnancy......................................................................................... 41 
4 
DATAMANAGEMENT AND QUALITY ASSURANCE ................................................. 42 
4.1.1 
Electronic Case Report Form .................................................................................. 42 
4.1.2 
Clinical Monitoring ................................................................................................. 42 
4.1.3 
Source Documentation ............................................................................................ 43 
5 
STATISTICAL METHODS ................................................................................................. 43 
5.1 
General Considerations .................................................................................................. 43 
5.2 
Analysis Populations ...................................................................................................... 43 


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5.3 
Statistical Hypotheses .................................................................................................... 43 
5.4 
Sample Size .................................................................................................................... 44 
5.5 
Missing Data .................................................................................................................. 44 
5.6 
Primary and Secondary Efficacy Analyses .................................................................... 44 
5.7 
Safety Analyses .............................................................................................................. 45 
6 
ADMINISTRATIVE AND SPECIAL PROCEDURES ...................................................... 45 
6.1 
Protocol Amendments .................................................................................................... 45 
6.2 
Investigator Responsibilities .......................................................................................... 45 
6.2.1 
Compliance with Protocol....................................................................................... 45 
6.2.2 
Good Clinical Practice (GCP) ................................................................................. 46 
6.2.3 
Institutional Review Board Review and Approval ................................................. 46 
6.2.4 
Informed Consent.................................................................................................... 46 
6.3 
Protocol Deviations ........................................................................................................ 46 
6.4 
Monitoring Activities ..................................................................................................... 46 
6.5 
Quality Assurance Audits and Regulatory Inspections .................................................. 47 
6.6 
Disclosure and Confidentiality ....................................................................................... 47 
6.7 
Changes in Study Personnel ........................................................................................... 47 
6.8 
Study Record Retention ................................................................................................. 47 
6.9 
Publication and Data Sharing Policy .............................................................................. 47 
7 
REFERENCES ..................................................................................................................... 48 
8 
APPENDICES ...................................................................................................................... 50 
8.1 
Laboratory Parameters ................................................................................................... 50 
8.2 
Schedule of Events ......................................................................................................... 51 
8.3 
Investigator Signature Page ............................................................................................ 52 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Listing of Tables  
 
Table 1.  Rat and Dog PK Half-Life and Clearance of TSC Dosed Once Daily for 14 Days and 
90 Days ........................................................................................................................ 17 
Table 2.  Mean Pharmacokinetic Parameters Correlated with Sample Radioactivity in Whole 
Blood and Plasma ......................................................................................................... 21 
Table 3.  Recovered Radioactivity through 168-hour Post-dose ............................................ 22 
Table 4.  Blood Parameters of Systemic Hypoxia Following Hemorrhagic Shock – Baseline, 
Post-Hemorrhage, and Following TSC vs. Saline Injection ................................... 24 
 
Listing of Figures 
 
Figure 1.  Trans Sodium Crocetinate ............................................................................................ 16 
Figure 2.  Effect of TSC On Blood Pressure After Hemorrhage When Therapy Is Delayed 20 
Minutes ......................................................................................................................... 24 
Figure 3.  Effect of TSC on Heart Rate After Hemorrhage When Therapy Is Delayed 20 Minutes
 ...................................................................................................................................... 24 
Figure 4.  Liver Transaminase Activity At Baseline and 24-Hours Post-Hemorrhagic Shock in 
TSC vs. Saline Treated Groups [A – aspartate aminotransferase (AST), B – alanine 
aminotransferase (ALT)] .............................................................................................. 25 
Figure 5.  Survival with TSC vs. Saline Following Second Hemorrhage .................................... 26 
 
 
 
LIST OF ABBREVIATIONS AND DEFINITIONS 
 
Abbreviation 
Definition 
ADME 
Absorption, distribution, metabolism and excretion 
AE 
Adverse event 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase (AST) 
AUC 
Area under the curve 
BMI 
Body mass index 
BMP 
Basic metabolic panel 
BZK 
Benzalkonium Chloride 
CBC 
Complete blood count 
CMP 
Clinical monitoring plan 
COT 
Claudication onset time 
CRA 
Clinical Research Associates 
CRF 
Case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DLT 
Dose Limiting Toxicities 
DSN 
Data Safety Navigator 
DVT 
Deep vein thrombosis 
EDC 
Electronic data capture 
EGC 
Electrocardiogram 
FDA 
Food and Drug Administration 


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Abbreviation 
Definition 
GCP 
Good Clinical Practice 
GBM 
Glioblastoma multiforme 
GOT 
Glutamate-oxaloacetic transaminase 
GPT 
Glutamate-pyruvate transaminase 
HBOT 
Hyperbaric oxygen therapy 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committees 
IRB 
Institutional Review Boards 
ITT 
Intention-to-Treat 
KPS 
Karnofsky Performance Score 
LAR 
Legally Authorized Representative 
LOQ 
Limit of quantitation 
mRNA 
Messenger ribonucleic acid 
NOAEL 
No observable adverse effect level 
NOEL 
No observable effect level 
O2 
Oxygen 
ONR 
Office of Naval Research 
OS 
Overall survival 
OTC 
Over the counter 
PAD 
Peripheral Artery Disease 
PFS 
Progression-free survival 
PH 
Proportional hazards 
PHI 
Protected Health Information 
PI 
Principal investigators 
PK 
Pharmacokinetic 
PCO2 
Partial pressure carbon dioxide 
PO2 
Partial pressure oxygen 
POC 
Point of care 
PWT 
Peak walking time 
QOL 
Quality of life 
RA 
Room air 
RT 
Radiation therapy 
SAE 
Serious adverse events 
SMC 
Safety Monitoring Committee 
SOC 
Standard of care 
SpO2 
Oxygen saturation 
TCOM 
Transcutaneous oximetry 
tcpO2 
Transcutaneous tissue oxygen tension 
TEAE 
Treatment emergent adverse event 
TSC 
Trans Sodium Crocetinate 
 
 
 
 


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PROTOCOL SYNOPSIS 
 
 
 
Protocol 
Number 
200-301 
Title of Study 
Randomized, double-blind, placebo-controlled, pharmacokinetic, 
pharmacodynamic study of Trans Sodium Crocetinate utilizing 
Transcutaneous Oximetry Measurement in healthy volunteers 
Study Phase 
1 
Study 
Objective 
Determine the dose response of Trans Sodium Crocetinate (TSC) on 
Transcutaneous Oximetry Measurements (tcpO2) following a single 
administration of TSC in subjects breathing  oxygen (O2) 
Study 
Population 
Healthy volunteers aged 18-50 
Number of 
Subjects 
Maximum of 30  
Participant 
Duration 
Maximum of 26 days, inclusive of screening, treatment and follow-up  
Study Centers 
1 
Eligibility 
Inclusion Criteria 
1. Healthy male or female, age 18-50 
2. Able and willing to lie quietly supine or semi-recumbent for up 
to 2.5 hours 
3. Abstinence from exercise, caffeine, alcohol, nicotine, and a 
heavy meal prior to testing on the day of the treatment visit 
4. Subject is able to communicate effectively with the Investigator 
and to comply with all study requirements, restrictions, and 
directions from the study staff 
5. Females of childbearing potential must have a negative blood 
pregnancy test at screening and agree to use one of the accepted 
contraceptive regimens, or a double method of birth control (e.g. 
condom and spermicide), during the study and at least 30 days 
after the last dose of study drug. Females of non-childbearing 
potential should be surgically sterile or at least one year post-
menopausal. 
6. Males who engage in sexual activity that has the risk of 
pregnancy must agree to use a double barrier method (e.g. 
condom and spermicide) and agree not to donate sperm during 
the study and for at least 90 days after the last dose of study drug 


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Exclusion Criteria 
1. Allergy to study medication 
2. Pregnant or breastfeeding 
3. Current smoker and/or any nicotine use within 4 hours of the 
start of tcpO2 procedures, to include e-cigarette vaping, snuff, 
chew, nicotine gum and nicotine patches 
4. Body Mass Index (BMI) > 30 
5. Positive test results for HIV-1/HIV-2 Antibodies (HIV), 
Hepatitis B surface Antigen (HBsAg), or Hepatitis C Antibody 
(HCVAb) 
6.  Blood donation (excluding plasma donation) of approximately 
500 mL within 56 days prior to screening  
7. Plasma donation within 7 days prior to screening 
8. Treatment with an investigational drug within 30 days or 5 times 
the half-life (whichever is longer) prior to screening 
9. Any skin condition on limbs to be tested that could impair 
testing (rash, wound, prior radiation therapy, other skin 
conditions, per Principal Investigator (PI) discretion) 
10. Known cardiovascular disease, including treated or untreated 
hypertension 
11. Significant respiratory disease and/or any other significant 
medical condition  
12. Subject has an acute illness (gastrointestinal, influenza, or 
known inflammatory process) at the Treatment Visit 
13. Urine screen positive for drugs or positive breathalyzer for 
alcohol (at screening and enrollment) 
14. Concomitant medications used to treat a diagnosed medical 
condition 
15. Subject who, for any reason, is deemed by the Investigator to be 
unsuitable for the study; or has any condition that would 
interfere with the evaluation of tissue oxygen measurements or 
PK of the investigational drug; or is otherwise unable to comply 
with the protocol 
Safety 
Assessment of adverse events, new medications, laboratory (complete 
blood count [CBC], basic metabolic panel [BMP] HIV, HBsAg, 
HCVAb), vital signs (blood pressure, heart rate, respiratory rate, 
temperature), and oxygen saturation (SpO2)   
Study Design 
Randomized, double-blind, placebo-controlled, pharmacokinetic, 
pharmacodynamic study 
 


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Subjects will be randomized to a single IV bolus dose of TSC (0.5, 
1.0, 1.5, 2.0, or 2.5 mg/kg) or placebo (normal saline).  
Study 
Overview 
A maximum of 30 healthy volunteers will be recruited to participate 
in this study. Following written informed consent, screening 
assessments will be obtained, including physical exam, laboratory 
(CBC, BMP, HIV, HBsAg, HCVAb), urine drug screen, alcohol 
screen, blood pregnancy test for all females, and vital signs. 
 
Following the screening visit, the subject will return to the clinic for 
study treatment procedures. If the site has Point of Care (POC) 
laboratory testing available, the screening and treatment procedures may 
occur on the same day. Subjects will refrain from exercise, caffeine, 
alcohol, nicotine, and a heavy meal prior to testing on the day of the 
treatment visit. 
 
Subjects will be randomized into one of 6 groups in a 1:1:1:1:1:1 
schema, to include the 5 TSC doses and one placebo arm. Each TSC 
dose will be calculated based on the subject’s body weight in kg at the 
dosing level they are randomized to. Each individual dose of placebo 
will be normal saline 7 mL. 
 
To maintain the double-blind, study drug administration will be 
performed by unblinded medical staff who will not be involved in other 
study procedures, including subject assessment.  Subjects, investigators 
and study coordinators will not see the injection or injection site or be 
aware of randomization. 
 
On the day of treatment, subjects will be maintained in a temperature-
controlled room (between 22.0 and 25.0˚C), and in a supine position 
with the head slightly raised on one pillow, or semi-recumbent. One 
blanket will be provided for comfort. One or two IV catheters will be 
placed for study drug administration and PK measurement. Direct 
venipuncture will also be allowed for PK draws, if needed. 
 
TcpO2 sensor electrodes will be applied to the left or right lower 
extremity, per PI discretion.  Sensor electrode temperature will be preset 
to 45˚C, to allow maximum vasodilation. Risk of sensor site superficial 
burn is minimal given the relatively brief testing period. Four (4) 
sensors will be applied to the following locations:  
 
• Sensor 1: Mid-dorsum of the foot  
• Sensor 2: 10 cm distal to the lateral femoral epicondyle  
• Sensor 3: 5 cm proximal to the anterior aspect of the lateral 
malleolus  


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• Sensor 4: 5 cm proximal from the center of the medial 
malleolus  
 
After the tcpO2 sensors have been applied and tested, subjects will be 
placed on O2 via simple face mask  at 6 L/minute, and  will remain 
on O2 for 70 minutes prior to study drug administration. The first 10 
minutes will allow for equilibration of O2 levels, and the subsequent 
60 minutes will serve as the baseline period. TcpO2 values and SpO2 
will be recorded every 5 minutes during the above periods. At the 
end of the 70-minute equilibration/baseline period, subjects will 
receive a single IV bolus injection of TSC at a dose of 0.5, 1.0, 1.5, 
2.0 or 2.5 mg/kg, or placebo.  
 
After study drug has been administered, subjects will continue on O2 
and be evaluated for an additional 60 minutes , with tcpO2 values 
and SpO2 recorded at 1, 2, and  5 minutes post-dose, and every 5 
minutes thereafter.   
 
In addition to assessment of tcpO2 as described, continuous tcpO2 
measurements will be provided by the TCOM machine in graphical 
format. 
 
Vital signs will be assessed prior to study drug dosing, and at 10, 30, 
and 60 minutes post-dosing. Adverse events will be assessed from the 
start of the equilibration period.  
 
Prior to and following study drug administration,  PK samples will be 
obtained at the below intervals. 
 
Pre-dose (within 10 minutes prior to injection) 
1 minute post end of injection (+1 min) 
10 minutes post end of injection (± 1 min) 
30 minutes post end of injection (± 1 min) 
1.5 hours post end of injection (± 2 min) 
 
After the 60 minute post-treatment evaluation period,  oxygen will be 
discontinued and the tcpO2 sensor electrodes removed. Subjects will 
remain in the procedure room for an additional 30 minutes to allow for 
collection of the 1.5-hour PK blood draw and repeat vital signs.  
Subjects will remain in the clinic overnight for observation. 
 
Subjects will be contacted by telephone at 48 hours (+ 2 days) for a 
safety follow up to assess adverse events and new medications. 


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Statistical 
Analysis Plan 
Subjects will be randomized using a non-stratified permuted block 
randomization scheme.  Subjects who fail to complete the study will not 
be replaced.  There will be a single analysis conducted at the end of the 
study; no interim assessments or analyses are planned for this fixed 
design.  All analyses will be based on the observed data; no data will be 
imputed for the statistical analyses.  The trapezoidal rule will be used for 
the construct of the area under the curve (AUC) analyses.  All probability 
values will be 2-sided and accepted at face value for this feasibility study; 
there will not be an adjustment for multiplicity.  For interpretation, 
probability values <0.05 will be considered significant; probability values 
<0.1 but ≥0.05 will be considered highly suggestive of a significant 
difference.  
 
The sample size selected for this clinical investigation with 6 treatment 
groups is based on clinical judgement.  While hypotheses will be 
evaluated at the end of the study, there is no pre-specified statistical 
power that was used to establish the sample size. 
 
The primary analysis will be based on the change over time in the tcpO2  
measurements following a single administration of TSC in subjects 
breathing  O2.  The time-matched changes from Period 1 (60 minute run-
in on  O2) to Period 2 (60 minute post-drug administration on  O2) will 
serve as the dependent variable in the repeated measures analysis.   The 
60 minute run-in on  O2 is intended to account for the intra-subject 
variability over time and the time-match differences represent the least 
biased estimate of the effect of the study drug.  With 5 subjects per 
randomized treatment arm, the change over time should provide 
sufficient clinical information to determine if there is a drug effect, and 
if the effect in an active drug treatment arm is different than placebo. 
   
To compare the time-match changes in tcpO2, a 2-factor (treatment and 
time) repeated measures (time) analysis of variance model will be used.  
Contrast statements within the model will facilitate comparisons between 
individual treatment arms over individual time points, providing 
guidance relative to the initial time and duration of separation. 
 
To compare the AUC based on the time-matched differences, a 1-factor 
(treatment) analysis of variance model will be used, followed by a 
Dunnett’s test to compare the least square mean AUC values from the 5 
active drug groups relative to the placebo group.  If the data within a 
treatment arm is skewed, suggesting that the least square mean value is 
not the best measure of central tendancy, the data may be ranked and the 
analyses will be conducted using the ranked scores.  Additional 
information regarding the examination of the distribution of the data will 
be presented in the Statistical Analysis Plan.  To compare the time to the 


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maximum increase in tcpO2, the event time distribution functions will be 
compared using a log rank test and presented using Kaplan-Meier curves. 
   
Summaries will be prepared for all parameters using descriptive statistics.  
For variables recored on a continous scale, results will be summarized by 
randomized treatment assignment and time. For categorical variables 
recorded on a multimonial or binomial scale, results will be presented 
using counts and percentages.  All recorded data will be presented in the 
data listings. 
 
Adverse events will be coded using MedDRA and summarized by system 
organ class and preferred term and presented by randomized treatment 
assignment, Period (1 or 2), and overall, independent of Period.         
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1 
INTRODUCTION 
1.1 
Study Rationale 
Hypoxia is a condition in which local, regional, or generalized oxygen delivery to tissues is 
either inadequate or disrupted.  If not reversed or corrected, tissue hypoxia can rapidly lead to 
tissue damage and cell death, which is irreversible. The global delivery of oxygen to tissues is 
dependent on both convection and diffusion. Convective delivery of oxygen is primarily driven 
by cardiac output and arterial oxygen content, while the diffusive component at the 
microcirculatory level (arterioles, capillaries, venules) is dependent on Fick’s Law of Diffusion.  
Stated simply, diffusing particles move from an area of high concentration to an area of low 
concentration when a gradient is present. This rate of diffusion is dependent not only on how 
large the concentration differences are across a membrane (the gradient), but also physical and 
chemical characteristics that can impede movement of particles. 
 
Transcutaneous Oximetry (tcpO2) is a well characterized and a non-invasive method of 
measuring the partial pressure of oxygen (oxygen tension) under the skin.  This is very different 
from pulse oximetry which uses infrared technology to measure hemoglobin saturation in the 
blood (not in the tissues). TSC’s unique mechanism of action is to enhance diffusion of oxygen, 
and tcpO2 methodology uniquely measures the amount of oxygen in the tissues that has diffused 
from the microcirculation.  tcpO2 is used extensively in cardiovascular patients being evaluated 
for revascularization for limb salvage or mapping for amputation.  tcpO2 also is used to 
determine if Hyperbaric Oxygen Therapy (HBOT) may help a patient’s particular condition. 
 
This clinical trial is designed to further clarify and optimize the dosing of TSC by directly 
measuring tcpO2 in multiple locations on the lower extremity of a healthy volunteer after a single 
dose of TSC, while breathing  O2.  
 
