Clinical trial NCT02106975
Conditions: Acute Lung Injury Sepsis
Sponsor: Virginia Commonwealth University
Start date: 2014-04-01; Completion date: 2018-01-08
Enrollment: 170 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Hypothesis 1A: Vitamin C infusion will significantly attenuate sepsis-induced systemic organ failure as measured by Sequential Organ Failure Assessment (SOFA) score,

Hypothesis 1B: Vitamin C infusion will attenuate sepsis-induced lung injury as assessed by the oxygenation index and the VE40

Hypothesis 1C: Vitamin C infusion will attenuate biomarkers of inflammation (C-Reactive Protein, Procalcitonin), vascular injury (Thrombomodulin, Angiopoietin-2), alveolar epithelial injury (Receptor for Advanced Glycation Products), while inducing the onset of a fibrinolytic state (Tissue Factor Pathway Inhibitor).

Detailed description:

Arms: arm 1: 200mg/kg/day divided over 4 doses. Administered every 6 hours for 96 hours arm 2: 50ml every 6 hours for 96 hours
Interventions: intervention 1: Ascorbic Acid intervention 2: Placebo: 5% Dextrose in water
Intervention descriptions: intervention 1: Intervention intervention 2: Placebo

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Title: Vitamin C Infusion for Treatment in Sepsis Induced Acute Lung Injury (CITRIS-ALI) 
 
PI: Alpha Berry Fowler, MD 
 
NCT #: NCT02106975 
 
Unique Protocol ID: HM20000917 
 
Document approval date: February 20, 2017 
 
Document Type: Protocol and SAP 
 
 
 


 
CITRIS Protocol Version 9 
 
1
 
 
 
 
 
 
Vitamin C Infusion for TReatment In Sepsis Associated Acute Lung Injury 
 
CITRIS-ALI 
 
 
Protocol Version 9 
 
 
  
 
Name 
Role 
Email 
Voice/Pager 
 
 
Alpha Fowler, III, MD 
 
Investigator 
alpha.fowler@vcuhealth.org 
804.519.4369  
(c) 
Jonathon D. Truwit, MD 
Investigator 
Jonathon.truwit@froedtert.com 
434.982.3500.4004  
434-243-7088 
434.227.1663  
(p) 
(o) 
(c) 
 
R. Duncan Hite, MD 
 
Investigator 
hited@ccf.org 
216.445.3098 
216.386.2099  
(o) 
(c) 
 
Chris DeWilde, MSN, 
RN, CCRC, TCN-A 
 
Lead Research 
Coordinator 
christine.dewilde@vcuhealth.org 804.628.5710 
804.519.0022 
(o) 
(c) 
 
Anna Priday 
 
Lead 
Regulatory 
Coordinator 
anna.priday@vcuhealth.org 
804.628.9612 
804.221.9258 
(o) 
(c) 
 
 
 
 
 


 
CITRIS Protocol Version 9 
 
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ABBREVIATIONS ............................................................................................................................................. 5 
DEFINITIONS ................................................................................................................................................... 6 
1. BACKGROUND .......................................................................................................................................... 12 
1.1. Introduction .............................................................................................................................................................................................. 12 
1.2. Preliminary Progress – Vitamin C Intervention in Experimental Septic Lung Injury ........................................................ 14 
1.3 Phase I Human Trial: Vitamin C Infusion In Human Sepsis: Preliminary Results From A Safety Trial ......................... 17 
1.4. Potential Mechanisms of Action of Ascorbic Acid in Sepsis ........................................................................................................ 19 
1.5. Ascorbic Acid Dose Selection ................................................................................................................................................................ 19 
1.6. Study Rationale ......................................................................................................................................................................................... 20 
2. OBJECTIVES .............................................................................................................................................. 20 
2.1. Primary Objectives .................................................................................................................................................................................. 20 
2.2. Hypothesis .................................................................................................................................................................................................. 21 
3. END-POINTS ............................................................................................................................................. 21 
3.1. Primary Endpoints .................................................................................................................................................................................. 21 
3.2. Secondary End Points ............................................................................................................................................................................. 21 
3.3. Focused Safety Analysis: ........................................................................................................................................................................ 22 
3.3.1 Renal Monitoring Plan .................................................................................................................................................................................................. 22 
3.3.2 Glucose Monitoring Plan .............................................................................................................................................................................................. 22 
4. STUDY POPULATION AND ENROLLMENT .................................................................................................. 23 
4.1. Number/Source/Screening ................................................................................................................................................................... 23 
4.2. Inclusion Criteria ...................................................................................................................................................................................... 23 
4.2.1 CITRIS-ALI Inclusion Criteria: ................................................................................................................................................................................... 23 
4.3. 
Enrollment, Randomization, and Study Initiation Time Window ........................................................................................ 25 
4.4. 
Informed Consent ................................................................................................................................................................................. 25 
4.5. 
Randomization ...................................................................................................................................................................................... 25 
4.7. 
Minorities and Women ....................................................................................................................................................................... 25 


 
CITRIS Protocol Version 9 
 
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5. STUDY PROCEDURES ................................................................................................................................. 25 
5.1. Vitamin C or Placebo Administration ............................................................................................................................................... 26 
5.2. Drug Level Specimens (venous blood) .............................................................................................................................................. 26 
5.3. Completion of Study Drug Administration ...................................................................................................................................... 26 
5.4. Premature Withdrawal from Treatment .......................................................................................................................................... 27 
5.5. Ventilator Procedures ............................................................................................................................................................................ 27 
5.6. On-Study Fluid Management ................................................................................................................................................................ 27 
6. DATA COLLECTION .................................................................................................................................... 28 
6.1. Background Assessments ...................................................................................................................................................................... 28 
6.2. Baseline/Hour 0 Assessments and Procedures .............................................................................................................................. 29 
6.3. 
Assessments after Enrollment ......................................................................................................................................................... 29 
7. STATISTICAL CONSIDERATIONS ................................................................................................................. 30 
7.1. Statistical Methods ................................................................................................................................................................................... 30 
8. DATA COLLECTION AND SITE MONITORING .............................................................................................. 31 
8.1. Data Collection .......................................................................................................................................................................................... 31 
8.2. Site Monitoring ......................................................................................................................................................................................... 31 
9. RISK ASSESSMENT ..................................................................................................................................... 32 
9.1. Risks of Active Study Drug ..................................................................................................................................................................... 32 
9.2. Risks of Blood Draws ............................................................................................................................................................................... 32 
9.3. Minimization of Risks .............................................................................................................................................................................. 32 
9.4. Potential Benefits ..................................................................................................................................................................................... 33 
9.5. Risks versus Benefits .............................................................................................................................................................................. 33 
10. HUMAN SUBJECTS ................................................................................................................................... 33 
10.1. Selection of Subjects .............................................................................................................................................................................. 33 
10.1.1. Equitable Selection of Subjects .............................................................................................................................................................................. 33 


 
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10.1.2. Vulnerable Subjects ................................................................................................................................................................................................... 33 
10.2. Informed Consent ................................................................................................................................................................................. 34 
10.3. Identification of Surrogates ............................................................................................................................................................... 34 
10.4. Justification of Surrogate Consent ................................................................................................................................................... 34 
10.5. Additional Safeguards for Vulnerable Subjects ........................................................................................................................... 35 
10.6. Confidentiality ....................................................................................................................................................................................... 35 
11. ADVERSE EVENT REPORTING/SAFETY REPORTING .................................................................................. 35 
12. APPENDICES ......................................................................................................................................... 38 
APPENDIX A: Guidelines for evidence of infection ................................................................................................................................. 38 
APPENDIX B:  Pleiotropic Effects of Vitamin C ........................................................................................................................................ 40 
APPENDIX C: Ventilator Procedures .......................................................................................................................................................... 42 
C.1. Ventilator Management .................................................................................................................................................................................................. 42 
C.2. Weaning ................................................................................................................................................................................................................................ 43 
C.3. Definition of Unassisted Breathing ........................................................................................................................................................................... 44 
C.4. Definition of Extubation ................................................................................................................................................................................................. 44 
C.5. Completion of Ventilator Procedures ...................................................................................................................................................................... 44 
C.6. Removal from the Ventilator Management Protocol ........................................................................................................................................ 45 
APPENDIX D: FACTT LITE Conservative Fluid Management Approach ........................................................................................... 46 
APPENDIX E: Time Events Schedule ........................................................................................................................................................... 47 
APPENDIX F:  Adverse Events ....................................................................................................................................................................... 48 
APPENDIX G: Modified SOFA Score Calculator ........................................................................................................................................ 53 
APPENDIX H: P/F from S/F  Calculator ...................................................................................................................................................... 54 
REFERENCES CITED: ....................................................................................................................................... 55 
 


 
CITRIS Protocol Version 9 
 
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ABBREVIATIONS 
 
ABG = Arterial blood gas 
AKI = Acute Kidney Injury 
ALI = Acute Lung Injury 
Ang-2 = Angiopoietin-2 
APACHE = Acute physiologic and chronic 
health evaluation 
AscA = Ascorbic Acid (Vitamin C) 
ARDS = Acute Respiratory Distress 
Syndrome 
BIPAP = Bi-level Positive Airway Pressure 
BMI = Body Mass Index 
CCC = Clinical Coordinating Center 
CK = Creatinine Kinase  
CPAP = Continuous Positive Airway 
Pressure 
CRP = C-reactive protein 
Day 0 = Day of Randomizations 
  DHA = Dehydroascorbic Acid 
DSMB = Data Safety Monitoring Board 
FACTT = Fluid and Catheter Treatment 
Trial 
FDA=Food and drug administration 
FiO2  = Fraction of Inspired Oxygen 
FIP = Feces Induced Peritonitis 
GCS = Glasgow Coma Scale 
ICU = Intensive care Unit 
IMV = Intermittent Mechanical Ventilation 
IRB = Institutional Review Board 
IVRS = Interactive Voice Response System 
 
 
LPS = Lipopolysaccharide 
MBW = measured body weight 
NFκB= Nuclear factor kappa B 
NHLBI = National Heart Lung and Blood 
Institute 
NIV=Non-invasive ventilation 
NOS= Nitric oxide synthase 
OI = Oxygenation Index = [mean airway 
pressure x Fi02]/Pa02 
PaCO2= Partial pressure of arterial carbon 
dioxide 
PaO2 = Partial pressure of arterial oxygen 
PBW = Predicted Body Weight 
PCT = Procalcitonin 
PCV = Pressure Control Ventilation 
PEEP = Positive End-Expiratory Pressure  
PIN = Personal Identification Number 
Pplat = Plateau pressure 
PSV = Pressure Support Ventilation 
PAOP = Pulmonary Artery Occlusion Pressure 
RAGE = receptor for advanced glycation end 
products 
RCT = Randomized Controlled Trial 
SBT = Spontaneous Breathing Trial 
SIRS = Systemic Inflammatory Response 
Syndrome 
SOFA =Sequential Organ Failure Assessment 
SpO2  = Oxygen Saturation 
TFPI = Tissue Factor Pathway Inhibitor 
TM = Thrombomodulin 
VFD = Ventilator-free Day 


 
CITRIS Protocol Version 9 
 
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DEFINITIONS 
 
Acute Kidney Injury: Acute kidney injury network Stage 3 disease, defined as a threefold increase in 
creatinine from baseline or the need for dialysis 
Asian: A person having origins in any of the original peoples of the Far East, Southeast Asia, or the Indian 
subcontinent including, for example, Cambodia, China, India, Japan, Korea, Malaysia, Pakistan, the 
Philippine Islands, Thailand, and Vietnam. 
Completing 48 hours of Unassisted Breathing (UAB) : Defined as the date (calendar day) that the 
subject reaches exactly 48 hours of UAB. Example: if subject meets UAB at 1900 on 6/1/14 and does not 
return to assisted breathing (AB), then the date of completing 48 hours of UAB would be 6/3/14. 
Date of first UAB: Defined as the first day that the subject is on UAB from midnight to midnight. 
Example: if subject meets UAB at 1900 on 6/1/14, then the date of first UAB would be 6/2/14, as long as 
subject does not return to AB on 6/2/14. 
Extubation: Removal of an orotracheal, nasotracheal tube, or unassisted breathing with a tracheostomy 
Home: Level of residence or health care facility where the patient was residing prior to hospital 
admission 
NYHA: New York Heart Association Class IV subjects (defined as subjects who have cardiac disease 
resulting in inability to carry out physical activity without discomfort.  Symptoms of cardiac 
insufficiency or an anginal syndrome may be present even at rest. If any physical activity is undertaken, 
discomfort is increased). 
Severe Sepsis: SIRS criteria plus suspected or known infection plus organ dysfunction.  Since intubation and 
hypoxemia is a requirement for enrollment into this trial, participants will, by definition, meet the SIRS 
criterion.   
Study hospital: Defined as the hospital where the patient was randomized and enrolled. 
Study withdrawal: Defined as permanent withdrawal from study before completion of study activities. 
This does not include those subjects who have completed the protocol procedures or stopped procedures 
because they have reached unassisted breathing. If a patient or surrogate requests withdrawal from the study 
the clinician should seek explicit permission to continue data collection. 
UAB (Unassisted Breathing):  Spontaneously breathing with face mask, nasal prong oxygen, or room air, 
T-tube breathing, tracheostomy collar (mask) breathing, or CPAP ≤ 5 without PSV or IMV assistance, or 
the use of noninvasive ventilation solely for sleep-disordered breathing.  Assisted breathing is any level of 
ventilatory support at pressures higher that the unassisted breathing thresholds. 
 
 


 
CITRIS Protocol Version 9 
 
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Part I: Study Summary 
 
Title: Vitamin C Infusion for TReatment In Sepsis Associated Acute Lung Injury 
(CITRIS-ALI) 
Objective:  To assess the efficacy of a 96-hour intravenous vitamin C infusion protocol (200 mg/kg per 24 
hours) in patients with established acute lung injury (ALI) from sepsis. In this course of performing this phase 
II trial we will explore three hypotheses: 
 
Hypothesis: 
Hypothesis 1A: Vitamin C infusion will significantly attenuate sepsis associated systemic organ failure as 
measured by Sequential Organ Failure Assessment (SOFA) score 
Hypothesis 1B: Vitamin C infusion will attenuate sepsis associated lung injury as assessed by oxygenation 
index and the VE 40 (see below) 
Hypothesis 1C: Vitamin C infusion will attenuate biomarkers of inflammation (C-Reactive Protein, 
Procalcitonin), vascular injury (Thrombomodulin, Angiopoietin-2), alveolar epithelial injury (Receptor For 
Advanced Glycation End Products), while inducing the onset of a fibrinolytic state (Tissue Factor Pathway 
Inhibitor).  
 
Study Design: 
1.   Multi-center, prospective, randomized, placebo-controlled clinical trial 
2.   A maximum of 170 patients will be enrolled 
3.  Participants will be randomized to receive either intravenous Vitamin C (mixed in 5%  dextrose in 
water) or placebo (5% dextrose in water) 
4.   Active treatment will continue for 96 hours, discharge from study hospital, discharge from the 
ICU, study withdrawal, or death, whichever comes first. 
5.   All participants will be followed for a total of 60 days.  
 
Analysis/Interim Monitoring: 
1. The principal analysis will be on the basis of the intention-to-treat. 
2.  Protocol compliance will be monitored by the study team by presentation of 1st two enrolled 
subjects per site to the team.  This will take place via investigator conference call and will 
address challenges encountered.  Trial progress will be monitored by an independent Data and 
Safety Monitoring Board to determine if the study should stop for safety reasons.  As an early 
phase study it is important to collect as much data as possible.  For this reason the study will only 
be halted by the DSMB for reasons of patient safety concerns.  The first scheduled analysis will 
occur after the enrollment of 80 patients or semi-annually, whichever happens first.  The next 
review will occur after enrollment of the last enrolled subject, or semi-annually, whichever comes 
first.  In the event that safety concerns arise prior to the scheduled analysis, the DSMB may 
request an unscheduled review at any time. The Data and Safety Monitoring Board (DSMB) will 
also monitor trial quality. 
3. Regulatory compliance, GCP, and risk-based monitoring will be provided by an independent 
CRO. 
 
 


 
CITRIS Protocol Version 9 
 
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CITRIS-ALI Inclusion and Exclusion Criteria:  
The inclusion and exclusion criteria for the CITRIS-ALI trial is listed here and in the full CITRIS-ALI protocol. 
The definition of severe sepsis for this study is derived and defined as previously published in the referenced 
literature.i,ii,iii 
CITRIS-ALI Inclusion Criteria: 
Patients must have suspected or proven infection, and meet 2 out of 4 of the criteria for Systemic Inflammatory 
Response (SIRS) due to infection, and be accompanied by at least 1 criterion for sepsis associated organ 
dysfunction, and meet all 5 criteria for Acute Respiratory Distress Syndrome (ARDS). 
1. Suspected or proven infection: (e.g., thorax, urinary tract, abdomen, skin, sinuses, central venous 
catheters, and central nervous system, see Appendix A). 
2. The presence of a systemic inflammatory response: Defined as: fever:  >38ºC (any route) or 
hypothermia: <36ºC (core temp only), tachycardia: heart rate > 90 beats/min or receiving medications 
that slow heart rate or paced rhythm, leukocytosis:  >12,000 WBC/µL or leukopenia:  <4,000 WBC/µL 
or >10% band forms.  Respiratory rate > 20 breaths per minute or PaCO2 < 32 or invasive mechanical 
ventilation. 
3. The presence of sepsis associated organ dysfunction: (any of the following thought to be due to 
infection) 
a. Sepsis associated hypotension (systolic blood pressure (SBP) < 90 mm Hg or an SBP decrease > 
40 mm Hg unexplained by other causes or use of vasopressors for blood pressure support 
(epinephrine, norepinephrine, dopamine =/> 5mcg, phenylephrine) 
b. Arterial hypoxemia (PaO2/FiO2 ≤ 300) or supplemental O2 > 6LPM. 
c. Lactate > upper limits of normal laboratory results 
d. Urine output < 0.5 ml/kg/hour for > two hours despite adequate fluid resuscitation 
e. Platelet count < 100,000 per mcL 
f. Coagulopathy (INR > 1.5) 
g. Bilirubin > 2 mg/dL 
h. Glasgow Coma Scale < 11 or a positive CAM ICU score 
4. ARDS characterized by all the following criteria 
a. Lung injury of acute onset, within 1 week of an apparent clinical insult and with progression of 
respiratory symptoms 
b. Bilateral opacities on chest imaging not explained by other pulmonary pathology (e.g. pleural 
effusions, lung collapse, or nodules) 
c. Respiratory failure not explained by heart failure or volume overload 
d. Decreased arterial PaO2/FiO2 ratio ≤ 300 mm Hg 
e. Minimum PEEP of 5 cmH2O  
 
 


 
CITRIS Protocol Version 9 
 
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CITRIS-ALI Exclusion Criteria: 
1. Known allergy to Vitamin C 
2. inability to obtain consent;  
3. age < 18 years; 
4. Not on a ventilator 
5. more than 48 hrs since meeting ARDS criteria; 
6. No indwelling venous or arterial catheter in patients requiring insulin in a manner that requires glucose 
being checked more than twice daily (e.g. continuous infusion, sliding scale)  
7. Presence of diabetic ketoacidosis 
8. patient or surrogate or physician not committed to full support (not excluded if patient would receive all 
supportive care except for cardiac resuscitation); 
9. pregnancy or breast feeding, 
10. moribund patient not expected to survive 24 hours; 
11. home mechanical ventilation (via tracheotomy or noninvasive) except for CPAP/BIPAP used only for 
sleep-disordered breathing; 
12. on home O2 > 2LPM, except for with CPAP/BIPAP 
13. diffuse alveolar hemorrhage (vasculitis); 
14. interstitial lung disease requiring continuous home oxygen therapy; 
15. Active kidney stone 
16. Non English speaking; 
17. ward of the state (inmate, other). 
Primary Objective: 
To assess the efficacy of a 96-hour high dose intravenous vitamin C infusion protocol (200 mg/kg per 24 
hours) in patients with established ALI/ARDS that results from severe sepsis. Patients will be randomized to 
receive either: 1) Placebo (50 ml of 5% dextrose in water) or Vitamin C (sterile L-ascorbic acid for injection at 
200 mg/kg per 24 hours with entire  calculated 24 hour dose diluted in 200 ml of 5% dextrose in water). One 
fourth of the 24 hour calculated dosage will be administered in 30 minute intravenous infusions will occur every 
6 hours.  
 
Endpoints: 
The CITRIS-ALI trial will depart from prior acute lung injury trials in that assessment of efficacy will not 
include 28-day all-cause mortality as a primary endpoint. As directed in RFA-HL-12-022, primary end points 
will focus on quantifiable measures of organ function and biomarker analysis (SOFA, CRP, Procalcitonin, 
Thrombomodulin). For this phase II trial we propose co-primary endpoints. 
 
Primary Endpoint #1: Change in SOFA score at 96 hours as compared to baseline when compared to placebo.  
Primary Endpoint #2: C-Reactive Protein and Thrombomodulin at study hours 0, 48, 96, 168 when compared 
to placebo. 
 
Secondary Endpoints: 
• Oxygenation Index (FiO2 x Mean Airway Pressure/PO2) at study hour 0, 48, 96, 168 if still intubated in 
ascorbate infused patient compared to placebo. 
• VE-40 (Vent RR x TV/Weight) x (PaCO2/40) at study hour 0, 48, 96, 168 if still intubated, in ascorbate 
infused patient compared to placebo 
• SOFA scores at hours 48, 96, 168 


 
CITRIS Protocol Version 9 
 
10
• SOFA Score Components at hours 48,96, 168 
o PaO2/FiO2 
o SpO2/FiO2 
o Platelets 
o Total Bilirubin 
o Vasopressor status 
o GCS 
o Creatinine or Urine Output 
• Procalcitonin, Receptor for Advanced Glycation End Products, Tissue Factor Pathway Inhibitor at 
study hour 0, 48, 96, 168  
• Ascorbate level at hour 0, 48, 96, 168 
• Ventilator Free Days to day 28 
• ICU-free days at day 28 
• All cause mortality to day 28 
• Hospital-free days at day 60 
 
Focused Safety Analysis: Given that L-Ascorbate is an “acid” the drug manufacturer adjusts the pH thus 
balancing pH at 7.4 and negating the acid effect of the drug.  Unexplained metabolic acidosis will be addressed 
per standard of care for each participating institution. 
 
Study Drug Dosing:  
 
1. First study drug dose (L-ascorbic acid or placebo) will be considered “Dose 1” and will be 
administered within 6 hours of randomization or the earliest available time post any clinically indicated 
procedure which requires the patient to be off the unit. All doses will be administered in the ICU.  
Patients receiving vitamin C will receive 25% of the total daily calculated dosing (200mg/kg/24 hours) 
and will be infused over 30 minutes for this first dosing. 
2. Subsequent doses which represent 25% of the day’s total dose will be infused every six hours through 
96 hours (+/- 3 hours).  
a. Timing of Dose 2 will be triggered by the physician order for q 6 hour administration and will 
therefore be listed on the bedside MAR.  As such, timing of Dose 2 may be out of the +/- 3 
hour window and will not trigger a protocol deviation. 
b. If for any reason any other maintenance dose is not administered within window, the dose will 
be skipped and the next scheduled dose will be given and documented in the data collection 
tool.  
 
Drug level specimens (venous blood): Septic patients exhibit subnormal plasma ascorbate levels. Phase I 
studies performed at Virginia Commonwealth University (VCU) show mean ascorbate levels of 17.5 µM 
(normal human ascorbate levels 60 to 70 µM). Entry ascorbate levels will be drawn.  
 
The target plasma range for modifying pro-inflammatory biomarkers and for attenuating vascular injury 
was obtained from the phase I safety trial and is greater than 500 µm as measured 24 hours after initiation 
of Vitamin C infusion. The day 2 – 7 plasma ascorbate levels are expected to be between 500 to 1000 µM 
based on pharmacokinetic studies generated during the phase I trial (VCU trial was entitled: Vitamin C 
Infusion in Human Sepsis).  
 
Blood drawn for ascorbate levels and biomarkers will occur at hour 0 (prior to the first infusion), hour 48 (+ 


 
CITRIS Protocol Version 9 
 
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or – 3 hours as long as it is drawn prior to Infusion 9), hour 96 (+ or – 3 hours as long as it is drawn at least 
3 hours post Infusion 16), and hour 168 (+ or – 6 hours).  If patient is moved out of the ICU to another 
hospital unit, DO continue to collect blood for biomarkers and ascorbate levels.   
 
If arterial line is removed prior to hour 168, do not collect arterial blood for PO2 analysis (SOFA Score 
component).   
 
Completion of study drug administration: Study drug administration will be stopped when one of the 
following conditions is met, whichever comes first: 
1. Final drug dose at 96 hours 
2. Discharge from study hospital 
3. Loss of indwelling venous or arterial catheter with no intent to replace the line, making it impossible to 
monitor glucose levels via central laboratory without multiple peripheral sticks.   
4. Discharge from the ICU 
5. Withdrawal from study 
6. Death 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
CITRIS Protocol Version 9 
 
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Part II: Study DescriptionVitamin C Infusion for 
TReatment In Sepsis Associated Acute Lung Injury 
CITRIS-ALI 
1. Background 
 
1.1. Introduction 
 
Pneumonia and extrapulmonary sepsis account for 50-65% of all ALI cases and mortality is high. Rising 
rates of hospitalization and death due to sepsis continue to be a worsening global health care problem.iv A large 
fraction of patients with severe sepsis develop acute lung injury (ALI) or its severe form, the acute respiratory 
distress syndrome (ARDS).v The pathogenesis of ALI is characterized by activation of tissue inflammation, 
oxidant mediated tissue injury and increased vascular leak. At a molecular level, sepsis is associated with 
activation of pro-inflammatory mediators driven by transcription factor nuclear factor kappa B (NFκB). The 
pathogenesis of ALI is characterized by activation of tissue inflammation, oxidant mediated tissue injury and 
increased vascular leak. These mediators are important for host defense against invading bacteria, but their 
uncontrolled and excessive production ultimately contributes to the pathogenesis of ALI. 
 