The rationale for enrolling healthy volunteers versus patients in the phase 1 trial is the 
components of oxygen delivery will be highly variable both intra- and inter-subject in patients to 
a much higher degree than healthy volunteers.  Patients already with some evidence of end organ 
disease (such as PAD mentioned in response) will have varying cardiac outputs, hemoglobin 
content, baseline oxygen saturation, and probable pulmonary function variability.  All of these 
physiologic variables will confound the ability to detect a specific signal we hope to confidently 
attribute to TSC in a small cohort of subjects that is appropriately powered for the intra- and 
inter-subject variances.  Additionally, the technicalities of TCOM measurements necessitates a 
person be quite motionless for accurate readings and any exertional test is not feasible utilizing 
this modality.  We feel a healthy volunteer population is a best model to reduce the confounders 
in oxygen delivery as in this early phase trial.   
 
The rationale to investigate TSC while on oxygen versus room air is based on two pre-clinical 
studies: 
• “Trans sodium crocetinate increases oxygen delivery to brain parenchyma in rats 
on oxygen supplementation.”  Okonkwo et al, Neuroscience Letters 352 (2003) 
97-100. 


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o Licox brain tissue probes placed within rats demonstrated enhanced 
oxygen delivery within 30 minutes after TSC was given versus saline 
placebo while ventilated with varying oxygen concentrations (21%, 60%, 
100%).  100% oxygen demonstrated the greatest change in brain 
parenchyma oxygenation versus the lower concentrations. 
• “Effect of trans sodium crocetinate on brain tumor oxygenation.”  Journal of 
Neurosurgery 111:226-229, 2009. 
o C6 glioma model rats with Licox probes inserted into the tumor and 
contralateral healthy cerebral tissue demonstrated improved oxygenation 
in the tumor tissue within 30 minutes after TSC administration versus 
saline placebo while the rats were sedated and breathing room air.  
 
Experience with TSC demonstrates there is a necessary diffusion gradient to demonstrate TSC’s 
efficacy and pre-clinical models also demonstrate that the maximal effect is likely when 
breathing 100% oxygen versus room air, but effects also potentially can be seen at lower 
concentrations. 
 
Oxygen is vital to sustain life and can be highly beneficial in certain illnesses. Too much oxygen 
can have strong unintended effects as well, and even cause oxygen toxicity.   
 
Oxygen toxicity is most likely to occur in high risk groups: 
o High concentrations (100% oxygen) for long duration (>24hrs) 
o Mechanical ventilation (ventilator) for long duration and high oxygen concentrations 
(>50%) 
o Hyperbaric oxygen therapy (patients in high pressure chambers and placed on 
supplemental oxygen) 
o Underwater divers  
 
Symptoms of Oxygen toxicity 
o Pulmonary – airway irritation, coughing, congestion, inflammation, shortness of 
breath 
o CNS (central nervous system) – headache, irritability, anxiety, dizziness, facial 
muscle twitching, ringing in ears, tingling in extremities, seizure 
 
In this study, volunteers will be place on low flow oxygen at 6 L/min via simple face mask for 
approximately 2 hours in total time and have clinical and research personnel at the bedside all 
times during oxygenation and monitoring phase.   
 
Each volunteer will be actively monitored by multiple clinical and research personnel at all times 
while in the clinical research unit, particularly with multiple clinical and research personnel 
present at all times at the bedside during the oxygenation, dosing, and TCOM monitoring period.  
After the oxygenation, dosing, and monitoring period, the volunteers will stay in the clinical 
research unit overnight for further safety and oversight monitoring before being discharged the 
next day per the principal investigator’s discretion. 
 


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1.1.1 TSC Background 
Diffusion Pharmaceuticals Inc. has developed a novel compound, trans sodium crocetinate 
(TSC), indicated as a drug to treat diffusion-limited hypoxia by selectively enhancing the re-
oxygenation of hypoxic tissues.  The ability of TSC to increase the diffusion (movement) of 
oxygen is the underlying mechanism by which TSC exerts its pharmacodynamic effects.  In vitro 
studies demonstrate that TSC increases the diffusion of oxygen by altering the molecular 
arrangement of the water molecules which constitute the bulk of blood plasma.  This effect 
creates a more ordered water structure which is less dense, reducing resistance to oxygen 
diffusion and allowing a more rapid movement of oxygen through the plasma (Laidig et al., 
1998; Stennett et al., 2006).  TSC exerts its pharmacodynamic effects via the ability to increase 
the diffusion of oxygen through blood plasma to hypoxic tissues.  Thus, it is based on physical-
chemical principles, unlike most drugs which are based on biochemistry based mechanisms. 
 
TSC was first made for a project between the University of Virginia and the Division of 
Battlefield Casualties of the United States Office of Naval Research (ONR). Blood loss is 
responsible for a significant proportion of battlefield casualties, and ONR supported the 
development of TSC to the limit of their capabilities, which included considerable preclinical 
studies plus a Phase 1 clinical trial for safety and dosing in TSC-treated normal subjects. At this 
point, Diffusion Pharmaceuticals took over the manufacture of TSC as well as the oversight of 
Phase 2 clinical trials, including the treatment of Peripheral Artery Disease (PAD) and as an 
adjunct to radiation therapy for glioblastoma multiforme (GBM).   
1.2 
TSC as a Therapeutic 
TSC (Disodium 2,6,11,15 Tetramethyl-hexadeca 2E,4E,6E,8E,10E,12E,14E-heptaene-1,16-
diotate) is a bipolar synthetic carotenoid having the chemical formula Na2C20H24O4, and a 
molecular weight of 372.  The Chemical Abstracts registry number is 64603-92-5. The chemical 
structure is shown in Figure 1.  Carotenoids are a diverse group of highly colored nonpolar, 
monopolar, and bipolar polyenes (conjugated double-bonded carbon chain backbone).  Crystals 
and concentrated solutions of TSC are orange-red, but in dilute solution, TSC is yellow.   
 
 
 
 
Figure 1.  Trans Sodium Crocetinate 
 
The carbon chain structure of TSC is identical to the naturally occurring dicarboxylic acid 
carotenoid, crocetin.  However, crocetin and TSC differ in that crocetin is comprised of a 
mixture of isomers.  TSC is a synthetically produced sodium salt of the single trans isomer.  TSC 
has been the subject of investigation in 5 clinical trials to date (Diffusion Protocols 100-001, 
100-201, 100-202, 100-301, 100-206).  
 
Based on in vitro and computer modeling studies, it has been proposed that TSC works by 
altering the molecular arrangement of the water molecules which constitute the bulk of blood 
plasma, thereby creating a more ordered water structure (Laidig et al., 1998; Stennett et al., 
2006).  This altered water structure is less dense, reducing resistance to oxygen diffusion and 


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allowing a more rapid movement of oxygen through the plasma.  More oxygen can thereby reach 
oxygen-deprived tissue (Giassi et al., 2003).  In vitro studies have shown that TSC also increases 
the diffusion of glucose in a manner similar to oxygen (Stennett et al., 2006).   
 
TSC administered intravenously does not appear to significantly affect oxygen levels in normal 
animals, but TSC increases oxygen levels in hypoxic animal models.  Healthy animals breathing 
normal ventilated air show little effect on brain PO2 after TSC administration, suggesting that 
TSC does not significantly affect oxygen tissue levels in normal animals (Okonkwo et al., 2003).   
When TSC is administered to animals ventilated with 100% oxygen, the animals show a 
significant increase in brain PO2 levels over a saline control.  It is hypothesized that a sufficient 
oxygen concentration gradient must be established in order to significantly increase the diffusion 
of oxygen with TSC.  This gradient appears to be present if the tissue is hypoxic, however, it can 
also be produced with hyper-oxygenation (such as 100% O2).  Such a gradient is not present 
under normal conditions.   
1.3 
Preclinical Experience 
1.3.1 Animal Pharmacokinetics 
Pharmacokinetic (PK) studies of TSC have been conducted in rats and dogs.  TSC has a 
relatively short half-life after a single intravenous injection.  The half-life of TSC in the rat after 
a single IV bolus injection of 5, 15, 45, or 100 mg/kg varied from 20 minutes to 2 hours.  
Clearance varied from 50 to 300 mL/hr/kg.  These data are shown below in Table 1 for both the 
low and high doses used in both 14- and 90-day studies.  In addition, the volume of distribution 
varied from 60 to 140 mL/kg. 
 
Table 1.  Rat and Dog PK Half-Life and Clearance of TSC Dosed Once Daily for 14 Days 
and 90 Days 
 
 
Half-Life 
Clearance 
 
Species 
Low Dose 
High Dose 
Low Dose 
High Dose 
 
 
 
 
 
 
Rat 14 Day 
5 mg/kg/day 
100 mg/kg/day 
5 mg/kg/day 
100 mg/kg/day 
 
~0.4 hr 
~2 hr 
300 ml/hr/kg 
70 ml/hr/kg 
Rat 90 Day 
5 mg/kg/day 
45 mg/kg/day 
5 mg/kg/day 
45 mg/kg/day 
 
~0.4 
~1.4 
233 ml/hr/kg 
59 ml/hr/kg 
 
 
 
 
 
Dog 14 Day 
2.6 mg/kg/day 
50 mg/kg/day 
2.6 mg/kg/day 
50 mg/kg/day 
 
~2 hr 
4 hr 
70 ml/hr/kg 
40 ml/hr/kg 
Dog 90 Day 
2.6 mg/kg/day 
50 mg/kg/day 
2.6 mg/kg/day 
50 mg/kg/day 
 
8 hr* 
6 hr 
115 ml/hr/kg 
52 ml/hr/kg 
*Half-lives for male dogs were 4, 1.91, 0.924 and 48.4 hours, and 4, 1.65, 0.0389 and 2.35 hours for 
female dogs. 
 


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The half-life of TSC in dogs after a single IV bolus injection of 2.6, 5, 25, or 50 mg/kg varied 
from 2 to 6 hours.  The half-life of 8 hr for the low-dose in the 90-day study is an average of 
values (listed below the table) that included an apparent erroneous value of 48.4 hours.  
Clearance varied from 40 to 115 mL/hr/kg.  Again, these data are included in Table 1.  In 
addition, the volume of distribution varied from 160 to 270 mL/kg.   
The PK parameters in rats and dogs in the 14-day and 90-day repeat-dose toxicology studies did 
not vary significantly after daily injections of TSC compared to a single dose of TSC in rats and 
dogs.  The pharmacokinetics in rats and dogs appears to be non-linear with greater than dose 
proportional increase for both Cmax and AUC.  The half-life increases with dose in rats and 
dogs.  The volume of distribution remained reasonably similar from low to high dose in rats and 
dogs, and did not exceed the total body weight for either species, indicating that TSC was not 
highly distributed into the tissues of rats and dogs after intravenous dosing. 
1.3.2 Safety Pharmacology 
Cardiovascular safety was tested in dogs.  Central nervous system and pulmonary safety studies 
were conducted in rats.  Single doses of 0.1, 1, or 10 mg/kg were tested in all safety 
pharmacology studies.  No effects were observed in the cardiovascular studies when assessed by 
body temperature, blood pressure, heart rate, and QRS, RR, PR, and QT intervals.  The no 
observable effect level (NOEL) was at least 10 mg/kg.  TSC did not have any adverse effects on 
pulmonary function as measured by respiratory rate, tidal volume, or minute volume.  
Neurobehavioral studies did not show any behavioral changes suggesting neurotoxicity.  The no 
observable adverse effect level (NOAEL) was at least 10 mg/kg in the pulmonary studies. 
1.3.3 Animal Toxicology 
Toxicology studies up to 14 days in one study and up to 90 days in another study were conducted 
in rats and dogs by the IV route of administration at maximum technically feasible doses based 
on limits of solubility and a clinically relevant dosing regimen.  In both the 14-day and 90-day 
rat studies, animals received a daily IV dose of TSC at levels of 5, 15, 45, or 100 mg/kg/day.  In 
the 14-day and 90-day dog studies, animals received a daily IV dose of TSC at levels of 2.6, 25, 
or 50 mg/kg/day. 
 
In the 14-day study, TSC did not result in any mortality.  In both species, TSC produced dose-
dependent yellow discoloration of urine, hair, skin, and eyes.  The discoloration generally 
disappeared after a 7-day recovery period indicating that the effects were reversible.  The 
discoloration was not considered adverse to the health of the animals and was considered related 
to the color of TSC, which is yellow at dilute concentrations.  Tissue discoloration appears to be 
a class effect of carotenoids because of their color in solution.  When given in sufficient doses to 
humans, carotenoids are known to color tissues and particularly contribute to a yellow cast of the 
skin (Alaluf et al., 2002; Micozzi et al., 1988).  
 
In the 90-day rat study, animals received a daily intravenous dose of TSC at 5, 15, or 45 
mg/kg/day.  There were no adverse effects noted following daily intravenous dosing of TSC for 
90 days in Sprague Dawley rats, so the NOAEL for this study is considered to be 45 mg/kg/day. 
The observations of yellow discolored hair and skin continued through the recovery period for 
both males and females at 45 mg/kg/day, with the incidence remaining constant for discolored 


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hair while the incidence of discolored skin reduced in incidence with time. The observations of 
discolored skin/hair are, again, attributed to the color of the test article, and since neither the 
discoloration nor sparse hair were associated with any impact on the health of the animals they 
were not considered adverse. 
 
In the 90-day dog study, animals received a daily intravenous dose of TSC of 2.6, 25 or 50 
mg/kg/day.  The NOAEL in dog was considered to be 25 mg/kg/day for the females due to the 
effects on body weight and euthanasia of 2 females at this level. The NOAEL is considered to be 
50 mg/kg/day for the males as there were no adverse effects noted for the males during the 
course of the study. Yellow discoloration of the body (minimal to severe) that occurred for males 
and females at ≥ 25 mg/kg/day was generally dose-related in incidence and severity and was 
attributed to the color of the test article. The yellow discoloration was not considered adverse, 
due to a general lack of microscopic correlates in most animals and an absence of clear toxicity 
associated with the color change.  
1.3.4 Hemocompatibility 
In vitro rat, dog, and human blood plasma and serum compatibility studies indicated that TSC 
was compatible with blood from each of these species since there was no evidence of 
coagulation or precipitation.  Hemocompatibility studies showed evidence of hemolysis in rat 
and dog blood, but not in human blood.  In animals, a small reduction (5 to 10%) in erythrocytes, 
hematocrit, and hemoglobin were found in the rat and dog 2-week studies in the highest dose 
groups tested; 100 mg/kg/day and 50 mg/kg/day, respectively in the rat and dog.  In addition, a 
46 to 56% reduction in reticulocytes was noted at 50 mg/kg/day in the dog study.  The increased 
bilirubin, when combined with an increase in urea nitrogen and the changes in the values of red 
cell variables described, may suggest a decreased red blood cell lifespan with impaired 
regeneration at the dose of 50 mg/kg/day in dogs.  The magnitude of effect on hematological 
variables was small and the findings were reversible.  
 
Consistent with the in vitro hemocompatibility results in human blood, there was no evidence of 
hemolysis or hemocompatibility issues in a Phase 1 study in 30 healthy human volunteers given 
a single IV bolus dose of TSC at a rate of 15 ml/min up to a maximum dose tested of 5 mg/kg.  
There were no laboratory reports of hemolysis or hemocompatibility issues in the Phase 1/2 
study in 40 PAD subjects who received multiple doses of 0.25 mg/kg to 2.0 mg/kg TSC with 
samples collected at multiple time points. 
1.3.5 Intravenous Irritation 
An IV irritation study was conducted in rabbits.  Using the marginal ear vein of rabbits, 
treatment with TSC resulted in very slight erythema in only 2 of 9 animals.  When administered 
perivascularly, TSC was considered to induce slight- to well-defined erythema and slight edema, 
with effects persisting for several days.   
1.3.6 Genotoxicity 
TSC was negative in the in vitro Ames assay and the L5178Y mouse lymphoma cells, in both 
studies in the presence and absence of metabolic activation. 


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1.3.7 Reproductive Toxicology 
The International Conference on Harmonisation (ICH) Stages C-D (Segment II) nonclinical 
reproductive toxicology studies in rats and rabbits using TSC as the formulated drug product 
administered intravenously to both species have been completed.  For the rat Segment II study, 
doses of 0 (0.9% saline), 25, 50, or 100 mg/kg/day TSC were administered intravenously in the 
tail vein.  The data demonstrated that the maternal NOAEL of TSC was 25 mg/kg/day.  The 50 
and 100 mg/kg/day TSC dosages caused reductions in body weight gain, and the 100 mg/kg/day 
TSC dosage also reduced food consumption.  Injection site reactions occurred at all dosages; the 
severity of these reactions at 50 and 100 mg/kg/day resulted in the early sacrifice of 2 and 1 rats 
at these dosages, respectively.  The developmental NOAEL was 50 mg/kg/day.  The 100 
mg/kg/day TSC dosage reduced fetal weight and increased the number of skeletal variations.  
Based on these data from the rat ICH Stages C-D (Segment II) reproductive toxicology study, 
TSC should not be identified as a developmental toxicant. 
 
In the rabbit Segment II study, doses of 0 (0.9% saline), 25, 50, or 75 mg/kg/day TSC using the 
formulated drug product were administered intravenously.  The maternal NOAEL of TSC was 50 
mg/kg/day.  The 75 mg/kg/day TSC dosage caused reductions in body weight gain and feed 
consumption.  The developmental NOAEL was greater than 75 mg/kg/day since no effects were 
observed at the highest dosage tested.  There were no adverse effects on embryo-fetal 
development as evaluated in this study.  Based on these data from the ICH Stages C-D (Segment 
II) study in rabbits, TSC should not be identified as a developmental toxicant.  
1.3.8 Absorption, Distribution, Metabolism, and Excretion 
The results from in vitro assessment of reaction phenotyping (enzyme identification) of human 
CYP450 enzymes by TSC indicate that TSC is not metabolized in large amounts by liver 
microsomal CYP450 enzymes.  Four in vitro metabolism studies within the full profile of 
absorption, distribution, metabolism and excretion (ADME) studies have been completed.  TSC 
has been evaluated in the following metabolism studies: Cytochrome (CYP) P450 enzyme 
inhibition and induction studies, reaction phenotyping for CYP P450 enzyme identification, and 
metabolic stability of TSC in hepatocytes from multiple species.  The in vitro studies testing the 
potential impact of TSC on liver direct enzyme inhibition in human microsomes showed no 
marked concentration-related increases at concentrations up to 40 µM in all tests done.  TSC did 
not have an effect on the time-dependent inhibition of the standard battery of agents tested and is 
not likely a mechanism-based inhibitor of these isoforms.  No significant induction responses (≥ 
40% of adjusted positive control) of CYP1A2, CYP2B6, and CYP3A4 enzyme activity were 
observed with any of the concentrations of TSC examined in any of the human donor hepatocyte 
preparations.  In addition, TSC treatment resulted in no marked CYP1A2, CYP2B6, and 
CYP3A4 mRNA induction.  The results from these studies suggest a low potential for drug-drug 
interactions with TSC due to enzyme inhibition or enzyme induction of CYP1A2, CYP2B6, and 
CYP3A4 at the concentrations examined.  A study was done to determine the in vitro metabolic 
stability of TSC in rat, dog, rabbit, and human cryopreserved hepatocytes.  The metabolic 
stability was evaluated based on analysis of the disappearance of TSC as a function of time.  The 
results from this multi-species metabolic stability study in cryopreserved hepatocytes suggest 
that TSC is not significantly (> 35%) metabolized via the particular CYP-mediated pathways 
present in the hepatocytes tested.  Ultimately, drug interactions will be dependent on multiple 
metabolism and PK factors encountered in vivo.  