The lung is an important target of inflammatory mediators in severe sepsisvi and increased pulmonary NFκB 
drives inflammatory mediators in severe sepsis.vii Reactive oxygen species (ROS) produced by lung cells 
oxidize vital lung proteins and activate redox-sensitive pathological signaling pathways. An extensive body of 
evidence shows a crosstalk between the cellular signaling pathways and the cellular redox state through 
multiple mechanisms. However, exuberant ROS synthesis may also damage cells and host tissues, and, thus, 
contribute to the pathogenesis of ALI. Although the potential role of antioxidant enzymes and scavengers of 
ROS in reducing the severity of ALI has been recognized, no single agent or treatment strategy has shown 
sufficient promise for use in routine clinical practice. 
 
Ascorbic acid is an essential vitamin for humans, primates, guinea pigs, and a few other animals and insects that 
lack the enzyme L-gulono-γ-lactone oxidase. Ascorbic acid is an essential vitamin for humans, primates, guinea 
pigs, and a few others.viii Ascorbic acid is transported into specialized cells as reduced ascorbic acid (AscA) by 
sodium dependent ascorbic acid transporters (SVCT-1 and SVCT-2) or in most cells in its oxidized form as 
dehydroascorbic acid (DHA) via facilitative glucose transporters.ix,x When DHA is transported via the glucose 
transporters, it is rapidly reduced and trapped inside the cell, where it accumulates as reduced ascorbic acid. 
Although ascorbic acid circulates in normal human plasma at approximately 60 - 70 µM, it accumulates in 
millimolar concentrations in host defense cells.xi Together with glutathione, AscA constitutes a primary line of 
defense against ROS and participates in the recycling of other antioxidants such as vitamin E. A growing body 
of evidence supports the notion that vitamin C is “negatively” involved in the pathogenesis of sepsis.xii 


 
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Subnormal ascorbate concentrations are common features of patients with sepsis. Furthermore, plasma 
ascorbate levels correlate inversely with multiple organ failurexiii and directly with survival.xiv Despite all the 
evidence, ascorbate is not used in a clinical setting. 
 
At physiological pH, AscA dissociates to form dehydroascorbic ascorbate.
xviii
xxiii
xxvii
xxviii
xxxii
xxxiii xxxiv
xv Ascorbate functions as an 
antioxidant by inhibiting cell death induced by hydrogen peroxidexvi and DNA damage induced by oxidative 
stress.xvii Ascorbate also functions as a cofactor for various enzymatic hydroxylation reactions and is involved 
in the biosynthesis of collagen, carnitine and norepinephrine.
,xix,xx As noted, circulating levels of ascorbate 
are low in patients with sepsis and plasma ascorbate correlates directly with survival and inversely with multiple 
organ failure. Similar results have been observed in animal models of sepsis. Ascorbate administration improves 
capillary blood flow, liver function and arteriolar responsiveness in experimental models of sepsis.xxi,xxii,
 In 
mice injected with pathogenic bacteria, prior ascorbate depletion results in decreased survival.xxiv Recently 
ascorbate was shown to regulate the stability of a master transcription factor HIF-1α.xxv As noted, circulating 
levels of ascorbate are low in patients with sepsis and plasma ascorbate may protect microvascular function by 
two distinct mechanisms: a) by inhibiting NADPH oxidase activation and b) by increasing endothelial nitric 
oxide synthase (eNOS) activity, and subsequently suppressing expression of NADPH oxidase, inducible nitric 
oxide synthase and tissue factor.xxvi However, little is known about the effects of ascorbate administration in the 
setting of sepsis-mediated ALI. Gram-negative sepsis is a leading cause of ALI/ARDS and multiple organ 
failure.
 Intra-peritoneal injection of a single bolus of bacterial LPS precipitates a systemic inflammatory 
response that resembles in many ways the observed clinical profile of sepsis including ALI and ARDS.
 It is 
well known that LPS activates inflammatory cells such as polymorphonuclear leukocytes, monocytes, 
macrophages and lymphocytes. Besides immune cells, microvascular endothelial cells in multiple organs also 
become activated in sepsis and may contribute to amplification of the inflammatory response. Moreover, it is 
generally agreed that it is not the bacterial infection itself, but rather the inflammatory response to infection that 
is the predominant determinant of outcome in sepsis.xxix,xxx In support of this hypothesis, it has been shown that 
septic stimuli initiate activation of transcription factors that transactivate multiple genes such as pro-
inflammatory cytokines, adhesion molecules and chemokines by endothelial cells.xxxi,
 Not surprisingly, 
efforts to block single components of the sepsis-associated inflammatory pathways have had little impact on 
patient survival and little progress has been made in improving outcomes.
,
 
 
Summary: Sepsis is a common clinical problem that frequently leads to widespread acute vascular injury that 
is clinically manifested by multiple organ failure. One of the most frequent organs injured following onset of 
sepsis is the lung.
xxxvi
xxxvii
xxxviii
xxxv At present, no truly specific therapy is available for acute lung injury (or other organ 
injury) that occurs in association with sepsis. The preliminary data presented below suggests that ascorbic acid 
may present a means by which sepsis-associated vascular injury can be interrupted or reversed. AscA has been 
intravenously infused in humans in high dosages previously. Nathens et al infused 1.5 grams of AscA every 8 
hours into surgical trauma ICU patients daily for 28 days with no adverse events.
 Muhlhofer et al infused 
high dose ascorbic acid (7500 mg) intravenously into human volunteers daily for 7 days and showed no 
adverse events.
 Finally, Tanaka et al infused high-dose ascorbic acid (66 mg/kg/hour, average 110 grams 
for 70 kg human) for 24 hours into patients with over 50% total body surface area burns.
 No 
abnormalities in hematologic, hepatic, or renal function was associated with ascorbate infusion at day 7 
following ascorbate administration. The scope of the study being presented in this phase II proof of concept 
trial involves the use of intravenously administered AscA as a future potential therapy for sepsis associated 
acute lung injury. Previous basic scientific research currently suggests that AscA can attenuate sepsis-
associated vascular injury. Further, prior data obtained from our phase I human safety studies suggests that 
high doses of AscA can be administered intravenously with little or no adverse events. Given these realities 
and the results of our phase I trials in human sepsis, we propose that intravenous AscA may present a unique 
therapy to improve the outcomes in human sepsis associated acute lung injury. 


 
CITRIS Protocol Version 9 
 
14
 
 
 
1.2. Preliminary Progress – Vitamin C Intervention in Experimental Septic Lung Injury 
 
We sought to examine the biological effects of ascorbic acid (AscA) infusion on systemic inflammatory 
responses and acute organ injury associated with bacterial sepsis. To accomplish this, we created a durable 
model of septic shock and acute lung injury in wild type C57BL6 mice. Beyond the creation of an animal model 
system of acute lung injury, a primary goal of these studies was to determine the extent to which ascorbic acid 
could be employed as an interventional therapy for bacterial sepsis. Multiple prior animal studies published 
over the years have examined pharmacological agents (e.g., methyl prednisolone, ibuprofen, simvastatin) and 
biological agents (e.g., monoclonal antibody to tumor necrosis factor alpha, interleukin-1 receptor antagonist) as 
potential sepsis therapies. Many agents have shown efficacy in sepsis when the agent was administered prior to 
induction of sepsis. In a “real world” setting, however, any intervention for sepsis will follow the development 
of symptoms and altered physiology. 
 
In preliminary studies described here, an interventional approach (agent administered after onset of sepsis) was 
employed to test the impact of ascorbic acid infusion on the course of 
sepsis associated acute lung injury. Sepsis was induced in mice by 
intraperitoneal (IP) administration of E coli lipopolysaccharide (LPS, 
0111:B4) at a concentration of 10 mcg/gram of body weight. Animals had 
average body weights of 30 grams. 
 
 Our first goal was to examine whether AscA infusion altered the course of 
murine sepsis. In these studies, mice 
received IP LPS at the stated dose. 
Thirty minutes following LPS infusion, 
animals received intraperitoneal either 
the reduced form of vitamin C (AscA) 
or the oxidized form of vitamin C 
dehydroascorbate (DHA)  at doses of 
200 mg/kg of body weight. Both the 
reduced form and the oxidized form 
were employed in separate 
experiments. Following LPS infusion, 
animals were then given free access to 
food and water. Mortality was observed 
over the ensuing 60 hours. Figure 1 
shows Kaplan-Meier survival curves of 
septic mice treated with AscA and 
DHA. These studies show that 
mortality induced by E coli sepsis in wild type mice was significantly improved by both ascorbic acid forms 
during the 60 hour observation period. 
 
Figure 1
Figure 2


 
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Sepsis is frequently accompanied by acute lung injury (ALI). Sepsis associated ALI is characterized by acute 
pulmonary edema and respiratory failure. Pulmonary edema results from loss of pulmonary microvascular 
endothelial integrity that leads to loss of endothelial “barrier function.” There is subsequent flooding of the dry 
airspaces of lung with plasma and cellular constituents. ALI is also 
characterized by intense sequestration of activated polymorphonuclear 
neutrophils (PMN). We assessed the loss of pulmonary microvascular 
barrier function using bronchoalveolar lavage fluid (BALF) protein 
analysis 16 hours following LPS infusion. Figure 2 shows that LPS treated 
mice exhibit significant increases in BALF protein, indicating a loss of 
barrier function. AscA treatment significantly attenuated microvascular 
injury as assessed by BALF protein. 
 
  In a second model of sepsis (fecal induced peritonitis, FIP), lungs were 
removed and total RNA isolated. Quantitative real time PCR (QPCR) was performed for myeloperoxidase 
mRNA (surrogate for assessing the extent of PMN sequestration). Figure 3 shows that untreated septic murine 
lung is characterized by significant PMN sequestration. Both AscA and dehydroascorbic acid (DHA) 
significantly attenuated PMN sequestration.  
 
Figure 4 shows H&E stains of 
sections of lungs removed at 16 hours 
following onset of feces induced 
peritonitis. As seen in this figure, 
intense cellular sequestration and 
septal edema is present in the lung of 
unprotected FIP-treated mice. In 
contradistinction, AscA treatment 
significantly attenuated the 
histological findings of murine sepsis.  
 
Sepsis is characterized by the development of 
a severe proinflammatory state with the rapid 
onset of expression of multiple NFκB driven 
genes. Figure 5 shows that septic murine 
lung is characterized by the presence of 
significant expression of two nuclear NFκB 
activated genes examined via quantitative 
real time PCR (KC [murine IL-8 homologue], 
LIX). AscA and DHA administered 
following onset of sepsis significantly 
reduced the expression of these genes. 
 
The image part with relationship ID rId23 was not found in the file.
The image part with relationship ID rId23 was not found in the file.
Figure 5
Figure 3
FIP
FIP-AA
Figure 4


 
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Bacterial sepsis is virtually always accompanied by disordered 
coagulation and is frequently   associated with disseminated 
intravascular coagulation (DIC). A large body of significant 
scientific literature has documented the disruption of microvascular 
function/integrity induced by DIC.xxxix Uncontrolled DIC uniformly 
induces activation of multiple proinflammatory coagulation-
associated proteases that activate both intrinsic and extrinsic 
coagulation pathways. The resulting “cascade effect” produces 
widespread 
microvascular thrombus 
formation and 
subsequent multiple 
organ injury and failure. 
DIC 
frequently produces 
acute lung injury.xl We examined the extent of microvascular 
thrombosis in the lungs of our LPS-treated  wild type mice. Sixteen 
hours after LPS infusion lungs were fixed, paraffin embedded, and 
H&E  stained sections examined for the presence of microthrombi. 
Figure 6, shows that untreated septic lungs exhibit extensive 
microvascular thrombosis (arrows). However, in AscA-treated 
septic lungs (Figure 6), virtually no microthrombi were observed. A 
Zeiss light microscope outfitted with a Axiovision counting software program was used to label and quantify 
microthrombi between multiple lung sections. Figure 7 shows that LPS (without treatment) produced highly 
significant numbers of micro thromboses. AscA intervention in septic mice abolished virtually all 
microthrombi. Similar findings were obtained in DHA treated lungs (data not shown). 
 
Following the onset of sepsis, multiple studies 
now show that sepsis-associated activation of 
Tissue Factor (factor III) is the sentinel event 
that induces the coagulation factor cascade 
leading to DIC. In further  preliminary studies, 
we examined the expression of tissue factor in 
the lungs of septic mice using  QPCR. Figure 8 
shows that both AscA and DHA dramatically 
attenuated the expression of tissue factor in septic 
lungs while inducing the inhibitor profibrinolytic 
peptide tissue factor pathway inhibitor (TFPI). 
 
Summary Of Preliminary Animal Studies: During the animal modeling studies, no untoward effects of AscA or 
DHA on animal subjects was observed. The preliminary data shows convincingly that ascorbic acid is capable 
of significantly altering the course of biological events which arise following the onset of bacterial sepsis that 
lead to lung injury. Our results show significant impacts on sepsis induced mortality with both the reduced and 
oxidized forms of AscA. 
 