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The in vivo ADME of TSC was also investigated using [14C]-labelled Trans Sodium Crocetinate 
([14C]-TSC) in the rat. Pharmacokinetic, excretion/balance and tissue distribution experiments 
were conducted and evaluated from samples collected following single oral and intravenous 
administration of [14C]-TSC combined with non-radioactive TSC for a total of 50 mg/kg doses to 
rats.  
 
Maximum mean plasma total radioactivity concentrations (Cmax) were observed at 0.5 hours 
(Tmax) post oral dose administration.  Radioactivity/concentrations declined slowly thereafter 
with an apparent half-life of approximately 18 hours.  Systemic exposure to the total 
radioactivity (AUC0-48) was calculated to be 1700 µg equivalents∙h/g (µg eq./g).  Mean plasma 
concentrations (radioactivity) were 8 µg eq./g at 48 hours post dose. 
 
Maximum mean whole blood total radioactivity concentrations were measured at 0.5 hours post 
oral dose administration.  Concentrations declined slowly thereafter with an apparent half-life 
of 25 hours.  Systemic exposure to the total radioactivity (AUC0-48) was 1230 µg eq∙h/g.  Mean 
whole blood concentrations/radioactivity equated to 7 µg eq./g at 48 hours post dose. 
 
Pharmacokinetics 
The mean pharmacokinetic parameters correlated with sample radioactivity obtained in whole 
blood and plasma following a single oral gavage dose of [14C]-TSC to male rats at 50 mg/kg, is 
displayed in Table 2. 
 
Table 2.  Mean Pharmacokinetic Parameters Correlated with Sample Radioactivity in 
Whole Blood and Plasma 
 
Parameter 
Male Rats (50 mg/kg ) 
Whole Blood 
Plasma 
Cmax (measured) 
(µg eq./g) 
196 
254 
Cmax (extrapolated to zero time) 
(µg eq./g) 
223 
279 
Tmax (measured) 
(hours) 
0.5 
0.5 
T½ 
(hours) 
25.10 
17.78 
AUC(0-48) 
(µg eq∙h/g) 
1228.53 
1675.27 
AUC(0-inf) 
(µg eq∙h/g) 
1508.4 
1861.25 
 
 


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Excretion balance 
Excretion of radioactivity was followed through 168 hours after a single intravenous 
administration of [14C]-TSC.  The main route of elimination was through feces as metabolites 
with a mean recovery of approximately 50% of the dose administered.  Expired air accounted for 
another 23% for each animal representing the next largest route of recovery. Urine accounted for 
a smaller proportion of radioactivity (approximately 6 % per each animal) with remaining 
quantitated concentrations of radioactivity measured in the carcass and cage washings.  The 
mean total recovery of administered radioactivity was 83%.  A summary of the relative amounts 
of recovered radioactivity through 168 hour post dose period following single intravenous 
administration of [14C]-TSC at 50 mg/kg, is shown in Table 3. 
Table 3.  Recovered Radioactivity through 168-hour Post-dose 
 
Sample 
% Recovery 
Male 
(50 mg/kg ) 
(n = 3) 
Urine 
5.90 
Faeces 
49.47 
Expired Air Trap 1 
10.43 
Expired Air Trap 2 
12.87 
Cage Wash 
0.05 
Carcass 
4.29 
Total 
83.01 
 
Normally, the minimum target for radioactive recovery is 90%.   The incomplete recovery may 
be explained by the fact that a large proportion of the radioactivity was eliminated in the first 24 
hours via expired air.  This could potentially be attributed to volatile components (i.e. CO2 based 
metabolites) not being trapped in the expired air trapping solutions. 
 
Tissue distribution 
Following a single intravenous dose of [14C]-TSC at 50 mg/kg to male Sprague Dawley rats, 
distribution of radioactivity was quick and widespread.  The highest concentrations of 
radioactivity at early time points were observed in the gastro-intestinal tract contents, implicating 
biliary excretion as a route of elimination.  Peak concentrations in remaining tissues generally 
occurred at 1 hour post dose, though some tissues (brain, spinal cord and some secretory glands) 
achieved peak concentrations at later times.   
 
Elimination of radioactivity from tissues was generally slow; at 168 hours post dose, 
radioactivity remained quantifiable in most of the tissues analyzed, with greatest concentrations 
associated with the liver, skin, glandular tissue and brown fat. 
 


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Tissue:  blood ratios indicate that up to 2 hours post dose, only the small intestine contents 
contained consistently greater concentrations of radioactivity than levels observed in blood.  Six 
(6) hours post dosing and beyond indicated secretory glands had equal to or greater than the 
corresponding blood concentrations.  Distribution of radioactivity in pigmented animals followed 
a similar pattern as non-pigmented animals, with no evidence of affinity for melanin-containing 
tissues observed. 
1.4 
TSC in Model of Hemorrhagic Shock 
The objective of this study was to determine if TSC could enhance survival in rats with severe 
hemorrhage in a time-dependent fashion, mirroring a battlefield scenario where there is often a 
treatment delay prior to resuscitation (Giassi et al. Shock 2002). A constant-volume hemorrhage 
protocol was used in Male Sprague-Dawley rats.  In the first set of experiments, the protocol 
involved removing 60% of the estimated blood volume, and 20 minutes elapsed before TSC (or 
saline) was first injected. TSC injections were repeated four times, every 10 minutes. After an 
additional 30 minutes, fluid resuscitation with saline was initiated. Blood pH, base excess, and 
lactate levels were monitored for 90 minutes after hemorrhage, prior to the point of initiation of 
fluid resuscitation. Possible liver damage was assessed 24 hours later by measurement of 
enzymes.  
 
Blood pressure decreased by 40-45% of its baseline value following hemorrhage, with a 
commensurate increase in heart rate. Following the 20 min delay and initial TSC administration, 
blood pressure rose immediately by around 10-15 mmHg, but there was no change in response to 
saline injections. This improvement declined over 10 min and TSC was injected again with a 
similar increase in blood pressure, with no change in the saline group. This discrepancy in blood 
pressure improvement between TSC and saline was reproduced at each of the serial five 
injections (Figure 2).  
 
 
 


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Figure 2.  Effect of TSC On Blood Pressure After Hemorrhage When Therapy Is Delayed 
20 Minutes 
 
Similarly, a significant decrease in heart rate was noted following TSC injection compared to 
saline, and with each serial injection following the 20 min delay from hemorrhage (Figure 3). 
 
 
 
 
 
Figure 3.  Effect of TSC on Heart Rate After Hemorrhage When Therapy Is Delayed 20 
Minutes 
 
Blood parameters of systemic hypoxia were taken at baseline, 15 min post-hemorrhage prior to 
injection, and 90 min post-hemorrhage following TSC (vs. saline) injection. TSC resulted in 
significant improvement in blood pH and base deficit compared saline, and demonstrated non-
significant improvements in PO2, PCo2, lactate, and bicarbonate (Table 4). 
 
Table 4.  Blood Parameters of Systemic Hypoxia Following Hemorrhagic Shock – Baseline, 
Post-Hemorrhage, and Following TSC vs. Saline Injection 
 
 
 
Liver injury was assessed by serum levels of glutamate-oxalacetic transaminase (GOT) and 
glutamate-pyruvate transaminase (GPT) (i.e. aspartate aminotransferase (AST) and alanine 


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aminotransferase (ALT)) before hemorrhage and 24 hours later. At the 24-hour mark post-
hemorrhage, liver enzymes doubled in the saline group but remained stable in the TSC group 
(p<0.05).   
 
 
 
Figure 4.  Liver Transaminase Activity At Baseline and 24-Hours Post-Hemorrhagic Shock 
in TSC vs. Saline Treated Groups [A – aspartate aminotransferase (AST), B – 
alanine aminotransferase (ALT)] 
 
In a second experiment, a smaller hemorrhage (10% blood volume) was incurred ten minutes 
after the initial severe hemorrhage (60% blood volume). TSC (vs. saline) was given immediately 
following the second hemorrhage and every ten minutes for a 60-minute period. No subsequent 
fluid resuscitation was provided. In the saline group, almost all of the animals died within 50 
minutes, whereas the majority treated with TSC survived.   
 


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Figure 5.  Survival with TSC vs. Saline Following Second Hemorrhage 
 
These sets of experiments in rats with hemorrhagic shock suggest that TSC has a measurable 
effect on markers of systemic hypoxia, including normalization of acidosis and acute liver injury. 
TSC’s effect on correcting hypotension and tachycardia following severe hemorrhage also 
supports the hypothesis that TSC enhances oxygenation and systemic oxygen transport. 
1.5 
Experience in Humans 
Phase 1 - Safety 
The safety and tolerability of the lyophilized injectable formulation of TSC in humans has been 
evaluated in a randomized, double-blinded, placebo-controlled Phase 1 trial in 40 normal healthy 
subjects (Diffusion Pharmaceuticals Clinical Study Report 100-001, 2008).  Tolerability and PK 
were assessed after a single intravenous dose of 0.1, 0.5, 1, 2.5, and 5 mg/kg (6 subjects per TSC 
dose) through a 1 month follow-up visit after a single IV bolus injection of 20 mg/ml TSC.  A 
total of 30 subjects received TSC and 10 received placebo.  A single IV dose of TSC was very 
well tolerated and the maximum tolerated dose was 2.5 mg/kg based on mild, transient, yellow 
visual field disturbances seen in 2 subjects at a dose of 5 mg/kg.  There were no clinically 
significant dose-dependent changes in laboratory parameters, vitals, or ECG up to the maximum 
dose evaluated of 5 mg/kg.  There were no serious adverse events (SAEs) or deaths and no 
subject withdrew from the study.   
 
Phase 2 
Peripheral Artery Disease 
A randomized, double-blinded, placebo-controlled Phase 1/2 study in 48 subjects has been 
conducted to evaluate the safety, PK, and dose response of TSC in subjects with symptomatic 
peripheral artery disease (PAD) and claudication (Diffusion Pharmaceuticals Clinical Study 
Report 100-301, 2010; Mohler et al., 2011).  A total of 40 subjects received TSC and 8 subjects 
received placebo.  Overall, multiple dosing by IV injection of TSC from 0.25 mg/kg to 2.00 
mg/kg in elderly subjects with PAD was well tolerated when compared to placebo.  The 
distribution of adverse events (AEs) observed in the study did not suggest predominance in any 
dosing arm compared to placebo.  No treatment-emergent or dose limiting toxicity was 


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discovered in this study and no subject withdrew due to an AE.  The 3 AEs reported by the 
principal investigators (PIs) as related to TSC were single incidents during dosing of mild to 
moderate burning or irritation at the injection site, and a yellow pigmented streaking at the 
injection site at the time of dosing; all resolved without treatment.  These were likely associated 
with the IV line catheter placement or dosing technique, since reconstituted TSC has a yellow 
color.  One subject receiving TSC at 2.00 mg/kg reported a single episode of a mild visual 
disturbance of “spots” that completely resolved without treatment.  The PI evaluated this event 
as not related to TSC.  The Sponsor determined this event was not a suspected adverse reaction, 
since it was not yellow in nature as was observed in the Phase 1 study and did not re-occur with 
repeated exposure to TSC.  There were no AEs considered by the PI or Sponsor to be associated 
to the highest TSC dose (2.00 mg/kg).   
 
In the 48 PAD subjects enrolled in Protocol 100-301, there were a total of 4 SAEs in TSC-treated 
subjects, including 1 death which occurred after the protocol-defined reporting period.  There 
were 2 SAEs, including 1 death, reported in the placebo group.  All of the SAEs were 
unexpected and considered unrelated to blinded study medication by the PI and Sponsor with the 
exception of the SAE of deep vein thrombosis (DVT).  The DVT occurred in a subject who was 
randomized to 0.50 mg/kg TSC, and it was considered possibly related by the PI to blinded study 
medication.  Although the DVT was an unexpected AE, the Sponsor determined this event was 
not a suspected adverse reaction since there was no evidence to suggest a causal relationship.  
While a causal attribution to study medication cannot be excluded, neither the PI nor the Sponsor 
can offer a probable pathophysiological mechanism by which TSC could result in a DVT.  Study 
blind was not broken for any of these AEs. 
 
The efficacy and dose response of TSC was evaluated in Protocol 100-301 using objective 
measurements from exercise treadmill tests in subjects with PAD and intermittent claudication 
symptoms.  The change in claudication onset time (COT) showed a significant increase in the 
0.25 mg/kg TSC dosing arm after Dose 1 and Dose 5 as well as lesser signals of improvement at 
TSC doses 1.25 mg/kg to 1.75 mg/kg.  Notable signs of clinical benefit were observed after Dose 
1 and Dose 5 of TSC above 1.0 mg/kg for both increased peak walking time (PWT) and COT on 
the exercise treadmill test, as well as subject-perceived increases in walking distance from the 
Modified Walking Improvement Questionnaire survey. 
 
The PK profile of TSC from Protocol 100-301 in the elderly PAD subject population after 
multiple doses was similar to the PK results observed in the Phase 1 study in normal healthy 
subjects.  TSC was eliminated quickly and the PK profile of TSC appears to be nonlinear after 
IV doses of 0.25 to 2.0 mg/kg in PAD subjects.  The mean elimination half-life (t½) increased 
with dose and at doses greater than 1 mg/kg, t½ appeared to fluctuate in the 1.5 hour range.  For 
the higher doses, 0.75 to 2.00 mg/kg, plasma concentrations appeared to decay at the same rate.  
Due to the short half-life of TSC relative to the 24-hour dosing interval, essentially 12.5 times 
the longest mean t½, and since only 4 subjects had a pre-dose plasma TSC concentration that 
was ≥ 10 ng/ml limit of quantitation (LOQ) and < 0.15% of corresponding maximal plasma 
concentration (Cmax), the Dose 3 PK data may be viewed as if a single dose. 
 
 
 


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Glioblastoma Multiforme (GBM) 
Diffusion Pharmaceuticals completed a phase 2 trial with a phase 1 safety lead-in (Diffusion 
Pharmaceuticals, Clinical Study Report 100-202, 2015) that evaluated the safety, tolerability, PK 
profile, efficacy, progression-free survival (PFS), quality of life (QOL), and overall survival 
(OS) in adults with GBM who are treated with TSC in addition to the standard of care (SOC) 
consisting of radiation therapy (RT) and temozolomide.  The Phase 1 safety lead-in portion of 
the protocol incorporated a TSC dose escalation approach.  The Phase 2 study portion enrolled 
56 subjects at the recommended dose based on the safety lead-in study results. A previously 
conducted study by Stupp et al (2005) was used as a historical control.  
 
During the conduct of Phase 1, monitoring for dose-limiting toxicity (DLT) events was 
performed. A Safety Monitoring Committee (SMC) evaluated safety data from the Phase 1 
cohort and recommended a dosage regimen for Phase 2. 
  
Safety and efficacy variables were evaluated at scheduled time points over 2 years after 
administration of study treatment. Investigators and independent reviewers assessed tumors and 
reported on tumor status.  Tumor scans were subsequently analyzed by a single, central group, 
Biomedical Medical Systems. 
 
The SOC treatment regimen, administered concomitantly with TSC treatment in both phases of 
the study, included RT and temozolomide, which were administered in a manner similar to that 
utilized in the Stupp et al (2005), which was used as a historical control study. Treatment with 
RT began within 5 weeks after tumor resection surgery or definitive biopsy. The RT treatment 
regimen included 5 sessions per week for 6 weeks (30 sessions total). Temozolomide 75 mg/m2 
was taken orally (capsule formulation) daily for 6 weeks (ie, 42 days) concomitantly with RT.   
 
• Phase 1: 3 weeks of TSC .25mg/kg with concomitant RT and temozolomide; TSC 
administered 45 to 60 minutes before RT (9 doses total).  
• Phase 2: 6 weeks of TSC .25 mg/kg with concomitant RT and temozolomide, TSC 
administered 45 to 60 minutes before RT (18 doses total).  
 
After completion of administration of the study treatment, efficacy and safety evaluations were 
continued for a total of 2 years (24 months). 
 
Overall survival (modified Intent-to-Treat [mITT] population; TSC 18 doses group)  
• Two years after initiation of treatment with TSC, 35 subjects (62.5%) had died while 21 
subjects (37.5%) remained alive. The probability (Kaplan-Meier analysis) of OS was 71.2% 
after 1 year and 36.3% after 2 years.  
• The mean standard deviation (SD) OS duration was 16.31 (7.313) months, and the median 
(95% confidence interval [CI]) OS duration (using a Kaplan-Meier analysis) was 16.3 
months (13.27, 23.66).  
 
Progression-free survival (mITT population; TSC 18 doses group)  
• The median (95% CI) PFS duration (using a Kaplan-Meier analysis) was 3.3 (3.15, 5.16) 
months. This may be a misleading value as many tumors that initially increased in size later 
decreased in size (see below). 


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Tumor size (mITT population; TSC 18 doses group)  
• A mean change from Baseline of 28.7% in tumor size (sum of tumor area) was seen at Week 
10. However, at the next time point (Week 18) mean tumor size was smaller, and 
subsequently mean tumor size remained smaller than at Baseline and continued to 
progressively decrease throughout the remainder of the 2-year treatment evaluation period. 
At the 1-year time point, mean tumor size in surviving subjects was approximately half of the 
mean tumor size at Baseline, and by the 2-year time point, 11 subjects had a 100% reduction 
in tumor size, indicating essentially complete disappearance of the GBM tumor. 
• Of 14 subjects who had a complete resection before Baseline, 6 were alive at 2 years and 
showed no tumor present.  
  
Quality of life and performance measures  
• Mean and median scores for the European Organization for Research and Treatment of 
Cancer (EORTC) QLQ-C30 and Karnofsky Performance Score (KPS) remained fairly 
constant over time, with subscale mean scores suggesting continued relatively good function 
and global health and generally low levels of symptoms.   
 
Safety  
A total of 56 subjects (94.9%) had a treatment emergent adverse event (TEAE), most of whom 
had more than one TEAE. The most frequently occurring TEAEs included fatigue (40.7%), 
alopecia (35.6%), nausea (27.1%), and constipation (27.1%). Most TEAEs were categorized as 
either common terminology for adverse events (CTCAE) Grade 1 (mild) or 2 (moderate); 10 
subjects (16.9%) had a Grade 3 (severe) TEAE and 4 subjects (6.8%) had a total of 6 Grade 4 
(life-threatening) TEAEs (ie, brain oedema, neutropenic sepsis, tooth abscess, pulmonary 
embolism, febrile neutropenia, drug hypersensitivity). There were no CTCAE Grade 5 (death 
related to AE) TEAEs.  
 
A total of 24 TEAEs in 12 subjects (20.3%) were assessed as related to TSC. The most 
frequently reported TSC-related AEs were fatigue (5 events in 4 subjects [6.8%]) and headache 
(3 events in 2 subjects [3.4%]). 
  
Eleven subjects (18.6%) had a total of 19 SAEs. The most frequently reported SAEs were 
hydrocephalus, pulmonary embolism, and muscular weakness, each of which occurred in 2 
subjects (3.4%). All other SAEs occurred in 1 subject each. None of the SAEs were considered 
related to TSC.  
  
No subject had an AE that resulted in death, and no subject had an AE that resulted in 
discontinuation of TSC.  
 
Overall, diastolic and systolic blood pressure and pulse rate mean values over time showed 
variability, but no concerning patterns of clinically meaningful TSC-related changes from 
Baseline were discerned for evaluated vital signs, ECGs, or clinical laboratory tests.  
 
In summary, TSC 0.25 mg/kg administered as an IV bolus 3 times per week for 6 consecutive 
weeks (18 doses total) as concomitant treatment along with SOC treatments of RT and 


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temozolomide was well tolerated in the subjects in this study with newly diagnosed GBM, and 
no safety findings were identified that would preclude further clinical development for the 
indication of treatment of GBM.  
 
2 
STUDY METHODS 
2.1 
Study Overview 
This is a randomized, double-blind, placebo-controlled, pharmacokinetic, pharmacodynamic 
study of Trans Sodium Crocetinate (TSC) utilizing Transcutaneous Oximetry Measurement 
(tcpO2) in healthy volunteers breathing  O2. Study assessments include tcpO2 levels, SpO2, and 
PK.   
2.2 
Study Objectives 
2.2.1 Primary Endpoint 
Determine the dose-response of TSC on tcpO2 following a single administration of TSC in 
subjects breathing  O2.  
2.2.2 Safety Endpoints 
Assessment of adverse events, new medications, laboratory (CBC, BMP), vital signs, and SpO2. 
2.3 
Study Population  
Subjects will be healthy adult volunteers meeting all inclusion and exclusion criteria.  Up to 30 
subjects will be randomized to a single IV bolus injection of TSC at a dose of 0.5, 1.0. 1.5, 2.0, 
or 2.5 mg/kg or placebo normal saline with concomitant O2 administration. 
2.3.1 Inclusion Criteria 
1. Healthy male or female, age 18-50 
2. Able and willing to lie quietly supine or semi-recumbent for up to 2.5 hours 
3. Abstinence from exercise, caffeine, alcohol, nicotine, and a heavy meal prior to testing on 
the day of the Treatment Visit 
4. Subject is able to communicate effectively with the Investigator and to comply with all 
study requirements, restrictions, and directions from the study staff 
5. Females of childbearing potential must have a negative blood pregnancy test at screening 
and agree to use one of the accepted contraceptive regimens, or a double method of birth 
control (e.g. condom and spermicide), during the study and at least 30 days after the last 
dose of study drug. Females of non-childbearing potential should be surgically sterile or 
at least one year post-menopausal. 
6. Males who engage in sexual activity that has the risk of pregnancy must agree to use a 
double barrier method (e.g. condom and spermicide) and agree not to donate sperm 
during the study and for at least 90 days after the last dose of study drug 


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2.3.2 Exclusion Criteria 
1. Allergy to study medication 
2. Pregnant or breastfeeding 
3. Current smoker and/or any nicotine use within 4 hours of the start of tcpO2 procedures, 
to include e-cigarette vaping, snuff, chew, nicotine gum and nicotine patches 
4. Body Mass Index (BMI) > 30 
5. Positive test results for HIV-1/HIV-2 Antibodies, Hepatitis B surface Antigen (HBsAg), 
or Hepatitis C Antibody (HCVAb)  
6. Blood donation (excluding plasma donation) of approximately 500 mL within 56 days 
prior to screening  
7. Plasma donation within 7 days prior to screening 
8. Treatment with an investigational drug within 30 days or 5 times the half-life (whichever 
is longer) prior to screening 
9. Any skin condition on limbs to be tested that could impair testing (rash, wound, prior 
radiation therapy, other skin conditions, per Principal Investigator (PI) discretion) 
10. Known cardiovascular disease, including treated or untreated hypertension 
11. Significant respiratory disease and/or any other significant medical condition  
12. Subject has an acute illness (gastrointestinal infection, influenza, or known inflammatory 
process) at the Treatment Visit 
13. Urine screen positive for drugs or positive breathalyzer for alcohol (at screening and 
enrollment) 
14. Concomitant medications used to treat a diagnosed medical condition 
15. Subject who, for any reason, is deemed by the Investigator to be unsuitable for the study; 
or has any condition that would interfere with the evaluation of tissue oxygen 
measurements or PK of the investigational drug; or is otherwise unable to comply with 
the protocol 
2.4 
Enrollment and Consent 
An investigator or delegated site staff will interview and examine the volunteer in the clinic, and 
will discuss inclusion in the clinical investigation, the details of the investigation, and other 
information contained in the informed consent document; and that the subject’s participation in 
the study may be discontinued at any time without penalty or loss of benefits to which the 
subject is otherwise entitled. 
2.5 
Withdrawal of Subjects 
2.5.1 Criteria for Early Withdrawal 
The following events are considered sufficient reason to discontinue a subject from the study: 
• Subjects are free to withdraw from the study at any time, for any reason, and without 
prejudice 
• The subject experiences an adverse event (AE) that in the investigator’s opinion 
precludes continued participation 


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• The subject incurs a significant protocol violation that constitutes a safety hazard or 
significantly confounds the interpretation of the data from that subject 
• At the investigator’s request 
• The study is terminated 
2.5.2 Screening Failures 
After the screening evaluations have been completed, the investigator or designee will review the 
inclusion and exclusion criteria and determine the subject’s eligibility for the study. Only the 
reason for ineligibility will be collected on screening failures. Subjects who are found to be 
ineligible will be told the reason for ineligibility.  
2.5.3 Subjects Lost to Follow Up 
The investigator will repeatedly attempt to contact any subject that is unreachable by telephone 
for the 48 Hour visit to obtain follow-up safety information.  All attempts to contact the subject 
should be documented, including the use of registered mail with return receipt.  
2.5.4 Replacement of Subjects 
Subjects who have received study drug will not be replaced. The analysis will be conducted on 
an intention-to-treat basis. 
2.5.5 Study Stopping Rules 
The Sponsor reserves the right to terminate the study at any time for reasonable medical or 
administrative reasons. Any premature discontinuation will be appropriately documented in 
compliance with applicable requirements and/or regulations (e.g., IRB/EC, regulatory 
authorities, etc.). 
2.6 
Study Costs  
There is no cost to subjects for the research tests, procedures, evaluations, and study drug while 
taking part in this study.  
2.7 
Study Drug 
2.7.1 TSC Packaging 
Study drug will be supplied as a sterile lyophilized powder in 10 mL vials and will be in bulk 
cardboard boxes stored at room temperature (15 to 30° Celsius or 59 to 86° Fahrenheit) under 
secure conditions with limited access.  Each vial will contain: 
 
 
10 mL Vial 
Trans Sodium Crocetinate 
100 mg 
Gamma cyclodextrin 
400 mg 
Glycine USP 
18.75 mg 


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Mannitol USP 
115 mg 
 
The label will contain appropriate labeling in accordance with all relevant labeling regulations. 
2.7.2 Reconstitution of TSC 
The 10 mL vial will be reconstituted with 5 mL of Sterile Water for Injection (USP).  TSC 
should not be reconstituted with any other diluent other than Sterile Water for Injection.  The 
lyophilized powder should rapidly reconstitute to a clear deep orange-red solution.  Each 
reconstituted vial will contain: 20 mg/mL TSC, 8% gamma cyclodextrin, 50 mM glycine, 2.3% 
mannitol at an approximate pH of 8.0 - 8.2.   
 
TSC is dosed based on the subject’s baseline weight, obtained on the day of screening, on a 
milligram per kilogram basis. Several vials may be needed depending on the subject’s weight.  
Reconstitution of multiple vials will be carried out per institutional Standard Operating 
Procedures relative to preparation of IV injectable drugs. 
 
The reconstituted study drug will be prepared separately for each subject at room temperature 
(15 to 30°C or 59 to 86°F).  TSC must be administered within 4 hours of reconstitution and any 
remaining TSC in the vial should not be used for further dosing of the same subject or another 
subject.  Reconstituted TSC should not be diluted prior to administration.  
2.7.3 Placebo 
Placebo normal saline will be supplied from the bulk stock of the Investigational Pharmacy. 
Placebo is dosed at 7 mL per administration. 
2.7.4 Randomization 
Volunteers will be randomized to one of 5 TSC doses or placebo in a 1:1:1:1:1:1 schema, with 5 
subjects randomized to each of the 6 cohorts. 
2.7.5 Blinding Protocol 
Because TSC is highly colorized, this study will utilize unblinded and blinded personnel as 
follows: 
• Unblinded study team: Pharmacist(s) and administering personnel  
• Blinded study team: Investigator(s) and study coordinators 
The unblinded study team will take specific steps to ensure that the subjects and blinded study 
personnel are not made aware of treatment assignment.  
• Prior to release from the pharmacy, the pharmacist will package the prepared syringe(s) 
in foil or acceptable masking material that masks the contents (i.e. cannot see through the 
material). The study drug should not be removed from the masking material at the time of 
administration to the subject. 
• The medical staff assigned to administer the masked study drug will do so using a shroud 
to insure that the patient is blinded to treatment.  The shroud may be made of any 
material and be of any size as long as it sufficiently blocks the patient’s view of the 


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injection site and injection syringe at the time of administration.  The shroud need not be 
in place at times other than during the time of study drug injection.   
• A cloth or paper drape will be added underneath the injection site in the event that a 
droplet falls from the syringe as part of the injection process. The drape will be removed 
and folded up following the injection and before the shroud is removed so that it is not 
visible to the study team or subject. 
• If in the treatment room at the time of injection, the blinded study team must position 
themselves so that the injection site and syringe(s) are not visible to them during study 
drug injection. The subject will also be asked to look away from the injection site. 
2.7.6 Dosing Regimen 
Study drug will be administered as a one-time IV bolus injection to 5 unique subjects per dose 
cohort, to include 5 TSC dose levels and placebo. Placebo will consist of 7 mL of normal saline.  
Subjects will be randomized to the following dose cohorts:  
 
Dosing 
Cohort 
# of Subjects in 
Cohort 
TSC Dose 
(mg/kg)  
Placebo 
(mL) 
1 
5 
0.5 
N/A 
2 
5 
1.0 
N/A 
3 
5 
1.5 
N/A 
4 
5 
2.0 
N/A 
5 
5 
2.5 
N/A 
6 
5 
N/A 
7 mL 
 
2.7.7 Drug Accountability 
It is the responsibility of the Principal Investigator to ensure that study drug at the site is 
inventoried upon receipt, accounted for throughout the study, and the results of drug 
accountability documented.  Return of drug to the sponsor or sponsor designee, or the destruction 
of study medication, will also be documented.  
 
Drug accountability tasks may be delegated to a pharmacist or other appropriately trained party 
at the site. 
2.8 
Concomitant Medications 
Subjects may only be enrolled in this trial if they are healthy and not taking any medication 
prescribed for a diagnosed medical condition. Over the Counter (OTC) medications and 
supplements will require a 7-day washout period. Concomitant medications, such as Tylenol, 
may be given prior to study drug administration at the PI’s discretion. 


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2.9 
Schedule of Events 
The charted schedule of events is included in Appendix 8.2  
2.9.1 Screening Evaluation (Day -21 to Day 0) 
Following completion of the informed consent process and signing of the Informed Consent 
Form (ICF), site staff will obtain the following.  
• Demography 
• Medical History 
• Concomitant medications  
• Physical exam 
• Vital signs (heart rate, blood pressure, respiratory rate, temperature) 
• Body Mass Index (BMI = kg/m2) 
• Oxygen saturation by pulse oximetry (SpO2) 
• 12-lead ECG 
• Complete blood count (CBC) 
• Basic metabolic panel (BMP) 
• HIV, HBsAg, and HCVAb 
• Urine drug screen 
• Alcohol screen 
• Serum pregnancy test for all females  
Subjects meeting all inclusion/exclusion criteria and who have completed the informed consent 
process may be enrolled in the study. 
2.9.2 Treatment Visit (Day 0)  
Following the screening visit, the subject will return to the clinic for study treatment procedures. 
If the site has Point of Care (POC) laboratory testing available, the screening and treatment 
procedures may occur on the same day. Subjects will refrain from exercise, caffeine, alcohol, 
nicotine, and a heavy meal prior to testing on the day of the treatment visit. 
 
Subjects will be randomized into one of 6 groups in a 1:1:1:1:1:1 schema, to include the 5 TSC 
doses and one placebo arm.  Each TSC dose will be calculated based on the subject’s body 
weight in kg at the dosing level they are randomized to. Each individual dose of placebo will be 
normal saline 7 mL. To maintain the double-blind, study drug administration will be performed 
by unblinded medical staff who will not be involved in other study procedures, including subject 
assessment.  Subjects, investigators, and study coordinators will not see the injection or injection 
site or be aware of randomization. Please refer to Section 2.7.5 for specific blinding protocols. 
2.9.2.1 Subject Arrival and TcpO2 Sensor Electrode Placement 
When subjects arrive to clinic for the treatment visit, medical history, physical exam, body 
weight, vital signs, concomitant medications, urine pregnancy screen, urine drug screen, and 
breathalyzer alcohol screen will be assessed (unless the Screening and Treatment visits are done 
on the same day). 
 


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Subjects will be maintained in a temperature-controlled room (between 22.0 and 25.0˚C), and in 
a supine position with the head slightly raised on one pillow, or semi-recumbent. One blanket 
will be provided for comfort. One or two IV catheters will be placed for study drug 
administration and PK measurement. Direct venipuncture will also be allowed for PK draws, if 
needed. 
 
TcpO2 sensor electrodes will be applied to the left or right lower extremity, per PI discretion.  
Sensor electrode temperature will be preset to 45˚C, to allow maximum vasodilation. Risk of 
sensor site superficial burn is minimal given the relatively brief testing period.  Four (4) sensors 
will be applied to the following locations:  
 
• Sensor 1: Mid-dorsum of the foot  
• Sensor 2: 10 cm distal to the lateral femoral epicondyle  
• Sensor 3: 5 cm proximal to the anterior aspect of the lateral malleolus  
• Sensor 4: 5 cm proximal from the center of the medial malleolus  
2.9.2.2 O2 Equilibration and Baseline Period 
After the tcpO2 sensors have been applied and tested, subjects will be placed on O2 via simple 
face mask at 6 L/minute, and will remain on O2 for 70 minutes prior to study drug 
administration. The first 10 minutes will allow for equilibration of O2 levels, and the 
subsequent 60 minutes will serve as the baseline period. TcpO2 values and SpO2 will be 
recorded every 5 minutes during the above periods. Baseline vital signs will be measured 
within 10 minutes prior to study drug dosing. 
 
Once the equilibration phase begins, it is important that subjects remain lying quietly and 
minimize bodily movement through the entire baseline and treatment periods. 
2.9.2.3 Treatment Evaluation Period 
At the end of the 70-minute equilibration/baseline period, subjects will continue on O2 and 
receive a single IV bolus injection of TSC at a dose of 0.5, 1.0, 1.5, 2.0 or 2.5 mg/kg, or 
placebo. After study drug administration, subjects will be evaluated for 60 minutes, with 
tcpO2 values and SpO2 recorded at 1, 2, and 5 minutes post-dose and then every 5 minutes 
thereafter. In addition to assessment of tcpO2 as described, continuous tcpO2 measurements 
will be provided by the TCOM machine in graphical format. 
 
Vital signs during the treatment and evaluation period will be assessed at 10, 30, and 60 minutes 
post-study drug dosing. Adverse events will be assessed from the beginning of the equilibration 
period. 
2.9.2.4 PK Measurements 
Prior to and following study drug administration, PK samples will be obtained at the below 
intervals. 
 
 


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Pre-dose (within 10 minutes prior to injection) 
1 minute post end of injection (+1 min) 
10 minutes post end of injection (± 1 min) 
30 minutes post end of injection (± 1 min) 
1.5 hours post end of injection (± 2 min) 
 
2.9.2.5 Post-Treatment Evaluation Period 
After the 60-minute treatment evaluation period, the tcpO2 sensor electrodes will be removed. 
Subjects will remain in the procedure room for an additional 30 minutes to allow for collection 
of the 1.5-hour PK blood draw and repeat vital signs.  
2.9.2.6 Overnight Stay in Clinic 
Each subject will remain in clinic overnight for observation. 
2.9.3 48 Hour Follow Up Telephone Visit 
Subjects will be contacted by telephone at 48 hours (+ 2 days) for a safety follow up to assess for 
adverse events and new medications. 
2.10 Laboratory and PK Assessment Parameters 
2.10.1 Laboratory 
• Clinical laboratory evaluations:  
o Fasting is not required before collection of laboratory samples 
o Blood will be collected at the time points indicated in the protocol 
 
• Venipuncture volumes: 
Day +/- Window 
Day -21 to Day 0 
CBC, BMP, HIV, 
HBsAg, HCVAb 
18 mL 
PK samples                
(5 draws X 6 mL) 
30 mL 
Total all study days  
48 mL  
 
2.10.2 PK Procedures 
Blood samples of approximately 6 mL will be collected at each timepoint for measurement of 
plasma concentrations of TSC.  
 