The image part with relationship ID rId23 was not found in the file.
LPS+AA
LPS
Figure 6
Figure 8
Control
LPS
LPS/AA
LPS/DHA
Figure 7


 
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1.3 Phase I Human Trial: Vitamin C Infusion In Human Sepsis: Preliminary Results From A Safety 
Trial 
Our animal modeling studies strongly suggested that vitamin C augmentation reverses detrimental septic 
biology that leads to lung injury. On the strength of these studies, we proceeded with a human trial. In 2010, 
following IRB approval, a phase I, randomized, double blind, placebo-controlled trial testing the safety of 
parenteral vitamin C in patients with severe sepsis was initiated at the VCU Medical Center. All patients 
enrolled, regardless of study arm, received full ICU standard of care. Patients were randomized to placebo (5% 
dextrose/water, D5W), low dose vitamin C (50 mg/kg/24hr), or high dose vitamin C (200 mg/kg/24hr). The 
calculated 24 hour vitamin C dose was divided into four equal doses and administered intravenously (in 50 ml 
D5W) over 30 minutes every 6 hours for 96 hours. Vital signs were monitored every 5 minutes during infusion 
and for 45 minutes afterwards by bedside ICU Nursing and the investigative team. Hypotension, tachycardia, 
and nausea/vomiting and hypernatremia were the primary safety outcomes assessed. A multi-departmental 
data safety monitoring board oversaw patient enrollment into the trial. Serum/plasma specimens were obtained 
every twelve hours for 2 days, then once daily for two days. Enrollment: Over a 1 year period, twenty-four 
patients were randomized to the three groups (Placebo, 4M, 4F, age 54-68 yrs.), (Lo-VitC, 5M, 3F, age 30-70 
yrs.), (Hi-VitC, 4M, 4F, age 44-92 yrs.). APACHE II score at Enrollment: Mean APACHE II scores between 
groups were: Placebo - 20.4 (range: 15-29), Lo-VitC: 20.2 (range: 12-33), and Hi-VitC: 24 (range: 17-33) 
respectively. The groups were statistically identical. 
Phase I Primary Outcomes: Safety of Vitamin C Infusion: Safety of vitamin C infusion was the primary focus 
of this trial. During the 96 hour infusion period, no patients were withdrawn due to identified negative outcomes 
(hypotension, tachycardia, nausea/vomiting, or hypernatremia). Infusions were halted in one patient (Hi-VitC) 
following infusion #14 (80hrs) for a ventricular arrhythmia later determined by Cardiology to be artifact. This 
patient is included in the analysis. One patient (Hi-VitC) was transferred to another facility at 48 hours at the 
insistence of family and was lost to follow up. 
Plasma Ascorbate Levels in Human Sepsis: The impact of 
Vitamin C Infusion: Vitamin C levels were quantified by 
HPLC in all patients at enrollment then at defined intervals to 96 
hours. Ascorbate levels in all septic patients at enrollment were 
subnormal (hyposcorbic) at 17.9 ± 2.4 µM (normal 50 – 70 µM) 
and were not significantly different at baseline. Figure 9 shows 
the change in plasma ascorbate levels through time across 
patient groups. Ascorbate levels rose rapidly in the two 
treatment groups and were significantly higher than placebo 
within twelve hours (Lo-VitC vs. placebo P<0.005, Hi-VitC vs. 
placebo p<0.0005) remaining consistently elevated for 96 hours. 
Ascorbate levels in the Hi-VitC group were significantly higher than the Lo-VitC group from the 12 hour point 
forward. These data show that an intermittent ascorbate infusion protocol (every 6 hour) produces sustained 
steady state levels. 
Figure 9


 
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Sequential Organ Failure Assessment (SOFA) Scores  SOFA 
scores obtained are robust indicators of mortality during critical 
illness.xli Increases in SOFA scores during the first 48 hours of ICU 
care predicts a mortality rate of at least 50%. Initial SOFA scores at 
enrollment were: placebo – 13.3 ± 2.9, Lo-VitC – 10.1 ± 2.0, and Hi-
VitC 10.8 ± 4.4 and were not significantly different  across groups. 
Figure 10 shows that patients treated with high dose of vitamin C 
exhibited significantly lower SOFA scores when compared to 
placebo over the 96 hour period. SOFA scores among treated patients 
did not exhibit any subsequent rise whereas patients in the placebo 
group exhibited a gradual rise in scores. Though the cohort size is 
limited, these early data suggest that vitamin C infusion attenuates 
systemic organ injury associated with sepsis. 
Phase I Trial Secondary Outcomes: Following enrollment 1) Days on vasopressor (DOVP), 2) ventilator free 
days (VFD), and 3) ICU days (ICUD), were monitored as 
secondary outcomes.  We observed trends for fewer DOVP and 
ICUD and more VFD in the Lo-VitC patients, but the numbers 
were small and statistically insignificant. Though this study was 
not powered to assess mortality, we present the results of 28 day 
all-cause mortality as a prospectively identified secondary 
outcome (Fig 11). In the placebo group we found a 63% mortality 
(5 of 8 patients died). In the Lo-VitC, 3 of 8 patients died for 38% 
mortality, and 4 of 7 patients died in the Hi-VitC for a mortality 
of 57%. The data showed in figure 11 further attests to the safety of the vitamin C dosing regimens in that there 
was no additional added mortality among the treatment group patients when compared to placebo. 
Vitamin C Infusion Attenuates Biomarkers of Inflammation and Endothelial Injury In Patients with Severe 
Sepsis Sera obtained from enrolled subjects was analyzed for three biomarkers: C-reactive protein   (CRP), 
procalcitonin (PCT), and thrombomodulin 
(TM). CRP and PCT were quantified as 
surrogates for inflammation while TM was 
employed as a surrogate for endothelial injury. 
At enrollment, biomarker levels across the three 
groups were not significantly different. CRP 
and PCT levels in all patient groups started high 
and trended down over time (Fig 12). Of 
importance, patients randomized to receive low 
or high dose vitamin C exhibited more rapid 
reductions in PCT and CRP levels than patients randomized to 
placebo, achieving significantly lower levels when compared to their 
own baseline by 48 hours (p<0.05). Thrombomodulin levels in 
patients randomized to placebo, though not different at baseline, 
began increasing, becoming significantly elevated beyond 36 hours 
remaining significantly elevated when compared to vitamin C treated 
patients (Fig 13). Vitamin C treated patients did not exhibit the 
increases in TM levels observed in placebo-infused patients. Our 
The image part with relationship ID rId23 was not found in the file.
The image part with relationship ID rId23 was not found in the file.
Figure 12
Figure 10
Figure 11
Figure 13


 
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preliminary results suggest for the first time that vitamin C infusion produces early reductions in 
proinflammatory mediators in patients with severe sepsis. The results further suggest that vitamin C infusion 
 
 Summary of Preliminary Phase I Safety Studies in Human Sepsis: During phase I human study/trial no 
untoward/adverse effects of intravenous vitamin C infusion were observed in any patient during the 96 hour 
treatment protocol. The preliminary data gathered suggests that vitamin C is capable of significantly altering the 
course of organ failure, which arises in humans following the onset of bacterial sepsis. Biomarker data also 
suggest that vitamin C infusion attenuates proinflammatory peptide expression, a process that contributes to 
sepsis mediated acute lung injury. Further, ascorbic acid infusion prevented subsequent increases in plasma 
thrombomodulin (an indicator of vascular injury). 
 
1.4. Potential Mechanisms of Action of Ascorbic Acid in Sepsis 
 
• Our experimental data in two murine models of sepsis induced lung injury suggest that vitamin C when 
infused acts in a pleotropic manner, attenuating NFκB inducible genes (chemokines, tissue factor) while 
boosting expression of genes leading to active fibrinolysis (i.e., tissue factor pathway inhibitor). 
• Humans lack L-gulono-γ-lactone oxidase, the final enzyme in vitamin C biosynthesis.xlii 
• Sodium-dependent vitamin C transporters move vitamin C into cells in reduced form or via facilitative 
glucose transporters in oxidized form as dehydroascorbic acid (DHA).xliii DHA is rapidly reduced and trapped 
intracellular as reduced vitamin C or L-ascorbic acid. 
• Though vitamin C circulates in normal human plasma at 60-70µM, it accumulates normally in millimolar 
concentrations in host defense cells (i.e., neutrophils, platelets, macrophages) and endothelium.xliv 
Together with glutathione, vitamin C constitutes a primary line of defense against ROS and promotes 
recycling of other antioxidants (e.g., vitamin E). 
• Subnormal plasma vitamin C concentrations in septic patients correlate inversely with multiple organ failure 
and directly with survival. Vitamin C depletion in sepsis results from: 1) ascorbate consumption by reduction 
of plasma free iron, 2) ascorbate consumption by the scavenging of aqueous free radicals, and 3) by 
destruction of DHA.xlv Sepsis associated vitamin C destruction permits uncontrolled oxidant activity.  
• Clinical protocols currently in use for hospitalized septic patients are inadequate to normalize plasma vitamin 
C levels.xlvi 
• Ascorbate infusion into septic animals: 1) improves survival, corrects hypotension,xlvii
xlviii
 improves capillary 
blood flow, 
 protects endothelial barrier function,xlix attenuates peroxynitrite formation,l attenuates ALI, 
and disrupts lung capillary microvascular thrombosis (see preliminary murine data above). 
 
1.5. Ascorbic Acid Dose Selection 
 
Dosing and bio-distribution data in humans show that pharmacological concentrations of vitamin C can only be 
attained following intravenous administration.li Dosage selection for this trial was determined both from animal 
modeling, examining the biological effectiveness in a lung injury model system and from the recently 
conducted randomized double blind phase I human sepsis safety trial. The 200 mg/kg/24 hour IV dosing 
protocol was determined from quantification of plasma ascorbate levels and from assessing the impact on 
SOFA scores. Further, the dosage was selected following observation of the 200 mg/kg/24 hour regimen on 
biomarker levels. 
 


 
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1.6. Study Rationale 
 
The purpose of this study is to assess the efficacy of intravenously infused ascorbic acid therapy for patients 
with sepsis associated ALI. By restricting the population to those we believe to have both infection and 
evidence for organ dysfunction (severe sepsis), this study targets a disease process and population that has 
been best studied in animal models and by a small RCT. By focusing on sepsis associated ALI, we have 
selected a group that has a higher disease burden than sepsis alone and thus likely to have both increased 
mortality and an increased opportunity for benefit, including a reduction in the requirement for mechanical 
ventilation. Given that the mortality and ventilator days are significant in patients with sepsis associated ALI, 
we believe there is real opportunity for improved clinical outcomes if the right interventional agent can be 
identified. In choosing the SOFA scores, and biomarkers of inflammation, vascular injury, and coagulation, as 
the primary outcomes, we will be able to detect changes in clinical outcomes that are important for proving 
proof of concept. 
 
2. Objectives 
2.1. Primary Objectives 
To assess the efficacy of a 96-hour high dose intravenous vitamin C infusion protocol (200 mg/kg per 24 
hours) in patients with established ALI/ARDS that results from severe sepsis. Patients will be randomized to 
receive either: 1) Placebo (50 ml of 5% dextrose in water) or Vitamin C (sterile L-ascorbic acid for injection at 
200 mg/kg per 24 hours with entire  calculated 24 hour dose diluted in 200 ml of 5% dextrose in water). One 
fourth of the 24 hour calculated dosage will be administered in 30 minute intravenous infusions will occur every 
6 hours.  
 
Clinical, physiological, and biomarker data will be collected at various time points while on study 
(See Appendix G). All data collected below will occur at the following timepoints: hour 0 (collected 
within the 24 hours prior to randomization, or post randomization, but pre-infusion), hour 48 (a 
timepoint prior to infusion 9), 96 (a timepoint close to hour 96 and after infusion 16), hour 168 (a 
timepoint close to hour 168) 
  : 
• VS (Body weight, blood pressure, heart rate, Temperature, mean arterial pressure, oxygen 
saturation, central venous pressure, glasgow coma score) 
• Vasopressor use (amount and type) (mcg/kg/min)  
• Ventilator data – Is the patient on or off vent? 
 If on mechanical ventilation: Mean Airway Pressure, tidal volume, Peak Inspiratory 
Pressure, FiO2, Respiratory Rate, Plateau Pressure, Positive End Expiratory Pressure 
(data recorded from chart at the 8am time point or closest time point to 8am available) 
• Arterial Blood Gases: pH, PaO2, PaCO2, SpO2  - for as long as subject has arterial line 
• Laboratory (Sodium, Potassium, Chloride, Metabolic Glucose, Hemoglobin, Hematocrit, 
Platelets, White Blood Cell Count, Creatinine, Blood Urea Nitrogen, Bilirubin (data recorded 
from chart at the 8am time point or closest time point to 8am available).  
• Biomarker blood sample 
• Sequential Organ Failure Assessment Score (SOFA): (if intubated use PaO2/FiO2 ratio for 
calculation.  If not intubated see Appendix H for caluculating FiO2 and PaO2/FiO2 from SpO2. 
Calculate using the worst value in the 24 hours preceding the Score time point for each 
component. See Appendix G for Modified SOFA Score Calculator. 
 


 
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2.2. Hypothesis 
Hypothesis 1A: Vitamin C infusion will significantly attenuate sepsis associated systemic organ failure as 
measured by Sequential Organ Failure Assessment (SOFA) score,  
Hypothesis 1B: Vitamin C infusion will attenuate sepsis associated lung injury as assessed by the oxygenation 
index and the VE40 
Hypothesis 1C: Vitamin C infusion will attenuate biomarkers of inflammation (C-Reactive Protein, 
Procalcitonin), vascular injury (Thrombomodulin, Angiopoietin-2), alveolar epithelial injury (Receptor for 
Advanced Glycation End Products), while inducing the onset of a fibrinolytic state (Tissue Factor Pathway 
Inhibitor).  
 
3. End-Points 
 
Analysis of the primary, secondary and other endpoints will be conducted on an intention-to- treat (as 
randomized) basis. 
 
3.1. Primary Endpoints 
 
Primary Endpoint #1: Change in SOFA score at 96 hours as compared to baseline when compared to placebo.  
Primary Endpoint #2: C-Reactive Protein and Thrombomodulin at study hours 0, 48, 96, 168 when compared 
to placebo. 
 
Explanation For The Choice of Primary Endpoints: The phase I trial was a safety trial and numbers of patients 
studied were small. The SOFA score was chosen as the "physiological primary endpoint" due to the prompt 
and significant reductions in the SOFA score observed in the high dose vitamin C group. The SOFA score, 
though not a primary lung function score, contains the PaO2/FiO2 ratio in its calculation. We have chosen C-
Reactive Protein and Thrombomodulin as broad indicators of inflammation and vascular injury to serve as the 
primary endpoint biomarkers. 
 
3.2. Secondary End Points 
 
Secondary Endpoints: 
• Oxygenation Index (FiO2 x Mean Airway Pressure/PaO2) at study hour 0, 48, 96, 168 if still intubated 
in ascorbate infused patient compared to placebo. 
• VE-40 (Vent RR x TV/Weight) x (PaCO2/40) at study hour 0, 48, 96, 168 if still intubated, in ascorbate 
infused patient compared to placebo 
• SOFA scores at hours 48, 96, 168 
• SOFA Score Components at hours 48,96, 168 
o PaO2/FiO2 
o SpO2/FiO2 
o Platelets 
o Total Bilirubin 
o Vasopressor status 
o GCS 


 
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o Creatinine or Urine Output 
• Angiopoietin-2, Procalcitonin, Receptor for Advanced Glycation End Products, Tissue Factor Pathway 
Inhibitor at study hour 0, 48, 96, 168  
• Ascorbate level at hour 0, 48, 96, 168 
• Ventilator Free Days to day 28 
• ICU-free days at day 28 
• All cause mortality to day 28 
• Hospital-free days at day 60 
 
VE40 is a bedside pulmonary dead-space calculation and is defined as the minute ventilation needed to 
bring PaCO2 to 40 mm Hg.lii Ventilator Free Days or VFDs to day 28 are defined as the number of days 
from the time of initiating unassisted breathing to day 28 after randomization, assuming survival for at least 
two consecutive calendar days after initiating unassisted breathing and continued unassisted breathing to 
day 28.  If a patient returns to assisted breathing and subsequently achieves unassisted breathing to day 28, 
VFDs will be counted from the end of the last period of assisted breathing to day 28.  A period of assisted 
breathing lasting less than 24 hours and for the purpose of a surgical procedure will not count against the 
VFD calculation. If a patient was receiving assisted breathing at day 27 or dies prior to day 28, VFDs will 
be zero.  Patients transferred to another hospital or other health care facility will be followed to day 28 to 
assess this endpoint. ICU- and Hospital-free days to day 28 and day 60 are defined as the number of days 
alive between day 1 and day 28 and day 1 and day 60 which were spent outside the ICU or outside of the 
hospital respectively. 
 
3.3. Focused Safety Analysis: 
 
The current trial will be enrolling patients with sepsis associated acute lung injury. We therefore expect that 
many of these patients will have some degree of organ dysfunction.  
3.3.1 Renal Monitoring Plan 
Patients with sepsis are at high risk of metabolic acidosis (including lactic acidosis). To prevent the possibility 
of metabolic acidosis due to drug administration, the study drug is formulated to a neutral pH of 7.4. Therefore, 
we do not anticipate the need for additional monitoring of acid/base balance beyond standard-of-care provided 
at each institution. Any observed abnormalities will be evaluated according to standard-of-care practice and 
documented in the research record. 
3.3.2 Glucose Monitoring Plan 
Guidance for blood glucose monitoring in patients enrolled in the CITRIS-ALI Trial: 
Ascorbic acid is known to artefactually raise POC blood glucose readings by all POC devices except the 
StatStrip glucometer. However, it does not raise blood glucose readings from a basic metabolic panel or glucose 
results using the gas lab.  Thus, extreme care must be taken to assure an accurate blood glucose level from a 
metabolic laboratory (BMP) or arterial blood gas panel before initiating any insulin therapy, including sliding 
scale or scheduled insulin. 
All study sites not using the StatStrip POC glucometer should follow these guidelines: 
Guidance for blood glucose monitoring in patients enrolled this study: 


 
CITRIS Protocol Version 9 
 
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• Critical care Nursing and Physician leadership at all study sites must be informed of vitamin C’s effect 
on point of care (glucometer) blood glucose and arterial blood gas glucose point of care values. 
• In-service training  will be documented in the Study Training Log 
• Bold signage will be displayed on all study instructions, data collection forms, and at the patient’s head 
of bed, stating: 
 STOP! Do not use Accuchek or other Point of Care devices to measure glucose on this patient 
 Use only metabolic or gas lab glucose screening methods 
 This patient is enrolled in a study with Vitamin C, which artefactually increases POC glucose 
testing 
 Do Not Initiate or Utilize Sliding Scale, Scheduled Insulin, or Continuous Insulin 
Infusion  Without Laboratory Confirmation of Blood Glucose 
• 
Those receiving insulin infusion or sliding scale insulin will have metabolic glucose screening on the 
schedule determined by the primary physician and paid for by the study 
• 
Blood glucose monitoring for insulin administration guidance should only be by a metabolic or blood 
gas laboratory measured blood glucose results, whether or not the study patient is receiving insulin  
• 
Study personnel will follow each study patient closely to monitor insulin use to ensure that point of 
care glucose screening is suspended for the research subject. 
• 
If subject loses central venous access (PICC line and arterial line acceptable), Vitamin C infusions are 
to stop but subject not withdrawn.  Data collected through end of study. 
• 
Point of care glucose testing may resume 36 hours after the last infusion of study drug. 
4. Study Population and Enrollment 
4.1. Number/Source/Screening 
 
The trial will accrue a maximum of 170 patients over a 2-3 year period. Patients with sepsis associated ALI 
will be recruited from intensive care units at Virginia Commonwealth University Health System, Medical 
College of Wisconsin and sub-site, Aurora St. Luke’s Medical Center, The Cleveland Clinic Health System 
and its sub-site, Fairview Hospital.  Study personnel will review patients within intensive care units daily to 
identify potential candidates for enrollment. Permission to approach patients and/or their families will be 
requested from the attending physicians in charge of patient care in the ICU. All patients meeting the 
inclusion/exclusion criteria will be approached with a consent and will be entered into a screening log. If the 
patient is not enrolled, the screening log will include information explaining why enrollment did not occur 
(exclusion criteria, attending physician denial, patient refusal, etc.).  
 
Patients will be documented in the Study Screening Log when all Inclusion Criteria are met.  A Screen 
Failure is defined as a patient meeting all Inclusion Criteria but not meeting Exclusion Criteria.  
4.2. Inclusion Criteria 
4.2.1 CITRIS-ALI Inclusion Criteria: 
CITRIS-ALI Inclusion Criteria: 
Patients must have suspected or proven infection, and meet 2 out of 4 of the criteria for Systemic Inflammatory 
Response (SIRS) due to infection, and be accompanied by at least 1 criterion for sepsis associated organ 
dysfunction, and meet all 5 criteria for Acute Respiratory Distress Syndrome (ARDS). 


 
CITRIS Protocol Version 9 
 
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1. Suspected or proven infection: (e.g., thorax, urinary tract, abdomen, skin, sinuses, central venous 
catheters, and central nervous system, see Appendix A). 
2. The presence of a systemic inflammatory response: Defined as: fever:  >38ºC (any route) or 
hypothermia: <36ºC (core temp only), tachycardia: heart rate > 90 beats/min or receiving medications 
that slow heart rate or paced rhythm, leukocytosis:  >12,000 WBC/µL or leukopenia:  <4,000 WBC/µL 
or >10% band forms.  Respiratory rate > 20 breaths per minute or PaCO2 < 32 or  invasive mechanical 
ventilation. 
3. The presence of sepsis associated organ dysfunction: (any of the following thought to be due to 
infection) 
a. Sepsis associated hypotension (systolic blood pressure (SBP) < 90 mm Hg or an SBP decrease > 
40 mm Hg unexplained by other causes or use of vasopressors for blood pressure support 
(epinephrine, norepinephrine, dopamine =/> 5mcg, phenylephrine, vasopressin) 
b. Arterial hypoxemia (PaO2/FiO2 < 300) or supplemental O2 > 6LPM. 
c. Lactate > upper limits of normal laboratory results 
d. Urine output < 0.5 ml/kg/hour for > two hours despite adequate fluid resuscitation 
e. Platelet count < 100,000 per mcL 
f. Coagulopathy (INR > 1.5) 
g. Bilirubin > 2 mg/dL 
h. Glasgow Coma Scale < 11 or a positive CAM ICU score 
4. ARDS characterized by all the following criteria 
a. Lung injury of acute onset, within 1 week of an apparent clinical insult and with progression of 
respiratory symptoms 
b. Bilateral opacities on chest imaging not explained by other pulmonary pathology (e.g. pleural 
effusions, lung collapse, or nodules) 
c. Respiratory failure not explained by heart failure or volume overload 
d. Decreased arterial PaO2/FiO2 ratio ≤ 300 mm Hg 
e. Minimum PEEP of 5 cmH2O  
 
CITRIS-ALI Exclusion Criteria: 
1. Known allergy to Vitamin C 
2. inability to obtain consent;  
3. age < 18 years; 
4. Not on a ventilator 
5. No indwelling venous or arterial catheter in patients requiring insulin in a manner that requires glucose 
being checked more than twice daily (e.g. continuous infusion, sliding scale)  
6. Presence of diabetic ketoacidosis 
7. more than 48 hrs since meeting ARDS criteria; 
8. patient or surrogate or physician not committed to full support (not excluded if patient would receive all 
supportive care except for cardiac resuscitation); 
9. pregnancy or breast feeding, 
10. moribund patient not expected to survive 24 hours; 
11. home mechanical ventilation (via tracheotomy or noninvasive) except for CPAP/BIPAP used only for 
sleep-disordered breathing; 
12. on home O2 > 2LPM, except for with CPAP/BIPAP 
13. diffuse alveolar hemorrhage (vasculitis); 
14. interstitial lung disease requiring continuous home oxygen therapy; 


 
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15. Active kidney stone 
16. Non English speaking; 
17. Ward of the state (inmate, other) 
 
4.3. 
Enrollment, Randomization, and Study Initiation Time Window 
 
All ALI criteria (4.2.3 a-d above) must occur within the same 24-hour period. The onset of ALI is when the 
last criterion is met.  Patients must be consented within 48 hours of ALI onset.  SIRS criteria must occur 
within the 48 hours before and 24 hours after ALI onset. Information for determining when these time 
window criteria were met may come from either the study hospital or a referring hospital report.  
Randomization must occur within the same 48 hours of ALI onset, as is for consent.  Dose 1 must be 
administered within 6 hours of randomization.  Following randomization, the low tidal volume protocol for 
mechanical ventilation (Appendix C) and the fluid management strategy protocol (Appendix D) may be 
initiated within one and four hours respectively (if not already being utilized), if clinically indicated. 
 
4.4. 
Informed Consent 
 
Informed consent will be obtained from each patient or surrogate (family or legal representative) before 
enrollment in the trial. No study procedures will be conducted before obtaining informed consent. 
 
4.5. 
Randomization 
 
After informed consent is given, a randomized assignment will be made by the Investigational 
Pharmacy of the Lead Site and Coordinating Center (VCUHS) to administer either Vitamin C therapy or 
placebo. Each participating pharmacy will have a pre-defined 
randomization chart by which to determine whether to 
administer the study drug or placebo to each particular 
subject. The randomization will be stratified by institution to 
one of the two study arms. 
 
4.7. 
Minorities and Women 
 
 The demographic profiles of the Centers selected for the study 
show that the aggregate patient population contains representative 
proportions of minorities and women (Table 1). Recruitment of 
minorities and women will be monitored by the Study Coordinating 
Center.  If necessary, additional recruitment efforts will be made at specific centers to ensure that the 
aggregate patient sample contains appropriate gender and minority subsets. Pregnant women will be excluded 
because of the lack of safety data for infused Vitamin C use during pregnancy. 
5. Study Procedures 
 
If a pregnancy test is not available before informed consent, blood or urine tests will be obtained after 
informed consent but before randomization to ensure eligibility. Patients excluded on the basis of tests 


 
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obtained in this manner will not be included in the study. 
 