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The actual date and time will be recorded by 24-hour clock time noting the hour and minute of 
the end of the TSC infusion. 
 
The same digital clock will be consistently used to record the actual hour and minute of the start 
of each blood specimen collection. 
 
Each sodium heparinized vacutainer blood collection tube will be clearly labeled with the patient 
identifier number, date and time point of the specimen collection. 
 
1. About 6 mL samples of blood will be collected in a sodium heparinized vacutainer tube 
and the blood will be centrifuged to separate plasma. 
2. The plasma will be separated into two approximately equal volumes in separate tubes.  The 
plasma should be approximately 1.5 mL in each tube. 
3. Protocol-specific instructions will be provided to the clinical site in a lab manual. 
4. The plasma samples will be frozen at -70°C. One aliquot will be shipped to the 
bioanalytical lab identified in the PK Laboratory Manual, and the other stored at -70°C as 
a back-up. 
5. PK samples that are remaining after all PK analyses have been completed, may be used for 
additional analysis.  Participant confidentiality will be maintained.  
 
3 
ADVERSE EVENT REPORTING 
3.1 
Definitions 
3.1.1 Adverse Event 
An adverse event (AE) is any untoward medical occurrence associated with the use of the drug in 
humans, whether considered drug related or not.  An AE is any unfavorable and unintended sign 
(e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of 
a drug, without any judgment about causality.  This includes changes in anatomical, 
physiological, or metabolic functions as indicated by physical examination signs, symptoms, 
and/or laboratory changes or medical occurrence which develops or worsens while enrolled in 
this study regardless of whether the event is considered related to the investigational drug.   
Signs and symptoms should be grouped into a single medical diagnosis and reported as a single 
AE when appropriate.   
Abnormal laboratory values or test results constitute AEs if they are deemed clinically 
significant by the PI and induce clinical signs or symptoms or require therapy.   
Adverse events will be collected from the start of the equilibration period through the 48 hour 
follow up phone visit.   


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3.1.2 Suspected Adverse Reaction 
A suspected adverse reaction means any AE for which there is a reasonable possibility that the 
drug caused the AE.  For the purposes of IND safety reporting, “reasonable possibility” means 
there is evidence to suggest a causal relationship between the drug and the AE.  The sponsor will 
evaluate the available evidence and make a judgment about the likelihood that the drug caused 
the AE.  
3.1.3 Serious Adverse Event 
Serious adverse events (SAEs) are AEs that pose a threat to the subject’s life or functioning 
based on the following outcome/actions regardless of whether the event is considered related to 
the investigational drug: 
• Results in death 
• Is life-threatening 
• Requires in-patient hospitalization or prolongation of existing hospitalization  
• Results in persistent or significant disability/incapacity  
• Is a congenital anomaly/birth defect  
• Other events, based on medical judgment, which jeopardize the subject and require 
medical/surgical intervention to prevent one of the outcomes above 
Events not considered to be SAEs are: 
• Treatment on an emergency outpatient basis for an event not fulfilling any of the definitions 
of serious and not resulting in hospital admission 
• A hospital admission of less than 24 hours in duration 
After the initial SAE report, the investigator is required to proactively follow each participant at 
subsequent visits/contacts. All SAEs will be followed until resolution, stabilization, until the 
event is otherwise explained, or the participant is lost to follow-up. 
3.1.4 Unexpected 
An unexpected adverse event is any adverse event where the nature, specificity or frequency of 
the event is not consistent with either: 1) the known or foreseeable risk associated with the 
procedures involved in the research that are described in the protocol or Investigator’s Brochure; 
or 2) the expected natural progression of any underlying disease, disorder, or condition of the 
subject(s) experiencing the adverse event and the subject’s predisposing risk factor profile for the 
adverse event.  
3.1.5 Unanticipated Problem (UP) 
An unanticipated problem is an event or outcome that meets all of the following criteria: 1) 
unexpected; 2) related or possibly related to participation in the research; and 3) places subjects 
or others at a greater risk of harm than was previously known or recognized. 


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3.2 
Adverse Events Previously Reported with TSC 
The Investigator’s Brochure for TSC should be used as a reference for up-to-date information 
regarding non-clinical and clinical data that are available.  As outlined in the subject informed 
consent form, the following risks and side effects may include the following: 
 
• TSC is a yellow-orange color and may cause irritation or burning and/or yellow streaking of 
the skin at the site of injection and may be due to the IV-line placement or dosing technique.   
• At the doses being tested in this study, 1 subject in another study reported a hot feeling in 
their leg which went away without treatment. 
• In another study, 2 subjects reported a brief and mild, reversible yellow visual effect at 5.0 
mg/kg, a dose that is higher than the doses used in this study, which resolved without 
treatment. 
 
In animals, the following have been reported at TSC doses 50 times the highest dose utilized in this 
study: 
• Mild, yellow skin discoloration 
• Mild, yellow to orange discoloration of urine 
• Mild changes in blood tests (liver enzymes, blood proteins, bilirubin, blood urea nitrogen) 
 
No subjects have withdrawn from any study as a result of an AE related to TSC.   
3.3 
Adverse Event Reporting Requirements 
Each subject will be evaluated for the development of AEs by the site study team. It is the 
responsibility of the investigator to document all AEs occurring during this investigation by 
entering them into the Case Report Form (CRF).  All AEs occurring from the start of the 
equilibration period  must be recorded, regardless of whether they are considered related to study 
drug.  The nature of each event, date and time of onset, outcome, frequency, intensity, action 
taken with respect to dosage, whether it was serious or non-serious, expected or unexpected, and 
relationship to treatment should be documented.  Signs and symptoms should be grouped into a 
single diagnosis and reported as a single AE when appropriate.   
SAEs require expedited review and must be reported by the site to the Sponsor or designee via 
the CRF and/or electronically within 24 hours of discovery. 
 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 5.0 will be used for AE reporting.  Adverse events will be 
coded using the MedDRA coding dictionary. 
3.3.1 Expedited Reporting Requirements for Unanticipated Problem SAEs 
SAEs will be reported to the appropriate regulatory authorities per the Sponsor Regulatory 
Reporting Requirements Matrix maintained by Drug Safety Navigator (DSN). For expedited 
cases, DSN will finalize the required documents on or before Day 7 or Day 15, as applicable, per 
FDA requirements as outlined in the Reporting Matrix.  


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3.4 
Research Related Injuries 
For any potential research related injury, the site PI or designee will assess the subject.   
Study personnel will try to reduce, control, and treat any complications from this study. 
Immediate medical treatment may be provided by the participating study site, such as giving 
emergency medications to stop immediate allergic reactions.   
 
As needed, referrals to appropriate health care facilities will be provided to the subject. The site 
PI should then determine if an injury occurred as a direct result of the tests or treatments that are 
done for this trial.  
3.5 
Contraception and Pregnancy 
A female study subject must meet one of the following criteria: 
• If of childbearing potential – agrees to use one of the accepted contraceptive regimens 
during the study, and for at least 30 days after the last dose of the study medication. An 
acceptable method of contraception includes one of the following: 
o Abstinence from heterosexual intercourse 
o Hormonal contraceptives (birth control pills, injectable/implant/insertable 
hormonal birth control products, transdermal patch) 
o Intrauterine device (with or without hormones)  
 
OR 
 
o Agrees to use a double barrier method (e.g. condom and spermicide) during the 
study and for at least 30 days after the last dose of the study medication 
• If of non-childbearing potential – should be surgically sterile (i.e. has undergone 
complete hysterectomy, bilateral oophorectomy, or tubal ligation/occlusion) or in a 
menopausal state (at least 1 year without menses), as confirmed by FSH levels (≥ 40 
mIU/mL). 
A male study subject that engages in sexual activity that has the risk of pregnancy must agree to 
use a double barrier method (e.g. condom and spermicide) and agree to not donate sperm during 
the study and for at least 90 days after the last dose of the study medication. 
 
A pregnancy of a female subject occurring within 28 days of the subject’s dose of study drug is 
considered a reportable event. If a pregnancy is reported or discovered, the investigator should 
inform the Sponsor or designee within 24 hours of learning of the pregnancy. Pregnancy itself is 
not considered an AE or SAE, however abnormal pregnancy outcomes (e.g., spontaneous 
abortion, fetal death, stillbirth, congenital anomalies, ectopic pregnancy) are considered SAEs 
and should be reported to the Sponsor. If a pregnancy is reported, the Sponsor or designee will 
follow up on pregnancy outcome 4 weeks after the projected due date. 
 


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4 
DATA MANAGEMENT AND QUALITY ASSURANCE 
4.1.1 Electronic Case Report Form 
Study data will be collected on a standardized case report form (CRF) and managed using an 
electronic data capture tool selected by the sponsor.  The EDC tool will be a secure, web-based 
software platform designed to support data capture for research studies, providing 1) an intuitive 
interface for validated data capture; 2) audit trails for tracking data manipulation and export 
procedures; 3) automated export procedures for seamless data downloads to common statistical 
packages; and 4) procedures for data integration. 
 
Clinical data will include subject demographics, medical history, vital signs, laboratory 
assessments, tissue oxygen measurements, and pulse oximetry. Additional clinical information 
including concomitant medications and adverse events will be included. 
4.1.2 Clinical Monitoring 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial subjects are 
protected, and that the reported trial data are accurate, complete, and verifiable. Clinical 
Monitoring also ensures conduct of the trial is in compliance with the currently approved 
protocol/amendment(s), ICH, GCP, applicable regulatory requirement(s) and sponsor 
requirements. Clinical monitoring will also verify that any critical study procedures are 
completed following specific instructions in the protocol.  
  
Monitoring for this study will be performed by qualified and trained Clinical Research 
Associates (CRAs) so contracted by the sponsor. Details of clinical site monitoring are 
documented in a clinical monitoring plan (CMP). The CMP describes in detail who will conduct 
the monitoring, at what frequency monitoring will be done, at what level of detail monitoring 
will be performed, and the distribution of monitoring reports. Monitoring visits will include, but 
are not limited to, review of regulatory files, drug accountability records, CRFs, ICFs, medical 
and laboratory reports, record storage, training records, and protocol and GCP compliance. Site 
monitors will have access to each participating site, study personnel, and all study documentation 
according to the sponsor approved clinical monitoring plan. Study monitors will meet with site 
PI, sub-Investigators and Study Coordinators to discuss any problems and outstanding issues and 
will document site visit findings and discussions. 
 
Study Coordinators will fill out a screening log for all study sites that documents all volunteers 
screened for the trial, and whether they screen-failed or were enrolled. For screen fails, the 
reason for exclusion will be recorded.  
 
Monitoring will be performed via remote or in-person monitoring. CRFs will be reviewed and 
compared with the data entered to the subject’s medical record (source documents).  If remote 
monitoring is necessary the Study Coordinator will collect the appropriate source documents, 
copy, and redact all PHI information and then scan and send them to the Study Monitor for 
review. Any inadequacies or errors will be reviewed with the PI at the site. Subsequently, on-site 
case report form monitoring visits (if in-person visits are cleared by the clinic) will be performed 
at regular intervals as appropriate. Otherwise remote monitoring procedures will continue. 
 


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Site investigators or Study Coordinators will inform the sponsor of every subject enrollment on 
the same or next business day, by entering the subject into the eCRF.    
4.1.3 Source Documentation 
Source data are all information in original records (and certified copies of original records) of 
clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial. Source data should be attributable, legible, 
contemporaneous, original, accurate, and complete. Each participating site will maintain 
appropriate medical and research records for this trial, in compliance with ICH, GCP, regulatory, 
and institutional requirements. Data recorded in the CRF derived from source documents should 
be consistent with the data recorded on the source documents.  
  
Interview of subjects is sufficient for obtaining medical history. Solicitation of medical records 
from the subject’s primary care provider is not required.  
 
5 
STATISTICAL METHODS 
5.1 
General Considerations 
Subjects will be randomized using a non-stratified permuted block randomization scheme.  
Subjects who fail to complete the study will not be replaced.  There will be a single analysis 
conducted at the end of the study; no interim assessments or analyses are planned for this fixed 
design.  The trapezoidal rule will be used for the construct of the area under the curve (AUC) 
analyses.  All probability values will be 2-sided and accepted at face value for this feasibility study; 
there will not be an adjustment for multiplicity.  For interpretation, probability values <0.05 will 
be considered significant; probability values <0.1 but ≥0.05 will be considered highly suggestive 
of a significant difference.  Summaries will be prepared for all parameters using descriptive 
statistics.  For variables recored on a continous scale, results will be summarized by randomized 
treatment assignment and time. For categorical variables recorded on a multimonial or binomial 
scale, results will be presented using counts and percentages.  All recorded data will be presented 
in the data listings. 
5.2 
Analysis Populations 
Two analysis populations will be used to summarize the results from this clinical investigation. 
 
• 
Intent-to-Treat Population: The intent-to-treat (ITT) population includes all randomized 
subjects. Subjects in the ITT population will be analyzed as randomized. 
• 
Per Protocol Population: The per protocol (PP) population includes subjects in the ITT 
population who do not have significant protocol deviations and who complete the study. Protocol 
deviations will be assessed prior to database lock and unmasking. The PP population will be 
analyzed using observed data only for efficacy variables. Subjects in the PP population will be 
analyzed as treated. 
5.3 
Statistical Hypotheses 
The primary endpoints will be tested in a hierarchical fixed sequence in the following order: 


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Based on the 2-factor (treatment and time) repeated measures (time) analysis of variance model 
using the time-match changes in tcpO2: 
 
H0: µ (active treatment) = µ (placebo treatment) 
H1: µ (active treatment) ≠ µ (placebo treatment) 
 
Separate examinations of each active dose will be compared to placebo.  
 
Based on the 1-factor (treatment) analysis of variance model using the AUC based on the time-
match changes in tcpO2: 
 
H0: µ (active treatment) = µ (placebo treatment) 
H1: µ (active treatment) ≠ µ (placebo treatment) 
 
Separate examinations of each active dose will be compared to placebo.  
 
Based on the comparison of the time to the maximum time-match increase in tcpO2: 
 
H0: exp(λ) (active treatment) = exp(λ) (placebo treatment) 
H1: exp(λ) (active treatment) ≠ exp(λ) (placebo treatment) 
5.4 
Sample Size 
The sample size selected for this clinical investigation with 6 treatment groups is based on clinical 
judgement.  While hypotheses will be evaluated at the end of the study, there is no pre-specified 
statistical power that was used to establish the sample size. 
5.5 
Missing Data 
All analyses will be based on the observed data; no data will be imputed for the statistical analyses. 
5.6 
Primary and Secondary Efficacy Analyses 
The primary analysis will be based on the change over time in the tcpO2  
measurements following a single administration of TSC in subjects breathing  O2.  The time-
matched changes from Period 1 (60 minute run-in on  O2) to Period 2 (60 minute post-drug 
administration on  O2) will serve as the dependent variable in the repeated measures analysis.   The 
60 minute run-in on  O2 is intended to account for the intra-subject variability over time and the 
time-match differences represent the least biased estimate of the effect of the study drug.  With 5 
subjects per randomized treatment arm, the change over time should provide sufficient clinical 
information to determine if there is a drug effect, and if the effect in an active drug treatment arm 
is different than placebo. 
   
To compare the time-match changes in tcpO2, a 2-factor (treatment and time) repeated measures 
(time) analysis of variance model will be used.  Contrast statements within the model will facilitate 
comparisons between individual treatment arms over individual time points, providing guidance 
relative to the initial time and duration of separation. 
 


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To compare the AUC based on the time-matched differences, a 1-factor (treatment) analysis of 
variance model will be used, followed by a Dunnett’s test to compare the least square mean AUC 
values from the 5 active drug groups relative to the placebo group.  If the data within a treatment 
arm is skewed, suggesting that the least square mean value is not the best measure of central 
tendancy, the data may be ranked and the analyses will be conducted using the ranked scores.  
Additional information regarding the examination of the distribution of the data will be presented 
in the Statistical Analysis Plan.  To compare the time to the maximum increase in tcpO2, the event 
time distribution functions will be compared using a log rank test and presented using Kaplan-
Meier curves. 
5.7 
Safety Analyses 
Adverse events will be coded using the MedDRA dictionary. Frequencies and percentages of 
subjects with treatment-emergent adverse events (TEAEs), serious TEAEs, and TEAEs causing 
premature discontinuation will be summarized by treatment group.  An adverse event is treatment 
emergent if it 1) occurs after the first dose of randomized study treatment or 2) if it is present prior 
to receipt of randomized study treatment but worsens in severity or increases in frequency after 
the dose of randomized study treatment. Frequencies will be presented for subjects with TEAEs 
by system organ class and preferred term; by system organ class, preferred term and maximal 
severity; by system organ class, preferred term for treatment-related AEs; by system organ class 
and preferred term for SAEs; and by system organ class, preferred term, day of onset and Period 
(1 or 2).          
 
6 
ADMINISTRATIVE AND SPECIAL PROCEDURES 
6.1 
Protocol Amendments 
Any changes to the protocol will be made in the form of an amendment.  Unless the changes are 
designed to eliminate an apparent immediate hazard to subjects, both the Sponsor and the 
governing IRB must grant approval of the amendment before any changes may be implemented 
in study conduct. 
6.2 
Investigator Responsibilities 
The investigator has overall responsibility for the conduct of the study at his/her site.  The 
investigator is responsible for ensuring that study staff has suitable qualifications, training, and 
authorization to perform any delegated study tasks.  The investigator is responsible for the care 
of the subjects throughout this study.  The investigator/authorized person will monitor the 
subjects for the occurrence of AEs throughout their participation in this study.  
6.2.1 Compliance with Protocol 
The investigator is responsible for the conduct of the study.  No alterations or changes in this 
protocol will be permitted without written approval from the Sponsor and the IRB unless the 
amendment is necessary to reduce immediate risk to trial participants.  The investigator should 
document and report to the Sponsor all deviations from the protocol. 


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6.2.2 Good Clinical Practice (GCP) 
The investigator agrees to conduct and monitor the clinical trial in accordance with GCP 
standards. 
6.2.3 Institutional Review Board Review and Approval 
It is the responsibility of the investigator to communicate with the IRB.  The IRB will be 
functioning in accordance with current regulations.  A copy of the IRB’s unconditional written 
approval of the protocol and the informed consent will be obtained and provided prior to the 
initial supply of test article and start of the trial.  All protocol amendments will be submitted to 
the IRB and approval sought prior to implementation, unless the amendment is necessary to 
reduce immediate risk to trial participants.  The IRB will be informed of any new safety 
information as it becomes available.  The investigator will provide reports to the IRB as 
requested, but at least annually, and after the trial is complete. 
6.2.4 Informed Consent 
The investigator is responsible for ensuring that a current informed consent is obtained from each 
subject or the subject’s LAR.  Subjects will be informed in simple terms and all questions 
answered about the objectives, procedures, and risks of study participation both verbally and in 
writing in accordance with current regulations.  A signed, dated, IRB-approved written informed 
consent form will be obtained prior to any study-specific procedure.   
6.3 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, any process that is 
noted in the protocol or GCP requirements or any critical study procedures with specific 
instructions in ancillary documents referenced in the protocol.  
  
The noncompliance may be either on the part of the subject, the investigator, or the study site 
staff. Following a deviation(s), corrective actions should be developed by the site and 
implemented promptly. All individual protocol deviations will be addressed in subject study 
records.  
  
It is the responsibility of the site PI and personnel to use continuous vigilance to identify and 
report deviations within five working days of identification of the protocol deviation, or within 
five (5) working days of the scheduled protocol-required activity. All deviations must be 
promptly reported per the protocol deviation reporting procedures. Protocol deviations must be 
sent to the local IRB/IEC per their guidelines. The site PI and personnel are responsible for 
knowing and adhering to their IRB requirements. A completed copy of the Protocol Deviation 
Form must be maintained in the Regulatory File, as well as in the subject’s chart if the deviation 
is subject specific.  
6.4 
Monitoring Activities 
The Sponsor and/or its designee will monitor the clinical trial.  At regular intervals, the monitor 
will visit the investigator’s site to assess the progress and conduct of the study.  CRFs, as well as 
source documents, must be available for review.  The monitor must have access to all trial-
related documents.   


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6.5 
Quality Assurance Audits and Regulatory Inspections 
The Sponsor, its designee, or a regulatory authority may audit the study.  Study-related 
documents must be made available for auditing purposes.   
The investigator is advised to contact the Sponsor immediately if a regulator contacts the site 
inquiring or requesting to inspect and/or audit the investigative study site and/or this clinical 
trial.  
6.6 
Disclosure and Confidentiality 
By signing the protocol, the investigator agrees to keep all information provided by the Sponsor 
or its designee in strict confidence and to request similar confidentiality from his/her staff and 
the IRB.  Study documents provided by the Sponsor (protocols, investigators’ brochure, 
CRFs/eCRFs, and other material) will be stored appropriately to ensure their confidentiality.  
Data generated by this study will be considered confidential by the investigator except to the 
extent that is included in a publication as provided in the investigator’s respective contractual 
agreements.  A description of this clinical trial will be available on 
http://www.ClinicalTrials.gov. 
6.7 
Changes in Study Personnel 
If there is a change of any personnel listed on the FDA form 1572, a new form reflecting the 
change must be completed and forwarded to the Sponsor or its designee including, when 
applicable, any new staff member’s signed and dated curriculum vitae, current medical license 
(as appropriate), and signed financial disclosure statement. 
6.8 
Study Record Retention 
Study related records, including the regulatory file, study drug accountability records, consent 
forms, subject source documents and electronic records should be maintained for a period of 2 
years following the date a marketing application is approved for the investigational product for 
the indication for which it is being investigated; or, if no application is to be filed or if the 
application is not approved for such indication, until 2 years after the investigation is 
discontinued and the regulatory authority is notified. These documents should be retained for a 
longer period, however, if required by local policies or regulations. No records will be destroyed 
without the written consent of the sponsor.  
6.9 
Publication and Data Sharing Policy  
Reporting and publication processes will follow applicable laws and guidelines and ensure that 
the design and results of the trial are reported in an accurate and complete manner.  Author lists 
and contributorship statements will accurately reflect all substantial intellectual contributions to 
the research and be in accordance with the policies associated with the journals where published. 
 
 
 
 


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7 
REFERENCES 
 
Blake DF, Crowe M, Lindsay D, Brouff A, Mitchell SJ, Pollock N. Comparison of tissue 
oxygenation achieved breathing oxygen from a demand valve with four different mask 
configurations. Diving and Hyperbaric Medicine. 2018; 48 (4): 209-217. 
 
Blake DF, Crowe M, Lindsay D, Brouff A, Mitchell SJ, Leggat PA, Pollock N. Comparison of 
tissue oxygenation achieved breathing oxygen using different delivery devices and flow rates. 
Diving and Hyperbaric Medicine. 2020; 50 (1): 34-42. 
 
Diffusion Pharmaceuticals, Inc. Investigators Brochure for Trans Sodium Crocetinate, Edition 
13, July 2017. 
 
Diffusion Pharmaceuticals LLC. A randomized, double-blinded, placebo-controlled phase 1/2 
dose-range-finding study to evaluate the safety, efficacy and pharmacokinetics of multiple once 
daily intravenous doses of trans sodium crocetinate (TSC) in patients with intermittent 
claudication (IC). Protocol 100-301. Clinical Study Unpublished Report. September, 2010. 
 
Diffusion Pharmaceuticals LLC. Open-label Phase 1/2 (Safety Lead-in) Study of Trans Sodium 
Crocetinate (TSC) with Concomitant Treatment of Fractionated Radiation Therapy and 
Temozolomide in Newly Diagnosed Glioblastoma (GBM) Patients to Evaluate Safety and 
Efficacy. Protocol 100-202. Clinical Study Unpublished Report. November, 2015. 
 
Diffusion Pharmaceuticals LLC. A double-blind, placebo-controlled phase 1 study to evaluate 
the tolerability and pharmacokinetics of a single intravenous injection of trans sodium 
crocetinate in healthy volunteers. Protocol 100-001. Clinical Study Unpublished Report. March, 
2008. 
 
Diffusion Pharmaceuticals LLC. The effects of trans sodium crocetinate on infarct volume in a 
permanent model of ischemic brain injury in the rat. Unpublished Report 1000-P018. January, 
2007. 
 
Ercengiz A, Mutluoglu M. Hyperbaric transcutaneous oximetry. StatPearls Publishing Jan 2020. 
 
Fife CE, Smart DR, Sheffield PJ, Hopf HW, Hawkins G, Clarke D. Transcutaneous Oximetry in 
Clinical Practice: Consensus statements from an expert panel based on evidence. Undersea and 
Hyperbaric Medicine. 2009; 36 (1): 43-53. 
 
Gainer JL. Trans-sodium crocetinate for treating hypoxia/ischemia. Expert Opin Investig Drugs. 
2008; 17(6):917-24. 
 
Greenwood TW, Gilchrist AG. Hyperbaric oxygen and wound healing in post-irration head and 
neck surgery. Brit J Surg. 1973; 60 (5): 394-397. 
 


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Hiatt W, Barz K, Gainer J, Mohler E. Evaluation of Trans Sodium Crocetinate on Safety and 
Exercise Performance in Patients with Peripheral Artery Disease and Intermittent Claudication. 
Circulation. 2010; 122:A12341. 
 
Huxley VH, Kutchai H. The effect of the red cell membrane and a diffusion boundary layer on 
the rate of oxygen uptake by human erythrocytes. J Physiol. 1981; 316:75-83. 
 
Laidig KE, Gainer JL, Daggett V. Altering diffusivity in biological solutions through 
modification of solution structure and dynamics. Journal of the American Chemical Society. 
1998; 120(36):9394-95 
 
Manabe H, Okonkwo D, Gainer JL, Clarke R, Lee KS. Protection against focal ischemic injury 
to the brain by trans-sodium crocetinate. J Neurosurg. 2010; 113: 802-809. 
 
Mohler ER, Gainer JL, Whitten KM, Eraso LH, Tanaporn PK, Bauer T.  Evaluation of trans 
sodium crocetinate on safety and exercise performance in patients with peripheral artery disease 
and intermittent claudication. Vascular Medicine. 2011; 16(5):346-353. 
 
Stennett AK, Dempsey GL, Gainer JL. Trans sodium crocetinate and diffusion enhancement. 
Journal of Physical Chemistry. 2006; 110(37):18078-80. 
 
Trinks TP, Blake DF, Young DA, Thistlewaite K, Vangaveti VN. Transcutaneous oximetry 
measurements of the leg: comparing different measuring equipment and establishing values in 
healthy young adults. Diving and Hyperbaric Medicine. 2017; 47 (2): 82-87. 
 
Weaver LK. Transcutaneous Oxygen and Carbon Dioxide Tensions Compared to Arterial Blood 
Gases in Normals. Respiratory Care Vol. 52 No 11, Nov 2007. 
 
Young DA, Blake DF, Brown LH. Transcutaneous oximetry measurement: normal values for the 
upper limb. Diving and Hyperbaric Medicine. 2012; 42 (4): 208-213.


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8 
APPENDICES 
8.1 
Laboratory Parameters 
 
CBC 
White blood cells (WBC), red blood cells (RBC), Hemoglobin, Hematocrit, mean corpuscular volume (MCV), mean 
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), 
Platelet Count, mean platelet volume (MPV) and Differential (Absolute and Percent - Neutrophils, Lymphocytes, 
Monocytes, Eosinophils, and Basophils)  
BMP 
BUN/Creatinine Ratio (calculated), Calcium, Carbon Dioxide, Chloride, Creatinine with GFR Estimated, Glucose, 
Potassium, Sodium, Urea Nitrogen (BUN) 
Serology 
HIV-1/HIV-2 Antibodies (HIV), Hepatitis B surface Antigen (HBsAg), Hepatitis C Antibody (HCVAb) 
Pregnancy 
Serum pregnancy test at screening, urine pregnancy test at clinic arrival 
Urine Drug Screen 
Amphetamines, barbiturates, benzodiazapenes, cannabinoids, cocaine, opiates, phencyclidine 
Alcohol Screen 
Breathalyzer 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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8.2 
Schedule of Events 
 
Screening 
Treatment (Day 0) 
Follow Up 
 
Day -21 to 
Day 0 
Clinic 
Arrival 
Equilibration/ 
Baseline: 
70 minutes  
on O2  
Treatment/ 
Evaluation:     
60 minutes on 
O2 
Post-
Treatment: 
30 minutes 
on Room Air 
Clinic 
Observa-
tion 
48 Hours (+ 2 
Days) 
Informed Consent 
X 
 
 
 
 
 
 
Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Medical history 
X 
X 
 
 
 
 
 
Concomitant medications  
X 
X 
X 
X 
X 
X 
X 
BMI (kg/m2) 
X 
 
 
 
 
 
 
Body weight (kg) 
 
X 
 
 
 
 
 
Physical Exam 
X 
X 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
Vital signs (HR, BP, RR, Temp) 
X 
X 
X 
X 
X 
X 
 
SpO2 b 
X 
 
X 
X 
 
 
 
12-lead ECG 
X 
 
 
 
 
 
 
Laboratory (CBC, BMP, HIV, Hep 
B, Hep C) 
X 
 
 
 
 
 
 
Pregnancy testa 
X 
X 
 
 
 
 
 
Urine drug screen 
X 
X 
 
 
 
 
 
Alcohol breathalyzer screen 
X 
X 
 
 
 
 
 
Subject resting quietly supine with 
one pillow, or semi-recumbent 
 
 
X 
X 
 
 
 
TcpO2 sensors in place 
 
X 
X 
X 
 
 
 
PK measurements 
 
 
X 
X 
X 
 
 
Oxygen:simple face mask at 6 
L/min 
 
 
X 
X 
 
 
 
TcpO2b 
 
 
X 
X 
 
 
 
Administer Study Drug  
 
 
 
X 
 
 
 
TcpO2 sensors removed 
 
 
 
 
X 
 
 
Overnight stay at clinic 
 
 
 
 
 
X 
 
Adverse Events 
 
 
X 
X 
X 
X 
X 
a For all females, with serum pregnancy test at screening and urine pregnancy test at clinic arrival 
b SpO2 and TcpO2 will be assessed every 5 minutes during the baseline period. After study drug administration they will be assessed at 1, 2, and 5 minutes, and then every 5 minutes thereafter. 


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8.3 
Investigator Signature Page 
 
DOCUMENT TYPE: 
Clinical protocol  
DOCUMENT NUMBER: 
200-301 
COMPOUND: 
Trans Sodium Crocetinate 
STUDY TITLE: 
Randomized, double-blind, placebo-controlled, 
pharmacokinetic, pharmacodynamic study of Trans 
Sodium Crocetinate utilizing Transcutaneous Oximetry 
Measurement in healthy volunteers 
 
CLINICAL PHASE: 
1 
INDICATION: 
Healthy volunteers  
IND NUMBER: 
68579  
 
SPONSOR: 
Diffusion Pharmaceuticals Inc. 
1317 Carlton Avenue, Suite 200 
Charlottesville, VA 22902 
DOCUMENT 
VERSION/STATUS: 
 
Final  
DOCUMENT 
RELEASE/AMENDMENT 
DATE: 
Amendment 2: March 9, 2021 
Amendment 1: February 12, 2021 
January 14, 2021 
 
This acknowledges receipt of the above protocol. 
 
Investigator Name: _____________________________ (Print) 
 
Investigator Signature: __________________________ Signature Date: ___________  
 

SAP_001.pdf: 
 
 
 
Title: 
Randomized, double-blind, placebo-controlled, pharmacokinetic, pharmacodynamic 
study of Trans Sodium Crocetinate utilizing Transcutaneous Oximetry Measurement in 
healthy volunteers 
 
NCT Number: 
NCT04808622 
 
Document Type: 
Statistical Analysis Plan  
 
Document Date: 
08 April 2021 


 
Protocol No. 200-301| Altasciences Project No. DIF-P3-945| Version Final 1.0 | 2021/04/08 
 
Page 1 of 31 
 
 
STATISTICAL ANALYSIS PLAN 
 
 
For: 
Diffusion Pharmaceuticals Inc. 
 
 
PROTOCOL No. 200-301 
 
Randomized, Double-blind, Placebo-controlled, Pharmacokinetic, Pharmacodynamic Study of 
Trans Sodium Crocetinate utilizing Transcutaneous Oximetry Measurement in Healthy 
Volunteers 
 
Altasciences Project No. DIF-P3-945 
 
Prepared by: 
Altasciences Company Inc. 
575 Armand-Frappier Boulevard 
Laval, Quebec 
Canada, H7V 4B3 
 
 
Version Final: 1.0 
Date: 2021/04/08 
 


Sanaa Boudhar
Digitally signed by Sanaa 
Boudhar 
Date: 2021.04.08 16:01:37 -04'00'
Dominique St-Fleur
Digitally signed by Dominique St-Fleur 
Date: 2021.04.08 16:09:02 -04'00'
Chris Galloway, MD
Digitally signed by Chris 
Galloway, MD 
Date: 2021.04.09 08:37:23 -06'00'


 
 
 
Protocol No. 200-301| Altasciences Project No. DIF-P3-945| Version Final 1.0 | 2021/04/08 
Page 3 of 31 
VERSION CONTROL 
Version 
Date 
Author 
Description of Changes 
1.0 
2021/04/08 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
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TABLE OF CONTENTS 
STATISTICAL ANALYSIS PLAN APPROVAL ..........................................................................2 
VERSION CONTROL ....................................................................................................................3 
TABLE OF CONTENTS .................................................................................................................4 
TABLE OF TABLES ......................................................................................................................6 
ABBREVIATIONS .........................................................................................................................7 
1 
INTRODUCTION ..................................................................................................................9 
2 
STUDY OBJECTIVES ........................................................................................................10 
3 
STUDY DESIGN .................................................................................................................11 
3.1 
General Description .........................................................................................................11 
3.2 
Treatments .......................................................................................................................11 
3.2.1 
TcpO2 Sensor Electrode Placement: ......................................................................11 
3.2.2 
O2 Equilibration and Baseline Period: ...................................................................11 
3.3 
Study Procedures .............................................................................................................11 
3.4 
Randomization and Unblinding Procedures ....................................................................11 
4 
ANALYSIS POPULATION ................................................................................................13 
5 
STUDY SUBJECTS ............................................................................................................14 
5.1 
Disposition .......................................................................................................................14 
5.2 
Protocol Deviations .........................................................................................................15 
6 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ...............................16 
7 
PHARMACODYNAMIC ANALYSIS ...............................................................................17 
7.1 
TcpO2 Data ......................................................................................................................17 
7.2 
Missing Values ................................................................................................................17 
7.3 
Measurements Below the Lower Limit of Quantitation ..................................................17 
7.4 
Nominal/Actual Record Times ........................................................................................17 
7.5 
Change from Baseline Calculation ..................................................................................17 
7.6 
Non-Compartmental Analysis .........................................................................................17 
7.7 
Statistical Methodology ...................................................................................................18 
7.7.1 
Summary Statistics.................................................................................................20 
7.8 
Analysis of the Primary Endpoint ...................................................................................20 
8 
SAFETY ANALYSIS ..........................................................................................................22 
8.1 
Adverse Events ................................................................................................................23 


 
 
 
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8.2 
Clinical Laboratory Evaluations ......................................................................................24 
8.3 
Vital Signs .......................................................................................................................24 
8.4 
Physical Examination Findings .......................................................................................24 
8.5 
12-Lead ECG Findings ....................................................................................................24 
9 
DATA HANDLING AND PRESENTATION ....................................................................25 
9.1 
Safety Analysis Presentation ...........................................................................................25 
9.2 
Pharmacokinetic Analysis ...............................................................................................26 
9.3 
Pharmacodynamic Analysis ............................................................................................26 
9.4 
Analysis Timepoints ........................................................................................................26 
9.5 
Methods for Handling Missing Data ...............................................................................27 
9.6 
Interim Analysis ..............................................................................................................27 
10 
CHANGES TO PROTOCOL-SPECIFIED ANALYSES ....................................................28 
11 
GENERAL INFORMATION RELATED TO DATA PRESENTATIONS ........................29 
APPENDIX A 
STUDY SCHEDULE ...................................................................................30 


 
 
 
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TABLE OF TABLES 
Table 1: 
Objectives and related endpoints .......................................................................... 10 
Table 2: 
Data presentations for study subject information ................................................. 14 
Table 3: 
Data presentations for demographic and other baseline characteristics ............... 16 
Table 4: 
Pharmacodynamic Parameters of tcpO2 and Change in tcpO2 Levels .................. 18 
Table 5: 
Data presentations for safety assessments ............................................................ 22 
  
 


 
 
 
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ABBREVIATIONS 
AE 
Adverse event 
ATC 
Anatomical therapeutic chemical 
AUEC 
Area under effect time curve 
BMI 
Body mass index 
BMP 
Basic metabolic panel 
CBC 
Complete blood count 
CI 
Confidence interval 
CRF 
Case report form 
CSR 
Clinical study report 
CV% 
Coefficient of variability 
DMP` 
Data management plan 
DTS 
Deviation tracking system  
ECG 
Electrocardiogram 
EOS 
End of study 
ET 
Early termination 
ICF 
Informed consent form 
ICH 
International Conference on Harmonisation 
ITT 
Intent-to-treat 
IV 
Intravenous 
LLOQ 
Lower limit of quantitation 
ln 
Natural log 
Max 
Maximum 
MedDRA 
Medical Dictionary for Regulatory Activities 
Min 
Minimum 
NCA 
Non-compartmental analysis 
O2 
Oxygen 
PD 
Pharmacodynamics(s) 
PK 
Pharmacokinetic(s) 
PP 
Per protocol 
PT 
Preferred term 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System organ class 
SOP 
Standard operating procedure 
SpO2 
Oxygen saturation 
TCOM 
Transcutaneous oximetry 
tcpO2 
Transcutaneous tissue oxygen tension 


 
 
 
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TEAE 
Treatment-emergent adverse event 
TFLs 
Tables, figures, and listings 
TSC 
Trans Sodium Crocetinate 
WHO-DDE 
World Health Organization Drug Dictionary- Enhanced 
 


 
 
 
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1 INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to prospectively outline the types of 
analyses and presentations of the data that will form the basis for conclusions regarding this 
clinical investigation.  The analyses defined in this plan should answer the safety, 
pharmacokinetic (PK), and pharmacodynamics (PD) objectives outlined in the protocol, and 
explain in detail how the data will be handled and analyzed, adhering to commonly accepted 
standards and practices for biostatistical analysis in the medical device industry. 
 