5.1. Vitamin C or Placebo Administration 
 
All study drug doses will be administered via central or peripheral line infusion. Should no central or 
peripheral line be available at scheduled time of infusion, a call should be placed to pharmacy to 
determine if study drug may be piggybacked into the line that is infusing a different drug.  If 
administering study drug via piggyback is contraindicated then study drug infusion may be delayed by a 
maximum of 6 hours. If clinical drug administration schedule is such that study drug will not have an 
available administration time beyond this delay, a dedicated new line (peripheral or central) should be 
inserted. Study drug will be blinded using an identical appearing placebo.   
 
The prepared IV bags will have the IV tubing attached and primed by the study pharmacist.  Amber shrouding 
will be used to cover the IV bag and the IV tubing in order to maintain the blind.  The product labeling will be 
blinded as to what the actual product is.  For example, the drug name and dose will be indicated as per the 
following:  "Ascorbic acid ____mg or placebo in 50cc D5W". 
 
 
1. First study drug dose (L-ascorbic acid or placebo) will be considered “Dose 1” and will be 
administered within 6 hours of randomization or the earliest available time post any clinically indicated 
procedure which requires the patient to be off the unit. All doses will be administered in the ICU.  
Patients receiving vitamin C will receive 25% of the total daily calculated dosing (200mg/kg/24 hours) 
and will be infused over 30 minutes for this first dosing. 
2. Subsequent doses which represent 25% of the day’s total dose will be infused every six hours through 
96 hours (+/- 3 hours).  
a. Timing of Dose 2 will be triggered by the physician order for q 6 hour administration and will 
therefore be listed on the bedside MAR.  As such, timing of Dose 2 may be out of the +/- 3 
hour window and will not trigger a protocol deviation. 
b. If for any reason any other maintenance dose is not administered within window, the dose will 
be skipped and the next scheduled dose will be given and documented in the data collection 
tool.  
 
5.2. Drug Level Specimens (venous blood) 
 
Preliminary studies performed during the phase I trial showed that an every 6 hour infusion protocol resulted 
in steady state plasma levels after 18 hours. In this phase II trial, plasma levels of Vitamin C will be 
obtained to determine the relationship, if any, of plasma levels to either the pleiotropic effects or the 
toxicities of Vitamin C or to biomarker levels. 
 
5.3. Completion of Study Drug Administration 
 
Completion of study drug administration: Study drug administration will be stopped when one of the 
following conditions is met, whichever comes first: 
1. Final drug dose at 96 hours or discharge from ICU, whichever comes first. 
2. Discharge from study hospital 
3. Loss of indwelling venous or arterial catheter with no intent to replace the line, making it impossible to 
monitor glucose levels via central laboratory without multiple peripheral sticks.   


 
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4. Withdrawal from study 
5. Death 
 
Note: If a patient is readmitted to the ICU after study drug has already been stopped per protocol, it does 
NOT get restarted when readmitted to the ICU. 
5.4. Premature Withdrawal from Treatment 
 
Loss of indwelling venous or arterial catheter will trigger the stopping of Vitamin C infusions but subjects will 
remain on study.  Blood glucose monitoring will continue via the central laboratory for 36 hours after the last 
infusion via peripheral IV draws or peripheral sticks.  Biomarker sampling via peripheral IV and/or peripheral 
stick is allowable as it occurs only 4 times throughout the study and likely only once (if at all) after the patient 
has been discharged from the unit and is without a central line. 
 
The study drug will be discontinued if a patient develops a metabolic acidosis unexplained by other 
etiologies (lactic acidosis secondary to septic shock). Determination of the presence of metabolic acidosis 
will be made by the site investigator.  Study drug will also be discontinued if primary care team or surrogate 
decision maker request withdrawal. Data collection will continue on these patients following withdrawal of 
study drug. 
 
Requests to unblind a patient's study treatment can be made to the study (investigational) pharmacist.  
Unblinding study treatment should occur only in the case of an emergency when knowledge of the study 
treatment is essential for subject care to treat a serious adverse event and prevent further harm or death. 
If possible, a decision to unblind should be discussed with the Principal Investigator or a sub-Investigator prior 
to unblinding the study treatment.  
If a blind is broken (either intentionally or unintentionally), the circumstances should be documented as 
to who, what, when, and why and all documentation shall be kept by the study pharmacist. 
 
5.5. Ventilator Procedures 
 
Ventilator management, including weaning, will follow the modified ARDS Network lower tidal volume (6 
ml/kg PBW) protocol (Appendix C).liii  If not already being utilized, this low tidal volume protocol for 
mechanical ventilation will be initiated within one hour of randomization, if possible. Since the time a patient 
achieves unassisted ventilation affects the secondary endpoint of ventilator free days (VFDs), and because 
recent evidence-based consensus recommendations have identified a best practice for weaning, weaning 
strategy will also be controlled by protocol rules in accordance with these evidence-based recommendations. 
This will assure similar weaning methods.  This newer weaning strategy is a simplified version of the 
weaning strategy used in prior ARDS Network study protocols (see Appendix C). 
 
5.6. On-Study Fluid Management 
 
Fluid management during shock will not be prescribed per study protocol. In subjects who are not in shock, a 
conservative fluid management approach will be administered, if possible. This conservative fluid 
management approach will represent a simplification of the algorithm utilized in the ARDS Network FACTT 
study (see Appendix D).liv  If not already being utilized, this conservative fluid management approach will be 
initiated, if possible, within four hours of randomization and continued until the subject has reached unassisted 


 
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breathing (UAB) or study hour 168, whichever occurs first. 
6. Data Collection 
 
6.1. Background Assessments 
 
1. Demographic:  
a. Gender 
b. Age 
c. Race/Ethnicity 
2. Insurance status 
a. Privately insured 
b. Medicaid 
c. Medicare 
d. Other public 
e. uninsured 
 
3. Pertinent Medical History and Physical Examination 
a. Etiology of Sepsis 
b. diabetic status – Hx of Diabetes 
Yes 
No  
i. Insulin received? 
 
Yes 
No 
c. patient place of residence: Home independently, home with help (supervision, direction, personal 
assistance), home with professional help (nursing/nursing service), intermediate care or rehab 
facility, skilled nursing facility, other (specify) 
d. Patient admitted directly from: OR, Recovery Room, ER, Floor, another special care unit, 
another hospital, direct admit, step-down unit 
e. Hx of alcohol use via the AUDIT-C Questionnaire: 
i. How often do you have a drink containing alcohol? 
Never 
Monthly or less 
2-4 times per month 
2-3 times per week 
4 or more times per week 
ii. How many standard drinks containing alcohol do have on a typical day? 
1 or 2 
3 or 4 
5 or 6 
7 to 9 
10 or more 
iii. How often do you have six or more drinks on one occasion? 
Never 
Less then monthly 
Monthly 
Weekly 


 
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Daily or almost daily 
 
4. Study enrollment date 
 
5. Acute or Chronic renal failure and use of dialysis 
 
6.2. Baseline/Hour 0 Assessments and Procedures 
 
The following information will be recorded during the 24 hour interval preceding randomization. If more than 
one value is available for this 24 hour period, the value closest to the time of randomization will be recorded. If 
no values are available from the 24 hours prior to randomization, then values will be measured post 
randomization but prior to initiation of study drug. 
 
Vital Signs: Blood Pressure (BP), Heart Rate (HR), Mean Arterial Pressure (MAP), Respiratory Rate 
(RR), Temperature., O2 saturations, Central Venous Pressure (CVP), Body weight, Glascow Coma 
Score 
Suspected or known site of sepsis 
SOFA Score 
VE40 = [Minute Ventilation ÷ Weight (kg)] x [PaCO2 ÷ 40] – closest one to time of randomization 
Oxygenation Index = [FiO2 x Mean Airway Pressure] ÷ PaO2 
Ventilator Data: tidal volume, FiO2, PEEP, inspiratory plateau pressure, Peak Inspiratory 
Pressure, and mean airway pressures.  
Arterial Blood Gasses: PaO2, PaCO2, pH, HCO3 and SpO2 
Serum Sodium, Potassium, Metabolic Glucose, BUN, Creatinine, Billirubin Total, WBC, Hgb, Hct, 
Platelets, PT/INR 
Vasopressors or inotropes (epinephrine, norepinephrine, phenylephrine, vasopressin, dopamine) 
Insulin received?  
Yes 
No 
In/Out – total – for first 7 days or for every day in ICU, whichever is shorter 
In/Out – Urine - for first 7 days or for every day in ICU, whichever is shorter 
Concomitant Medications: use of steroids 
Blood for biomarkers will equal approximately 12ml. Blood samples will be processed and 
divided per the Laboratory Instructions Manual 
 
6.3. 
Assessments after Enrollment 
 
The following data will provide the basis for assessing protocol compliance and safety as well as between-
group differences in several efficacy variables.  Data for each of the variables will be recorded on the days 
shown in the Time-Events schedule (Appendix E) or until death or discharge from the intensive care unit. 
 
 
Hours:  
 
Required: 
 
Q 24 hours for as 
long as patient is in 
ICU or 7 days 
(whichever comes 
first 
I/O Total 
I/O Urine 
Insulin Receipt 
0, 48, 96, 168  
VS, SOFA, ABGs, Routine Labs, Con Meds, AE/SAE Assessments, Blood draw for 


 
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30
Biomarkers, Insulin Receipt 
 
As Available: 
VE40, OI, Weight, Bilirubin, Ventilator Data, known site of sepsis 
28 Days 
Required: 
Ventilator Free Days to Day 28, All-Cause Mortality to Day 28, ICU Free Days to 
Day 28 
 
Use of dialysis 
60 Days 
Hospital Free Days at Day 60 
 
 
Reference Measurements 
All data collected below will occur at the following timepoints: hour 0 (collected within the 24 hours prior to 
randomization, or post randomization, but pre-infusion), hour 48 (a timepoint prior to infusion 9), 96 (a 
timepoint close to hour 96 and after infusion 16), hour 168 (a timepoint close to hour 168) 
 
1. Ventilator Data - The following conditions will be ensured prior to measurements:  no endobronchial 
suctioning for 10 minutes; no invasive procedures or ventilator changes for 30 minutes. 
2. Arterial Blood Gases when Arterial line is in place for clinical reasons 
3. Vital Signs 
4. Labs 
5. Vasopressor  
6. Use of Steroids 
7. Blood samples for biomarkers.  
 
Blood specimens will be batch-sent to the VCU Central Repository to be stored. Specimens will be identified 
by a unique number.  All data released by the Clinical Coordinating Center for studies will be linked to the 
specimen but will be de-identified. Plasma collected for this trial will be frozen and stored at the VCU bio-
repository for future research. 
7. Statistical Considerations 
 
7.1. Statistical Methods 
 
CITRIS-ALI Data Analysis Plan: We plan to enroll 170 patients in the CITRIS-ALI study (85 per 
group) to allow for the possibility of approximately 10% dropouts. The projected sample size for this study 
should provide adequate power to detect meaningful differences between the study groups over time. All tests 
will be two-sided at the α = .05 level of significance. Effects will be reported with a point estimate and 95% 
confidence intervals in addition to p-values. We will examine the distributions of all measures and identify 
possible outliers; outliers will be thoroughly checked for collection or data entry errors before being used in the 
analysis. All hypotheses will be tested and data analysis will be done using a variety of statistical methods with 
the most common method expected to be a Mixed Linear Model (MLM) for continuous repeated measures. To 
assess the effect of the treatment on continuous outcome measures that repeat over time the MLMlv,lvi will be 
used to fit a series of repeated measures ANOVA (RMANOVA) models. These models will have one between 
subject factor (Group; Placebo, Hi-VitC), one within subject factor (Time; Baseline, 12 hours, 24 hours, 36 
hours, 48 hours, 72 hours, 96 hours, 7 days) and the interaction between “Group and Time.” The Group by 


 
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Time interaction term will allow us to test the hypothesis that the difference between the treatment groups is the 
same over time. The MLM that will be used for these analyses differs from the usual general linear model (i.e. 
ANOVA) in two ways. The MLM allows for the inclusion of both fixed and random effects at the same time 
and thus allows for the complete analysis of repeated measures designs. Second, observations in the MLM are 
not required to be independent, as is the case with ANOVA, so that correlated observations that arise from 
repeated measurements made on the same subjects can be accommodated. For the repeated within subjects 
measures, a variety of variance-covariance structures will be evaluated to determine which provides the best fit 
to the observed data. Further, MLMs do not require complete repeated measurements data on all subjects when 
used to estimate the course of the outcome variable over time. Incomplete or missing data are handled by the 
model, providing that the missing data are assumed to be “missing at random.”lvii While it is expected that the 
randomization process will prevent any group differences with respect to factors that could impact the outcome 
measures we can also fit models that include these measures as additional covariates to determine whether any 
Group by Time interactions remain significant. Early Stopping: An early stopping determination will be made 
by the Data Safety Monitoring Board 
Power Estimates Sample Size Calculation: The primary goal of this study is to examine the efficacy of 
vitamin C infusion on organ failure and selected biomarkers. We have chosen the co-primary variables of (1) 
SOFA score, (2) plasma c-reactive protein and (3) thrombomodulin. The sample size for the proposed study was 
calculated using observed organ failure data and the biomarker analyses from the phase I clinical trial at VCU. 
Using data from two biomarkers and the SOFA scores, a power/sample size calculation was conducted. Using 
the RMANOVA model and assuming an alpha level of 0.05, and using a Holm-Bonferroni correction to 
accommodate the multiple tests, the 
following table shows the required 
sample sizes to detect a significant 
Group by Time interaction effect at 96 
hours. 
 