This document contains information to support the generation of a Clinical Study Report (CSR) 
for Clinical Protocol 200-301, including detailed descriptions of the statistical methods to be 
applied, as well as the analysis summary tables and figures and subject data listings intended to 
present the analysis results. 
 
This clinical trial is designed to further clarify and optimize the dosing of trans sodium 
crocetinate (TSC) by directly measuring tcpO2 in multiple locations on the lower extremity of a 
healthy volunteer after a single dose of TSC, and determine if subjecting the subjects to 100% 
O2 or Room Air (RA) is a more sensitive for evaluating the effect of TSC. 
 
The sample size selected for this clinical investigation with 6 dose groups is based on clinical 
judgement.  While hypotheses will be evaluated at the end of the study, there is no pre-specified 
statistical power that was used to establish the sample size. 
 
The planned analyses identified in this SAP may be included in regulatory submissions, medical 
presentations and manuscripts.  Exploratory analyses, not identified in this SAP, may be 
performed to support the clinical development program.  Any post-hoc or unplanned analyses 
that are performed but not identified in this SAP will be clearly identified in the CSR.  The 
structure and content of this SAP provides sufficient detail to meet the requirements identified by 
the Food and Drug Administration (FDA) and the International Conference on Harmonization 
(ICH) Guidance on Statistical Principles for Clinical Trials. 
 
The analyses described in the SAP are based upon the final protocol version Amendment 2: 
dated 2021/03/09. 
 
 


 
 
 
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2 STUDY OBJECTIVES 
The objectives of the study and corresponding study endpoints are detailed in Table 1. 
Table 1: 
Objectives and related endpoints 
Objective 
Endpoint 
Analysis 
Primary  
To determine the dose-
response of TSC on tcpO2 
following a single 
administration of TSC in 
subjects breathing O2.  
 
The primary PD endpoints are: 
 
Time-matched change in tcpO2 
levels 
 The following PD parameters will 
be calculated for time-matched 
change in tcpO2: 
o AUEC, Emax, and TEmax 
Refer to Section 7 
Secondary  
To evaluate the safety and the 
tolerability of TSC on tcpO2 
following a single 
administration of TSC in 
subjects breathing  O2. 
Assessment of adverse events, new 
medications, laboratory (CBC, 
BMP), vital signs, and SpO2. 
Refer to Section 8 
Abbreviations: BMP = basic metabolic panel; CBC = complete blood count; O2 = oxygen; PD = pharmacodynamic; 
SpO2 = oxygen saturation; tcpO2 = transcutaneous tissue oxygen tension; TSC = trans sodium crocetinate 
 
 


 
 
 
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3 STUDY DESIGN  
3.1 
General Description 
This single center clinical trial is designed to determine the dose response of TSC on tcpO2 
following a single administration of TSC in subjects breathing  O2. Study assessments include 
tcpO2 levels, SpO2, and PK.   
This is a randomized, double-blind, placebo-controlled study. A maximum of 30 healthy 
volunteers, aged 18-50 will be randomized to a single intravenous (IV) bolus dose of TSC 
(0.5, 1.0, 1.5, 2.0, or 2.5 mg/kg) or placebo (normal saline).  
 
3.2 
Treatments 
The TSC will be administered as a one-time IV bolus injection to 5 unique subjects per dose 
cohort, to include 5 TSC dose levels and placebo. Placebo will consist of 7 mL of normal saline.   
3.2.1 TcpO2 Sensor Electrode Placement: 
The TcpO2 sensor electrodes will be applied to the left or right lower extremity. Four (4) sensors 
will be applied to the following locations:  
 
 Sensor 1: Mid-dorsum of the foot  
 Sensor 2: 10 cm distal to the lateral femoral epicondyle  
 Sensor 3: 5 cm proximal to the anterior aspect of the lateral malleolus  
 Sensor 4: 5 cm proximal from the center of the medial malleolus  
 
3.2.2 O2 Equilibration and Baseline Period: 
After the tcpO2 sensors have been applied and tested, subjects will be placed on O2 via simple 
face mask at 6 L/minute, and will remain on O2 for 70 minutes prior to study drug administration. 
At the end of the 70-minute equilibration/baseline period, subjects will continue on O2 and 
receive a single IV bolus injection of TSC at a dose of 0.5, 1.0, 1.5, 2.0 or 2.5 mg/kg, or placebo.  
After study drug administration, subjects will be evaluated for 60 minutes, with tcpO2 values and 
SpO2 recorded at 1, 2, and 5 minutes immediately post-dose and then every 5 minutes thereafter. 
In addition to the assessment of tcpO2 as described, continuous tcpO2 measurements will be 
recorded by the TCOM machine in graphical format. 
 
3.3 
Study Procedures 
For complete details on the study assessments to be performed for each study period, refer to 
APPENDIX A.  
3.4 
Randomization and Unblinding Procedures 
Volunteers will be randomized to one of 5 TSC doses or placebo in a 1:1:1:1:1:1 schema, with 5 
subjects randomized to each of the 6 cohorts. 


 
 
 
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Subjects will be randomized to the following dose cohorts:  
 
Dosing 
Cohort 
# of Subjects in 
Cohort 
TSC Dose 
(mg/kg)  
Placebo 
(mL) 
1 
5 
0.5 
N/A 
2 
5 
1.0 
N/A 
3 
5 
1.5 
N/A 
4 
5 
2.0 
N/A 
5 
5 
2.5 
N/A 
6 
5 
N/A 
7 mL 
N/A: Not Applicable 
Each TSC dose will be calculated based on the subject’s body weight in kg at the dosing level 
they are randomized to. Each individual dose of placebo will be normal saline 7 mL. 
To maintain the double-blind, study drug administration will be performed by unblinded medical 
staff who will not be involved in other study procedures, including subject assessment.  Subjects, 
investigators, and study coordinators will not see the injection or injection site or be aware of 
randomization. 
Because TSC is highly colorized, this study will utilize unblinded and blinded personnel as 
follows: 
 Unblinded study team: Pharmacist(s) and administering personnel  
 Blinded study team: Investigator(s) and study coordinators 
The unblinded study team will take specific steps to ensure that the subjects and blinded study 
personnel are not made aware of treatment assignment.  
 Prior to release of the study drug from the pharmacy, the pharmacist will package the 
prepared syringe(s) in foil or acceptable masking material that masks the contents (i.e. 
cannot see through the material). The study drug should not be removed from the 
masking material until the time of administration to the subject. 
 The medical staff assigned to administer the masked study drug will use a shroud to 
ensure that the subject is blinded to treatment.  The shroud may be made of any material 
and be of any size as long as it sufficiently blocks the subject’s view of the injection site 
and injection syringe at the time of administration.  The shroud only needs to be in place 
at the time of study drug injection.   
 A cloth or paper drape will be added underneath the injection site in the event that a 
droplet falls from the syringe as part of the injection process. The drape will be removed 
and folded up following the injection and before the shroud is removed so that it is not 
visible to the study team or subject. 


 
 
 
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 At the time of injection, the blinded study team must position themselves so that the 
injection site and syringe(s) are not visible during study drug injection. The subject will 
be asked to look away from the injection site at the time of study drug administration. 
 
4 
ANALYSIS POPULATION 
Safety population will include healthy adult volunteers meeting all inclusion and exclusion 
criteria.   
The following analysis populations will be used to summarize the results: 
• 
Intent-to-Treat Population: The intent-to-treat (ITT) population includes all randomized 
subjects. Subjects in the ITT population will be analyzed as randomized. 
• 
Per Protocol Population: The per protocol (PP) population includes subjects in the ITT 
population who do not have significant protocol deviations and complete the study. 
Protocol deviations will be assessed prior to database lock and release of the 
randomization codes.  
The PP population will be analyzed using the observed data for efficacy variables. 
Subjects in the PP population will be analyzed as treated, in the case there is an issue with 
the randomization assignment for the subject. 
• 
Safety Population: The Safety population is synonymous with the ITT population. 
• 
PK Population: The PK Population will include all subjects who have received at least 
one dose of the study drug and had at least 1 post-dose PK sample collection. 
• 
PD Population: The PD Population will include all subjects in the ITT population who 
receive the study drug and have at least 1 pre-dose PD measurement prior to study drug 
dosing and at least 1 time-matched PD measurement after study drug dosing. 


 
 
 
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5 STUDY SUBJECTS 
All recorded data will be presented in the data listings and summarized in table format.  The 
disposition and protocol deviations will be presented for the safety population as described in 
Table 2.   
Table 2: 
Data presentations for study subject information  
Data 
Variables 
Presentation 
Disposition and analysis 
population 
Subject, completion status (i.e., 
completed or withdrawn), reason for 
withdrawal, analysis population 
determination 
Listings: 
 
Disposition, 
 
Analysis population, 
 
Randomization,  
Summary table including: 
 
Number (N) of subjects 
enrolled, 
 
Number (N) of subjects 
randomized, 
 
N and %of subjects who 
completed the study, 
 
N and % of subjects who 
discontinued from the study 
by primary reason for 
discontinuation and the 
overall number, independent 
of the reason for 
discontinuation. 
 
N and % of subjects included 
in each of the study 
populations. 
Protocol deviations 
Protocol deviations 
Listing: 
 
General deviations 
 
5.1 
Disposition 
Subject disposition will be summarized for all subjects enrolled in this study. The percentages 
will be calculated using the number of subjects randomized in each treatment group as the 
denominator. The number of subjects who complete the study, the number of subjects who 
prematurely withdraw from the study, and the reason for withdrawal, will be summarized by the 
randomized dose group and overall, independent of the randomized treatment assignment. 
 
A listing of the disposition of all consented subjects will be provided. A listing of subjects 
included in each of the analysis populations will also be provided. A listing of the randomization 
assignment for each subject will also be presented. 


 
 
 
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5.2 
Protocol Deviations 
Deviations will be recorded in the clinic deviation tracking system (DTS) and presented as 
entered in a general protocol deviation listing and in a summary table as well.  
 
 


 
 
 
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6 DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS 
All recoded data will be presented in the listings and summarize in table format for 
demographics and other baseline characteristics as described in Table 3.  
Table 3: 
Data presentations for demographic and other baseline characteristics  
Data 
Variables 
Presentation 
Demographics, and 
Baseline characteristics 
Sex, age, ethnicity, and race. 
Height, body weight at screening, 
and body mass index (BMI) 
Listing and summary table 
Lifestyle 
Drinking and smoking habits 
Listing 
Medical history 
All medical history findings 
Listing 
Note: includes coded terms 
(system organ class [SOC] 
and preferred term [PT]) 
Prior medications 
All medications taken prior to 
study drug administration  
(including prescription 
medications, nonprescription 
medications, dietary supplements, 
vitamins, or herbal medications) 
Listing 
Note: includes coded terms 
(anatomic therapeutic 
chemical [ATC] level 1 and 
preferred name) 
Contraception 
Contraceptive method 
Listing 
 
 


 
 
 
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7 PHARMACODYNAMIC ANALYSIS 
The PD analysis will be performed on tcpO2 pre dose and post dose values, and according to 
Altasciences SOPs. 
7.1 
TcpO2 Data 
TcpO2 in subjects breathing oxygen (O2) will be measured prior to study drug administration 
(baseline; Period 1) and following a single administration of TSC (Period 2) to determine the 
dose response of TSC on tcpO2 levels. 
Individual subject and summary of tcpO2 levels will be listed.  
7.2 
Missing Values 
The lack of tcpO2 values due to failure to record the tcpO2 measurement, a lost or compromised 
record or due to the subject’s early termination from the study will be considered “missing” in 
the dataset, and no imputation will be done. 
If the actual record time of a PD measurement is unknown, but a valid tcpO2 value has been 
recorded, the nominal record time will be used for the PD analysis and will be presented in 
listing excluded from descriptive statistics. 
7.3 
Measurements Below the Lower Limit of Quantitation 
A non-detectable tcpO2 value associated with baseline and postdose record times will be 
replaced with zero for PD analysis.  
7.4 
 Nominal/Actual Record Times 
For the time-matched change in tcpO2 analysis, the non-compartmental analysis (NCA) on PD 
data will be based on the nominal record time. For the separate analysis of each study period, the 
NCA on PD data will be based on the actual record time.   
The time-matched change in tcpO2 levels vs time profiles, the mean tcpO2 levels vs time profiles, 
the mean time-matched change in tcpO2 levels vs time profiles and the tables presenting 
summary statistics of tcpO2-time or time-matched change in tcpO2 level-time data will be 
presented using nominal record times. The individual raw tcpO2 levels vs time data will be 
presented using actual record times. 
7.5 
Change from Baseline Calculation 
The baseline period is defined as the 60 minutes (following a 10-minute equilibration period) of 
O2 delivery via a simple face mask prior to study drug administration. 
The time-matched tcpO2 change from baseline will be calculated by time point and subject using 
the following equation: 
Time-matched change in tcpO2 = ([tcpO2, postdose, Time x] - [tcpO2, baseline, Time x]). 
Where Time x is the matching record time baseline and postdose. 
7.6 
Non-Compartmental Analysis 
The following configuration for the NCA of tcpO2 levels of each period and time-matched 
change in tcpO2 levels (with Phoenix® WinNonlin® version 8, or higher) will be used: 


 
 
 
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 Data: 
 
 
Serial sampled data 
 Model/Dose options Type:  
Plasma (200 -202) / Extravascular  
 AUEC Calculation Method: 
Linear Trapezoidal/Linear Interpolation  
The  NCA will be performed using the median value from all sensors (calculated using the 
median value from each sensor). 
The PD parameters for tcpO2 levels of each period and time-matched change in tcpO2 levels are 
defined in Table 4. 
Table 4: Pharmacodynamic Parameters of tcpO2 and Change in tcpO2 Levels 
PD parameter 
Definition  
Emax  
Maximum observed effect  
TEmax  
Time of maximum observed effect; if it occurs at more than one 
time point, TEmax is defined as the first time point with this value 
AUEC 
Area under the effect-time curve from time 0 through 60 minutes 
postdose 
7.7 
Statistical Methodology 
Specific algorithms are discussed for imputing missing or partially missing data, if deemed 
appropriate, under specific data topics.  Imputed or derived data will be flagged in the individual 
subject data Listings.  Imputed data will not be incorporated into any raw or primary datasets.  
The imputed data will be retained in the derived / analysis datasets. 
 
The total duration for a subject on-study will be calculated as the difference between the date of 
the start of Period 1 to the last day of observation plus 1 day.  All calculations for defining the 
duration on-study will follow the algorithm DURATION = [STUDY COMPLETION OR 
WITHDRAW DATE – PERIOD 1 START DATE + 1].  Extra measurements (such as 
unscheduled or repeat assessments) will not be included in the descriptive statistics, but will be 
included in subject listings.  
Demographic and safety summary statistics will be presented for all subjects in the ITT / Safety 
Population. Data from subjects excluded from an analysis population will be included in the data 
listings, but not in the summaries.   
For changes from baseline (pre-administration of study drug), only changes from baseline (pre-
administration of study drug) to the defined post-administration time point will be shown in the 
listings and tabulations.  
Listings will include all assessments (including repeated and unscheduled measurements) in 
chronological order with the scheduled measurements. Unscheduled and/or repeated 
measurements (unless used as the baseline measurement) will not be included in the tables.        
If any baseline (pre- administration) value is missing, the change from baseline will not be 
calculated.   
This section details general conventions to be used for the statistical analyses.  The following 
conventions will be applied to all data presentations and analyses. 


 
 
 
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 Summary statistics will consist of the count, percentage, and exact 95% and 97.5% 
binomial confidence intervals, depending on the variable, at each level for categorical 
variables, and the sample size (n) mean, median, standard deviation (SD), minimum, and 
maximum values for continuous variables. 
 
 All mean and median values will be formatted to one more decimal place than the 
measured value.  Standard deviation values will be formatted to two more decimal places 
than the measured value.  Minimum and maximum values will be presented with the 
same number of decimal places as the measured value. 
 
 The number and percentage of responses will be presented in the form XX (XX.X%). 
 
 All probability values will be rounded to four (4) decimal places.  All p-values that round 
to 0.0000 will be presented as ‘<0.0001’ and p-values that round to 1.0000 will be 
presented as ‘>0.9999’.  P-values <0.05 will be considered to be statistically significant.   
 
 All summary tables will include the analysis population sample size (i.e., number of 
subjects with values for the analysis). 
 
 Study Day 1 is defined as the day the subject receives the study drug.  All study days are 
determined relative to the day of the administration of study drug.  
 
 Baseline values will be defined as those values recorded closest to, but prior to, the 
administration of study drug. 
 
 Change from baseline will be calculated as follows:  
o Change = Post-baseline value  baseline value. 
 
 Date variables will be formatted as DDMMMYYYY for presentation. 
 
 Tables, figures, and Listings will be presented in landscape orientation. 
 
 SAS Version 9.4 will be the statistical software package used for all data analyses.   
 
 All data from this study will be presented in Listings.  All Listings will be sorted by 
clinical site, subject number, and visit date, as applicable.   
 
Table and Listing numbering will follow ICH guidelines for post-text table and Listing 
numbering. 


 
 
 
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The header of each table and Listing will include the sponsor’s name and the study number.  The 
information and explanatory notes to be provided in the “footer” or bottom of each table and 
Listing will include the following information: 
1. 
Date and time of output generation. 
2. 
SAS® program name, including the path that generates the output. 
3. 
Any other output specific details that require further elaboration. 
 
In general, tables will be formatted with a column displaying findings for all subjects combined.  
Row entries in tables are made only if data exist for at least one (1) subject (i.e., a row with all 
zeros will not appear).  The only exception to this rule applies to tables that list the termination 
status of subjects (e.g., reasons for not completing the study).  In this case, zeros will appear for 
study termination reasons that no subject satisfied.  The summary tables clearly indicate the 
number of subjects to which the data apply and unknown or not performed are distinguished 
from missing data.  Tables, Listings, and figures will provide the units of measurement, unless 
not applicable. 
All summaries of AEs will be based on events that occurred during the study.  AEs will be 
mapped to preferred terms and body systems using the Medical Dictionary for Regulatory 
Activities (MedDRA) coding dictionary. The number and percentage of subjects experiencing 
AEs will be summarized by system organ class and preferred term.  Summaries by maximum 
severity and relationship to the study device and procedure will also be provided.  SAEs and AEs 
leading to discontinuation from the study will be presented by system organ class and preferred 
term. 
All tables, figures and listings, when appropriate, will be stratified by treatment. 
7.7.1 Summary Statistics 
Summary statistics of the individual tcpO2 levels, time-matched tcpO2 change from baseline 
levels and derived parameters will be calculated with the built-in Phoenix® WinNonlin® platform 
for the PD population. 
Summary statistics will be calculated for tcpO2 levels and time-matched tcpO2 change from 
baseline levels at each individual time point and for all PD parameters. Individual tcpO2 levels, 
time-matched tcpO2 change from baseline levels, and derived PD parameters obtained from the 
NCA will be summarized using the following descriptive statistics: number of observations (N), 
minimum, arithmetic mean, median, maximum, standard deviation (SD), and CV%. 
7.8 
Analysis of the Primary Endpoint 
Analysis of Change Over Time in tcpO2: 
The primary analysis will be based on the change over time in the tcpO2 measurements following 
a single administration of TSC in subjects breathing O2.  The time-matched changes in 5 minute 
increments for Period 1 (60 minute run-in on O2) to Period 2 (60 minute post-drug administration 
on O2) in 5 minute increments will calculated for each subject.  The first 5 minute interval in 
Period 1, following the 10 minute equilibration period, will be matched to the first 5 minute 
interval in Period 2.  This same approach will be followed for the remaining eleven 5 minute 
intervals.    
 


 
 
 
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The median value for each sensor will be calculated using the 10 second readings within a 5 
minute interval, calculated separately for Period 1 and Period 2.  The median will be calculated 
using the median value from each sensor. 
 
The intra-subject changes (Period 2 minute Period 1 in 5 minute intervals) will serve as the 
dependent variable in the repeated measures analysis. The 60 minute run-in on O2 is intended to 
account for the intra-subject variability over time and the time-match differences represent the 
least biased estimate of the effect of the study drug.  With 5 subjects per randomized treatment 
arm, the change over time should provide sufficient clinical information to determine if there is a 
drug effect, and if the effect in an active drug treatment arm is different than placebo. 
 
To compare the time-match changes in tcpO2, a 2-factor (treatment and time) repeated measures 
(time) analysis of variance (ANOVA) model will be used. Contrast statements within the model 
will facilitate comparisons between individual treatment arms over individual time points, 
providing guidance relative to the initial time and duration of separation. 
 
Analysis of AUEC Comparison:  
The trapezoidal rule will be used for the construct of the area under the curve (AUEC) analyses.   
The AUEC based on the time-matched differences, a 1-factor (treatment) analysis of variance 
model will be used, followed by a Dunnett’s test to compare the least square mean AUEC values 
from the 5 active drug groups relative to the placebo group.  
 
A separate AUC analysis will be performed, calculating the AUC for Period 1 and Period 2 for 
each subject.  The intra-subject difference (AUC from Period 2 minus the AUC from Period 1) 
will be analyzed using a repeated measures (Period) analysis of covariance (Period 1 AUC) 
followed by a Dunnett’s test to compare the least square mean AUC values from the 5 active 
drug groups relative to the placebo group.  
 
If the data within a treatment arm is skewed, suggesting that the least square mean value is not 
the best measure of central tendency, the data may be ranked and the analyses will be conducted 
using the ranked scores.   
Information regarding the examination of the distribution of the data   
To compare the time to the maximum increase in tcpO2, the event time distribution functions will 
be compared using a log rank test and presented using Kaplan-Meier curves. 
   
All probability values will be 2-sided and accepted at face value for this feasibility study; there 
will not be an adjustment for multiplicity.  For interpretation, probability values <0.05 will be 
considered significant; probability values <0.1 but ≥0.05 will be considered highly suggestive of 
a significant difference.  
 
 
 


 
 
 
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8 SAFETY ANALYSIS 
All data will be listed and summary tables for safety assessments will be presented for the safety 
population as detailed in Table 5. 
Table 5: Data presentations for safety assessments  
Data 
Variables 
Presentation 
Adverse events 
Adverse event (AE) description, 
date and time, severity, 
relationship to study drug, action 
taken, and outcome 
Listings, summary tables by randomized 
treatment ,  time, and overall, including 
number and percentage of subjects 
experiencing: 
 
Reported AEs, 
 
Treatment-emergent adverse events 
(TEAEs), 
 
Drug-related TEAEs (i.e. those with a 
relationship classified asRelated),  
 
TEAEs by relationship to study treatment 
(i.e. related or not related),  
 
TEAEs by severity,  
 
Serious TEAEs,  
 
Study drug-related serious TEAEs, 
 
TEAEs leading to withdrawal, and  
 
TEAEs with an outcome of death. 
Note: includes coded terms (system organ 
class [SOC] and preferred term [PT]) 
Concomitant 
medications 
All medications taken during the 
study (as defined in the protocol 
and including prior medications 
that are continued), dose, units, 
frequency, formulation, route, 
indication/ reason taken, and start 
and end date and time 
Listing 
Note: includes coded terms (anatomic 
therapeutic chemical [ATC] level 1 and 
preferred name) 
Extent of exposure 
Category of treatment, Study drug 
administration dose, date, time, 
formulation, frequency, route and 
compliance questions (if 
applicable) 
Listing 
Clinical laboratory 
evaluations 
Laboratory results (refer to section 
8.2 for parameters) 
Listings:  
 
All laboratory values by category,  
 
All out-of-range laboratory values, and 
 
All clinically significant laboratory values  


 
 
 
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Data 
Variables 
Presentation 
Vital signs 
Blood pressure, pulse, respiratory 
rate and body temperature 
Listings:  
 
All vital signs values, and  
 
Clinically significant vital signs values  
Summary tables by randomized treatment and 
overall. 
Physical 
examination 
Physical examination findings 
Listings: 
 
All physical examination findings, and  
 
Clinically significant physical 
examination findings 
Electrocardiograms 
(ECGs) 
ECG interpretations and findings 
Listings: 
 
All ECGs, and  
 
Clinically significant ECGs 
Note: Sub-bullets denote individual listings or tables to be generated. 
8.1 
Adverse Events 
An AE is defined as any untoward medical occurrence in a clinical investigation subject 
administered an study drug and which does not necessarily have a causal relationship with the 
treatment. An AE can therefore be any unfavorable and unintended sign (including a clinically 
significant abnormal laboratory finding, for example), symptom, or disease temporally 
associated with the use of a study drug, whether or not related to the study drug. 
Treatment emergent adverse events (TEAEs) are AEs not present prior to the exposure to study 
treatment or AEs already present that worsen in intensity or frequency following exposure to 
study treatment. All AEs reported following exposure to study treatment are considered TEAEs. 
All TEAEs will be assigned to a treatment using the following rules:  
 A TEAEs will be assigned to the last drug taken by the subject where the date and time of 
the last drug dosing is on or before of the start date and time of the event. Such 
assignment will be performed irrespective of any washout period between the start and 
stop dates of the TEAE.  
 Any TEAE started during the follow-up period will be assigned to the last treatment that 
the subject has taken.  
In case that the time of onset or time of resolution is unknown, worst case scenario will be 
considered. For the onset, 00h01 will be the time considered. For the resolution time, if the time 
is unknown, 23h59 will be the time considered.  
Frequency tables summarizing all TEAEs and all study drug-related TEAEs (including SOC and 
PT) will be presented by treatment and overall as described in Error! Reference source not 
found.. 
Adverse events will be coded using MedDRA and summarized by system organ class and 
preferred term and presented by randomized treatment assignment, Period 1 (60 minute run-in on  
O2) or Period 2 (60 minute post-drug administration on O2), and overall, independent of Period. 


 
 
 
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8.2 
Clinical Laboratory Evaluations 
Laboratory data will be listed for abnormal results and clinically significant (CS) abnormal 
results.  
Laboratory data will be presented using units as reported by the clinical laboratory. 
The following clinical laboratory tests listed will be performed as scheduled in 11APPENDIX A. 
CBC 
White blood cells (WBC), red blood cells (RBC), Hemoglobin, Hematocrit, mean corpuscular 
volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin 
concentration (MCHC), red cell distribution width (RDW), Platelet Count, mean platelet 
volume (MPV) and Differential (Absolute and Percent - Neutrophils, Lymphocytes, 
Monocytes, Eosinophils, and Basophils)  
BMP 
BUN/Creatinine Ratio (calculated), Calcium, Carbon Dioxide, Chloride, Creatinine with GFR 
Estimated, Glucose, Potassium, Sodium, Urea Nitrogen (BUN) 
Serology 
HIV-1/HIV-2 Antibodies (HIV), Hepatitis B surface Antigen (HBsAg), Hepatitis C Antibody 
(HCVAb) 
Pregnancy 
Serum pregnancy test at screening, urine pregnancy test at clinic arrival 
Urine Drug Screen 
Amphetamines, barbiturates, benzodiazapenes, cannabinoids, cocaine, opiates, phencyclidine 
Alcohol Screen 
Breathalyzer 
 
8.3 
Vital Signs 
Vital signs will include systolic and diastolic blood pressure, heart rate, and temperature. 
Values will be listed and summarized by time points for each treatment group, and overall. 
Changes from baseline will also be presented. 
Subject listing of vital signs values, clinically significant on-study vital signs and abnormal on-
study vital signs values will also be provided. 
 
8.4 
Physical Examination Findings 
Physical examination includes a review of the following: head, eyes, ears, nose, throat (HEENT), 
neck, chest, back, abdomen, extremities and neurological function. All physical examination 
records will be presented in a listing, by subject, and visit. Similarly, clinically significant on-
study physical examinations will be presented in a second listing. 
 
8.5 
12-Lead ECG Findings 
Subject listings will be provided for all ECG parameters, and clinically significant (CS) 
abnormal results.  


 
 
 
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9 DATA HANDLING AND PRESENTATION 
All safety and statistical outputs will be generated using SAS software, version 9.4. 
All programs used to generate statistical analyses will be validated according to Altasciences’s 
standard operating procedures (SOPs). 
The analyses described in this plan are considered a priori, in that they have been defined prior to 
database lock and prior to breaking the blind.  Any analyses performed subsequent to database 
lock and breaking the blind that are not described within the present plan will be considered post 
hoc and exploratory.  Post hoc analyses will be labeled as such in the corresponding statistical 
output and identified in the clinical study report (CSR). 
9.1 
Safety Analysis Presentation 
Adverse events and medical history will be classified using the Medical Dictionary for 
Regulatory Activities (MedDRA) terminology version 23.1 as defined in the study data 
management plan (DMP) dated 2021/03/01.  
Prior and concomitant medications will be coded with the World Health Organization Drug 
Dictionary- Enhanced (WHO-DDE) as defined in the study DMP. 
In general, all safety summary tables will be presented for the safety population.  Summaries for 
AEs will be presented by treatment and Overall.  Summaries for other safety endpoints will be 
presented by treatment if the endpoints are measured at the end of each period.  
In general, the data listings will include all randomized subjects up to the point of study 
completion or discontinuation; exceptions will be listings pertaining to a subset of subjects only 
(e.g., subjects with protocol deviations) or a subset of records/events (e.g., abnormal laboratory 
values). 
Categorical variables will be summarized using the PROC FREQ procedure. Continuous 
variables will be summarized using the PROC UNIVARIATE procedure. For natural log (ln)-
transformed endpoints, geometric mean, geometric standard deviation (SD), and coefficient of 
variation (CV%) will also be presented. 
The following general comments also apply to all statistical analyses and data presentations: 
 Duration variables will be calculated using the general formula: (end date - start date) +1. 
 If the reported value of a clinical laboratory parameter cannot be used in a statistical 
summary table (e.g., a character string is reported for a parameter of the numerical type), 
a coded value must be appropriately determined and used in the statistical analyses.  In 
general, a value or lower and upper limit of normal range such as ‘<10’ or ‘≤5’ will be 
treated as ‘10’ or ‘5’ respectively, and a value such as ‘>100’ will be treated as ’100’.  
However, the actual values as reported in the database will be presented in data listings. 
 When assessments are repeated for a given time point or performed at unscheduled times, 
only the result which is the closest to the dosing time will be included in summary tables. 
In general, summary statistics for raw variables (i.e., variables measured at the study site or 
central laboratory) will be displayed as follows:  


 
 
 
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 Minima and maxima will be displayed to the same number of decimal places as the raw 
data.  
 Means, medians, and quartiles will be displayed to 1additional decimal place.  
 Standard deviations will be displayed to 2 additional decimal places.   
 Percentages will be displayed to 1 decimal place. Percentages between 0 and 0.1 
(exclusive) will be displayed as ‘<0.1’.   
 P-values will be displayed to 3 decimal places. P-values that are less than 0.001 will be 
displayed as ‘<0.001’. 
The numbers of decimal places for summary statistics of derived variables (i.e., variables that are 
not measured by the study site but are calculated for analysis based on other measured variables) 
will be determined on a case by case basis.  In general:  
 Minima and maxima will be displayed to the commonly used unit of precision for the 
parameter.  
 Means, medians, quartiles, and confidence limits will be displayed to 1 additional 
decimal place.  
 Standard deviations will be displayed to 2 additional decimal places.  
 
9.2 
Pharmacokinetic Analysis 
Individual raw PK concentrations will be displayed with the same precision as received from the 
bioanalytical laboratory. 
Summary statistics for concentration will be displayed with the same precision as the individual 
values, with the exception of number of observations (N) and CV% which will be presented with 
0 and 1 decimal place, respectively. 
 
9.3 
Pharmacodynamic Analysis 
Precision for individual PD parameters will be displayed as follows: 
• 
Emax, AUEC with the same precision as the raw PD data; 
• 
PD parameters associated with time (i.e. TEmax) with 2 decimal places; 
Summary statistics for tcpO2 levels, time-matched change in tcpO2 levels and PD parameters will 
be displayed with the same precision as the individual values unless specified otherwise, with the 
exception of N and CV% which will be presented with 0 and 1 decimal place, respectively. 
 
9.4 
Analysis Timepoints  
Unless otherwise specified, the baseline value will be defined as the last non-missing evaluation 
prior to the first dose of study medication. 
 


 
 
 
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9.5 
Methods for Handling Missing Data  
All analyses will be based on the observed data; no data will be imputed for the statistical 
analyses. 
 
9.6 
Interim Analysis 
No interim assessments or analyses are planned for this study.   
 
 
 


 
 
 
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10 CHANGES TO PROTOCOL-SPECIFIED ANALYSES 
Addition of the PD and PK population in the analysis population. Clarification of the PD analysis 
to be performed. 
 
 


 
 
 
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11 GENERAL INFORMATION RELATED TO DATA PRESENTATIONS 
The formats and layouts of TFLs are provided in a separate document and are common displays.  
Their numbering and general content follow the International Conference on Harmonisation 
(ICH) E3 guidelines.  Actual formats and layouts may be altered slightly from those presented as 
necessary to accommodate actual data or statistics.  Minor format changes will not require 
updates to the SAP, rather they may be documented in a Note to SAP. 
 
 


 
 
 
 
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APPENDIX A STUDY SCHEDULE 
 
Screening 
Treatment (Day 0) 
Follow Up 
 
Day -21 to 
Day 0 
Clinic 
Arrival 
Equilibration/ 
Baseline: 
70 minutes  
on O2  
Treatment/ 
Evaluation:     
60 minutes on 
O2 
Post-
Treatment: 
30 minutes 
on Room Air 
Clinic 
Observation 
48 Hours (+ 2 
Days) 
Informed Consent 
X 
 
 
 
 
 
 
Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Medical history 
X 
X 
 
 
 
 
 
Concomitant medications  
X 
X 
X 
X 
X 
X 
X 
BMI (kg/m2) 
X 
 
 
 
 
 
 
Body weight (kg) 
 
X 
 
 
 
 
 
Physical Exam 
X 
X 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
Vital signs (HR, BP, RR, Temp) 
X 
 
X 
X 
X 
X 
 
SpO2 
b 
X 
 
X 
X 
 
 
 
12-lead ECG 
X 
 
 
 
 
 
 
Laboratory (CBC, BMP, HIV, Hep 
B, Hep C) 
X 
 
 
 
 
 
 
Pregnancy testa 
X 
X 
 
 
 
 
 
Urine drug screen 
X 
X 
 
 
 
 
 
Alcohol breathalyzer screen 
X 
X 
 
 
 
 
 
Subject resting quietly supine with 
one pillow, or semi-recumbent 
 
 
X 
X 
 
 
 
TcpO2 sensors in place 
 
X 
X 
X 
 
 
 
PK measurements 
 
 
X 
X 
X 
 
 


 
 
 
 
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Oxygen: simple face mask at 6 
L/min 
 
 
X 
X 
 
 
 
TcpO2
b 
 
 
X 
X 
 
 
 
Administer Study Drug  
 
 
 
X 
 
 
 
TcpO2 sensors removed 
 
 
 
 
X 
 
 
Overnight stay at clinic 
 
 
 
 
 
X 
 
Adverse Events 
 
 
X 
X 
X 
X 
X 
a For all females, with serum pregnancy test at screening and urine pregnancy test at clinic arrival 
b SpO2 and TcpO2 will be assessed every 5 minutes during the baseline period. After study drug administration they will be assessed at 1, 2, and 5 minutes, and then every 5 minutes thereafter.