 
 
 
Subgroup Analyses from data at enrollment: 
Shock presence or absence 
8. Data Collection and Site Monitoring 
 
8.1. Data Collection 
 
Research coordinators will collect data and enter it directly into the web-based data entry system managed by 
the Clinical Coordinating Center and record on paper data forms. Data will be transferred to the Clinical 
Coordinating Center on a prescribed basis through a web-based data entry program. 
 
8.2. Site Monitoring 
 
Remote monitoring will be used and augmented with site visits performed by a contracted monitoring service 
to ensure that all regulatory requirements are met and to monitor the quality of the data collected. Records of 
Institutional Review Board approvals and patients’ charts will be examined on a spot check basis to evaluate 
Power Estimates for a variety of samples sizes, α=0.05 significance level, for multiple 
primary endpoints using the Holm-Bonferroni Correction
Empirical Power
75/Group
80/Group
85/Group
90/Group
95/Group
100/Group
Co-Primary Variables: 
SOFA, CRP, TM
77%
77%
80%
80%
82%
83%


 
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the accuracy of the data entered into the database. 
 
9. Risk Assessment 
 
9.1. Risks of Active Study Drug 
 
Intravenous Ascorbic Acid Infusion: High dose ascorbic acid therapy is a powerful anti-oxidant and 
micronutrient and a safe therapy in normal subjects and in critically ill patients.lviii Muhlhofer and colleagues 
examined for the presence of adverse effects following intravenous infusion of 7500 mg Ascorbic Acid daily for 
6 days in normal subjects (n=6). No abnormalities in laboratory analysis (fasting state; hemoglobin, leukocytes, 
platelets, sodium, potassium, calcium, chloride, glucose, creatinine, urea, bilirubin, ALT, AST, ggGT, alkaline 
phosphatase, and prothrombin time were found. Nathens et al infused high dose ascorbic acid (1500 mg three 
times daily) into critically ill surgical patients for 28 days and found no serious adverse events.lix Hoffer et al 
infused dosages as high as 1.5 grams/kg body weight three times weekly into patients with advanced cancer.lx  
Adverse events were infrequent consisting of nausea, vomiting, dizziness, and headache.  A systematic review 
conducted by Hans K. Biesalski lxion the safety of the long term low dose parenteral administration of ascorbic 
acid in patients on haemodialysis revealed it to be safe with frequent monitoring of oxalate following dialysis.  
Finally, in the safety study conducted here at VCU testing the safety of infusing high doses of ascorbic acid in 
patients with sepsis, in which approximately 8 of 24 subjects were receiving dialysis, no adverse events 
occurred that could be related to the ascorbic acid.  
 
Potential Physical Risks of Ascorbic Acid Infusion: As noted above, the risks associated with ascorbic acid 
infusion are few. Potential risks include: dry mouth, nausea, vomiting, dizziness, headache. 
 
Potential Psychological, Social, Legal Risks of Ascorbic Acid Infusion: No psychological, social or legal 
risks are identifiable from an extensive literature search. The recently completed phase I trial: Vitamin C 
(Ascorbic Acid) Infusion in Human Sepsis where up to 16 grams of ascorbic acid was infused daily for 4 
days identified no further risk that that identified. 
 
9.2. Risks of Blood Draws 
 
All patients will have blood drawn for research purposes. Most blood will be drawn through indwelling 
catheters. Risks of drawing blood percutaneously are uncommon and include bleeding and bruising. 
 
9.3. Minimization of Risks 
 
Federal regulations at 45 CFR 46.111(a) (1) requires that risks to subjects are minimized by 
using procedures which are consistent with sound research design.  There are several elements of study design 
in the present protocol that meets this human subject protection requirement. 
 
Exclusion criteria prohibit participation of patients who might be at increased risk from the effects Vitamin C.  
Additionally, no adverse events occurred during the pilot study found to be related to the study drug.  Finally, 
vigilant clinical monitoring is standard of care for ICU patients. 
 


 
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9.4. Potential Benefits 
 
Most observational studies suggest a mortality benefit from prior or in-patient Vitamin C use after 
hospitalization for serious infections.  None of the observational trials have reported significant Vitamin C-
related toxicity.  An animal model of acute lung injury with intravenous LPS and feces induced peritonitis 
demonstrate significantly less lung injury with Vitamin C, which may result in shortening the time patients 
require mechanical ventilation. 
 
9.5. Risks versus Benefits 
 
The identifiable risks arising from exposure to intravenous ascorbic acid infusion are low. In our preliminary 
data, we extensively outlined the potential benefits brought by attenuation of acute lung injury and organ failure 
associated with bacterial sepsis. Given the low risk associated with ascorbic acid infusion and the potential high 
likelihood of benefit we assess the risk/benefit ratio to be low (i.e., that benefit far outweighs risk). 
 
10. Human Subjects 
 
Each study participant or a legally authorized representative must sign and date an informed consent form.  
Institutional review board approval will be required before any subject is entered into the study. 
 
10.1. Selection of Subjects 
 
Screening for patients to be enrolled in the CITRIS-ALI trial will occur in the ICUs at VCU Health System, 
The Medical College of Wisconsin and sub-site, Aurora St. Luke’s Medical Center , The Cleveland Clinic and 
its sub-site, Fairview Hospital. 
  
10.1.1. Equitable Selection of Subjects 
 
Federal regulations at 45 CFR 46(a)(3) require the equitable selection of subjects.   The ICUs will be 
screened to determine if any patient meets the inclusion and exclusion criteria. Data that have been collected 
as part of the routine management of the subject will be reviewed to determine eligibility.  No protocol-
specific tests or procedures will be performed as part of the screening process.  If any subjects meet criteria 
for study enrollment, then the attending physician will be asked for permission to approach the patient or 
his/her surrogate for informed consent.  Justifications of exclusion criteria are given in Section 4.3. These 
exclusion criteria neither unjustly exclude classes of individuals from participation in the research nor 
unjustly include classes of individuals from participation in the research.  Hence, the recruitment of subjects 
conforms to the principle of distributive justice. 
 
10.1.2. Vulnerable Subjects 
 
The present research aims to investigate the safety and efficacy of a type of treatment for patients with ALI 
and ARDS secondary to severe sepsis.  No vulnerable subjects will be entered into this phase II trial. 
 


 
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10.2. Informed Consent 
 
Federal regulations 45 CFR 46.111(a)(5) require that informed consent will be sought from each prospective 
subject or the subject’s legally authorized representative.  The investigator is responsible for ensuring that the 
patient or patient’s legal representative understands the risks and benefits of participating in the study, and 
answering any questions the patient may have throughout the study and sharing any new information in a 
timely manner that may be relevant to the patient’s or the legal representative’s willingness to continue his or 
her participation in the trial. All study participants or their surrogates will be informed of the objectives of 
the study and the potential risks.  The informed consent document will be used to explain the risks and 
benefits of study participation to the patient in simple terms before the patient is entered into the study, and to 
document that the patient is satisfied with his or her understanding of the risks and benefits of participating in 
the study and desires to participate in the study.  The investigator is responsible for ensuring that informed 
consent is given by each patient or legal representative. This includes obtaining the appropriate signatures 
and dates on the informed consent document prior to the performance of any protocol procedures and prior to 
the administration of study agent. 
 
 
10.3. Identification of Surrogates 
 
Many of the patients approached for participation in this research protocol will have limitations of decision-
making abilities due to their critical illness. Hence, most patients will not be able to provide informed 
consent.  Accordingly, informed consent will be sought from the potential subject’s legally authorized 
representative. 
 
Regarding proxy consent, the existing federal research regulations (‘the Common Rule’) state at 
45 CFR 46.116 that: “no investigator may involve a human being as a subject in research…unless the 
investigator has obtained the legally effective informed consent of the subject or the subject’s legally 
authorized representative”; and defines at 45 CFR 46 102 (c) a legally authorized representative (LAR) as: “an 
individual or judicial or other body authorized under applicable law to consent on behalf of a prospective 
subject to the subject’s participation in the procedures(s) involved in the research.”  OHRP defined examples 
of “applicable law” as being state statutes, regulations, case law, or formal opinion of a State Attorney General 
that addresses the issue of surrogate consent to medical procedures.  Such “applicable law” could then be 
considered as empowering the surrogate to provide consent for subject participation in the research. 
Interpretation of “applicable law” is therefore state specific and hence, will be left to the discretion of the 
individual IRBs of the respective clinical centers involved in the CITRIS-ALI trial. 
 
According to a previous President’s Bioethics Committee (National Bioethics Advisory Committee), an 
investigator should accept as an LAR…a relative or friend of the potential subject who is recognized as an 
LAR for purposes of clinical decision making under the law of the state where the research takes 
place.
lxiii
lxii Finally, OHRP has opined in their determination letters that a surrogate could serve as a LAR for 
research decision making if such an individual is authorized under applicable state law to provide consent 
for the “procedures” involved in the research study.
 
 
10.4. Justification of Surrogate Consent 
 
According to the Belmont Report, respect for persons incorporates at least two ethical convictions; first, that 
individuals should be treated as autonomous agents, and second, that persons with diminished autonomy are 
entitled to protection. One method that serves to protect subjects is restrictions on the participation of 


 
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subjects in research that presents more than minimal risks. Commentators and Research Ethics Commission 
have held the view that it is permissible to include incapable subjects in research that involves more than 
minimal risk as long as there is the potential for beneficial effects and if the research presents a balance of 
risks and expected direct benefits similar to that available in the clinical setting.lxiv  Several U.S. task forces 
have deemed it is permissible to include incapable subjects in research.  For example, the American College 
of Physicians’ document allows surrogates to consent to research involving incapable subjects only “if the 
net additional risks of participation are not substantially greater than the risks of standard treatment.”lxv   
Finally, the National Bioethics Advisory Committee (NBAC) stated “that an IRB may approve a protocol 
that presents greater than minimal risk but offers the prospect of direct medical benefits to the subject, 
provided that…the potential subject’s LAR gives permission…” 
 
Consistent with the above ethical sensibilities regarding the participation of decisionally incapable subjects in 
research and the previous assessment of risks and benefits in the previous section, the present trial presents a 
balance of risks and potential direct benefits that is similar to that available in the clinical setting, with the 
exception of the additional blood draws. 
 
10.5. Additional Safeguards for Vulnerable Subjects 
 
The present research will not involve subjects who might be vulnerable to coercion or undue influence.  
 
10.6. Confidentiality 
 
Federal regulations at 45 CFR 46 111 (a) (7) requires that when appropriate, there are adequate provisions 
to protect the privacy of subjects and to maintain the confidentiality of data. To maintain confidentiality, all 
laboratory specimens, evaluation forms, and reports will be identified only by a coded number.  The coded 
number will be generated at random by a computer, and only the study investigators will have access to the 
codes. All records will be kept in a locked, password protected computer. All computer entry and 
networking programs will be done with coded numbers only. All paper case report forms will be maintained 
in a locked cabinet inside a locked office. Clinical information will not be released without the written 
permission of the patient, except as necessary for monitoring by the National Heart, Lung, and Blood 
Institute, the Federal Drug Administration or other authorized Federal Agencies. 
 
11. Adverse Event Reporting/Safety Reporting 
Investigators will determine daily if any clinical adverse experiences occur during the period from informed 
consent through study hour 168 and will be followed up through resolution, resolved with sequelae, 
unresolvable or death.   
 
It is expected that Diseases/Illnesses/Symptoms associated with the SEPSIS ALI study population will occur 
in the study population, independent of investigation product exposure.  These associated 
diseases/illnesses/symptoms will be considered as part of the study inclusion processes and/or study 
assessments and as such will not be considered ‘reportable’ Adverse Events (AE)/Serious Adverse Events 
(SAE) unless the Investigator has a reasonable doubt regarding the relatedness of the event to the 
investigational product.  


 
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The investigator will evaluate any changes in laboratory values and physical signs and will determine if the 
change is clinically important and different from what is expected in the course of treatment of patients with 
ALI.  If clinically important and unexpected adverse experiences occur, they will be recorded on the adverse 
event case report form. 
 
 The following will be considered reportable adverse events: 
 
For this trial, a reportable adverse event is defined as: 
 
1. 
Any clinically important untoward medical occurrence in a patient receiving study drug or 
undergoing study procedures which is different from what is expected in the clinical course of a 
patient with severe sepsis associated ALI, or, 
2. 
Any clinically important, untoward medical occurrence that is thought to be associated with the 
study drug or procedures, regardless of the “expectedness” of the event for the course of a patient 
with severe sepsis associated ALI. 
    3. 
Investigators will report all serious, unexpected, AND study-related adverse events from the time 
of informed consent through study hour 168 that are considered to be harmful and unintended 
responses to the investigational product and/or study related procedures in the participants’ case 
report forms. ‘Responses to investigational product’ means that the causal relationship between an 
investigational product and an adverse event cannot be ruled out.   
 
Expected Events For ALI considered unreportable: refer to Appendix F Table 
1. Defined as: Untoward clinical occurrences perceived by the investigator to occur with 
reasonable frequency in the day to day care of patients with ALI treated in an intensive care 
unit with mechanical ventilation.                                   
2. Examples of untoward clinical occurrences that are expected in the course of ALI include: 1) 
transient hypoxemia, 2) agitation, 3) delirium, 4) nosocomial infections, 5) skin breakdown, 
and 6) gastrointestinal bleeding. Such events, which are often the focus of prevention efforts 
as part of usual ICU care, will not be considered reportable adverse events unless the event is 
considered by the investigator to be associated with the study drug or procedures, or 
unexpectedly severe or frequent for an individual patient with ALI. Examples of 
unexpectedly frequent untoward clinical occurrences would be repeated episodes of 
unexplained hypoxemia. This would be in contrast to an isolated episode of transient 
hypoxemia (e.g., Sp02  ~85%), related to positioning or suctioning.  This latter event would 
not be considered unexpected by nature, severity or frequency. 
3. Adverse events occurring from the time of informed consent through study hour 168 or until 
discharged from the hospital, withdrawal from the study or death, will not be considered ‘reportable’ 
Adverse Events (AE)/Serious Adverse Events (SAE) unless the Investigator has a reasonable doubt 
regarding the relatedness of the event to the investigational product. 
 
The following will be reported as adverse events:  
 
Investigators will report all unanticipated problems that involve risk or harm to a research participant 
AND was not anticipated or foreseen (e.g., not described in the consent form) AND is probably or 
definitely related to or caused by the research, as defined in Appendix F, to the Data Coordinating Center 
by phone and email within 24 hours of becoming aware of event. The Institutional Review Board for the lead 


 
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37
site will be notified within 5 business days of receiving notice of the unanticipated problem.  Participating 
sites shall report to their Institutional Review Board in accordance with their institution’s rules and 
regulations. 
 
The Data Coordinating Center (VCU) will report all, unanticipated problems, defined as problems that 
involve risk or harm to a research participant AND was not anticipated or foreseen (e.g., not described 
in the consent form) AND is probably or definitely related to or caused by the research, to the DSMB 
within 7 calendar days of the CCC being notified of the event. The Data Coordinating Center will distribute 
the written summaries of the DSMB’s periodic reviews to participating centers. 
 
The Data Coordinating Center will also determine if the serious adverse event is unexpected for Vitamin C.  
Unexpected for Vitamin C is defined as any event not listed in the Vitamin C package insert. If the  Data 
Coordinating Center determines that any serious and study-related adverse event is unexpected for Vitamin C, 
the FDA will be notified within 7 calendar days. Such events may also meet the definition of Unanticipated 
Problems as described below. 
 
Investigators must report Unanticipated Problems, regardless of severity, associated with the study drug or 
study procedures within 24 hours.  An unanticipated problem is defined as follows:  
 
Unanticipated Problem (UP): any incident, experience, or outcome that meets all of the following criteria 
will be reported from the time of consent through study hour 168 until resolved, withdrawn from the study, 
death occurs or lost to follow up: 
• Unexpected, in terms of nature, severity, or frequency, given the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document; and the characteristics of the subject population being studied; 
• Related or possibly related to participation in the research, in this guidance document, possibly 
related means there is a reasonable possibility that the incident, experience, or outcome may have 
been caused by the procedures involved in the research; 
• Suggests that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
1. Safety Reporting: 
Investigator safety reports are prepared for suspected unanticipated serious adverse reactions according to local 
regulatory requirements and are forwarded to investigators as necessary.  An investigator who receives an 
investigator safety report describing an SAE(s) or other specific safety information (e.g., summary or listing of 
SAEs) from the Clinical Coordinating Center will file it and will notify the IRB/IEC, if appropriate, according 
to local requirements. 
 
 


 
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12. APPENDICES 
  
APPENDIX A: Guidelines for evidence of infection 
 
1.  Infections of the thorax: 
a) Chest x-ray or CT scan showing a new or progressive infiltrate, consolidation, 
cavitation, collection, or pleural effusion, and a clinical presentation consistent with 
pneumonia or empyema 
b) Pneumonia can be defined as the presence of new infiltrate (s), absence of a noninfectious 
explanation and either signs of SIRS as per protocol or purulent sputum production with 
an identifiable pathogen. 
c) Aspiration Pneumonitis in the acute phase is not considered an infection. 
d) However, if SIRS persists > 24 hours after aspiration, then an infectious etiology can be 
presumed. 
2.  Abdominal infection: 
a) Perforated viscus or ischemic bowel with either localized peritonitis  
b) Peritoneal fluid with > 250 PMNs 
c) Clinical signs of cholangitis or appendicitis 
d) Clostridium difficile toxin positive with evidence of colon dilation 
e) Suspicion of peritonitis by clinical examination only 
3.  Skin or soft tissue infection: Acute onset infection of the skin, such as erysipelas, or 
infection involving deeper soft tissue 
4.  Bacterial meningitis: cerebrospinal fluid analyses if available and a clinical presentation 
consistent with bacterial meningitis 
5.  Urinary Tract: 
a) Positive test for granulocyte esterase or nitrate in urine, or a positive culture (defined as >105 
CFU/mL) 
b) Urinalysis with increased WBC count or positive Gram stain 
6.  Central Line infections: 
a) Catheter-related bloodstream infections (CR-BSIs) are defined as bacteremia/fungemia in a 
patient with an intravascular catheter with at least one positive blood culture obtained from a 
peripheral vein, clinical manifestations of infection (i.e., fever, chills, and/or hypotension), and 
no apparent source for the bloodstream infection except the catheter. The catheter must be in 
place for at least 48 hours prior to development of the bloodstream infection. 
 
7.  Sinusitis 
a) Air fluid levels in sinus seen on CT scan 
** Use of antibiotics at time of consent (provided the antibiotics are not for prophylaxis) is considered 
evidence of suspected infection. Examples of prophylactic antibiotics include: pre-surgical incision, 
antibiotic for the prevention of pneumocystis jiroveci (aka carinii), herpes simplex, cytomegaloviryus, 
and latent mycobacterial disease. 
 
The following are not considered evidence of infection: 
a) Fever of unknown origin 
b) Blood cultures that are considered positive only because of the isolation of a likely 
contaminant organism 


 
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c) Postoperative hypotension within 24 hours of incision and/or fever without a verified 
infectious focus. 
d) Leukocytosis alone in the presence of steroid usage is insufficient evidence of infection. 
e) Leukocytosis alone in the presence of connective tissue disorder is insufficient evidence of 
infection. 
 
 


 
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APPENDIX B:  Pleiotropic Effects of Vitamin C 
  
1.  Cell culture/in vitro studies 
• 
Reduced human neutrophil adhesion to endothelial cellslxvi 
• 
Protects neutrophils against intracellular effects of superoxide generation.lxvii 
• 
Protects monocytes against oxidative damagelxviii 
• 
Reduced PMA induction of NF-κB 
• 
Protects against oxidized-LDL-induced expression of MCP-1 in cultured human umbilical vein 
endothelial cellslxix 
• 
Modulates the inhibition of platelet aggregation by neutrophilslxx 
• 
Inhibits expression of platelet expression of CD40 ligand which promotes thrombosislxxi 
• 
Inhibits NADPH oxidase subunit p47phox expression in microvascular endotheliumlxxii 
• 
Influences dendritic cell function.lxxiii 
• 
Effects of vitamin C on intra cytoplasmic cytokine production in human whole blood monocytes and 
lymphocytes. exposed to LPS or immune complexeslxxiv 
• 
Inhibition of the induction of inducible nitric oxide sythetase (iNOS) and TNF-α, IL-1β and IL-6 in 
astrocytes, microglia and macrophages stimulated with LPS or cytokines  
• Vitamin C inhibits NO-induced stabilization of HIF-1alpha in HUVECslxxv 
• 
Cobalt-induced oxidant stress in cultured endothelial cells: prevention by ascorbate in relation to HIF-
1alpha.lxxvi 
 
2.  Intact animal studies  
• Attenuates LPS induced acute lung injury.lxxvii  
• Attenuates lung injury in a cecal ligation and puncture model of peritonitis.lxxviii 
• Corrects capillary blood flow in septic skeletal musculature.lxxix 
• Inhibits iNOS expression in septic vasculature.lxxx 
• Attenuates iNOS expression in IFN gamma-stimulated rat skeletal muscle endothelial cellslxxxi 
• Attenuates hepatic fibrosis by upregulating peroxisome proliferators-activated receptor-gammalxxxii 
• Inhibits both flow- and agonist-induced EDHF in the rat mesenterylxxxiii lxxxiv
,
 
• Multiple molecular transporters responsible for movement of ascorbate intracellularlxxxv 
• Attenuates peroxidative damage and tissue edema in ischemia/reperfused gut.lxxxvi 
• Ascorbate supplementation significantly decreases plasma IL-6 levels in an animal model of 
hemorrhagic shock.lxxxvii 
• 
Vitamin C deficiency causes the collagen-disassembly disease scurvy  
• 
Ascorbic acid prevents testosterone-induced hyperplasia of rat prostate by down-regulating HIF-
1alphalxxxviii 
• 
Plasma AA maintains the stability of “acellular Hb” susceptible to oxidationlxxxix 
• Endogenous ascorbate on oxidation, oxygenation, and toxicokinetics of cell-free modified 
hemoglobin after exchange transfusion in rat and guinea xc 
Human studies 
• Normalizes monocyte adhesion to endothelium in vitamin C deficient subjects. xci 
• Supplementation in chronic hemodialysis patients reduce lymphocyte 8-OHdG levels and 
intracellular ROS production xcii 
• Increase in muscle blood flow during dynamic exercise with acute AA administration in older 
adult humansxciii 
• Intravenous ascorbate improves outcomes during percutaneous myocardial interventionxciv 
• Plasma vitamin C level positively associated with serum pre-albumin levels and negatively 
associated with high sensitivity C-Reactive Protein levels in patients with chronic renal 


 
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41
failure.xcv,xcvi 
• Low plasma ascorbate levels are associated with enhanced proinflammatory responses and 
impaired vascular function in lean and obese men.xcvii 
• Reduced inflammatory tissue damage in patients subjected to cardiac surgery with 
extracorporeal circulationxcviii 
• Ascorbate promotes iron utilization for erythropoiesis in patients with chronic renal failurexcix 
• High dose vitamin C infusion into surgically critically ill daily for 28 days attenuated the 
incidence of acute lung injuy/ARDS.c 
 
 


 
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APPENDIX C: Ventilator Procedures 
 
C.1. Ventilator Management 
 
A modified, simplified version of the ARDS Network lung protective lower tidal volume 
strategy will be used in this trial.  This strategy, which was associated with low mortality rates in three 
previous ARDS Network trials (ARMA, ALVEOLI, and FACTT), will ensure that study subjects receive the 
beneficial effects of lung protection while participating in this trial.ci cii The PI/PDs (Drs. Fowler, Truwit, 
Hite, Martin) and professionals at the CITRIS-ALI consortium medical centers have significant experience 
with the application of ARDS Network ventilation protocols. 
 
1. Any mode of ventilation capable of delivering the prescribed tidal volume (VT, 6ml/kg predicted body 
weight, +/- 2ml/kg) may be used, provided the VT target is monitored and adjusted appropriately. If 
airway pressure release ventilation (APRV) is used, tidal volume is defined as the sum of the volume 
that results from the ventilator pressure- release and an estimation of the average spontaneous VT. 
2. VT Goal: 6 ml / kg predicted body weight. 
3. Predicted body weight (PBW) is calculated from age, gender, and height (heel to crown) 
a. according to the following equations: 
b. Males: PBW (kg) = 50 + 2.3 [height (inches) – 60] 
c. Females:  PBW (kg) = 45.5 + 2.3 [height (inches) – 60] 
4. Measure and record inspiratory plateau pressure (Pplat) according to ICU routine (at least every four 
hours and after changes in VT and PEEP recommended) 
5. If Pplat > 30 cm H2O, reduce VT to 5 ml/kg and then to 4 ml/kg PBW if necessary to decrease Pplat to 
≤ 30 cm H2O. 
6. If VT < 6 ml/kg PBW and Pplat < 25 cm H2O, raise VT by 1 ml/kg PBW to a maximum of 6 ml/kg. 
7. If “severe dyspnea" (more than 3 double breaths per minute or airway pressure remains at or below 
PEEP level during inspiration), then raise VT to 7 or 8 ml/kg PBW if Pplat remains below 30 cm H2O. 
If Pplat exceeds 30 cm H2O with VT of 7 or 8 ml/kg PBW, then revert to lower VT and consider more 
sedation. 
8. If pH < 7.15, VT may be raised and Pplat limit suspended (not required). 
9. Oxygenation target: [55 mm Hg <  PaO2  < 80 mm Hg] or [88% < SpO2  < 95%].  When both PaO2  
and SpO2 are available simultaneously, the PaO2 criterion will take precedence. 
10. Minimum PEEP = 5 cm H2O 
11. Adjust FiO2 or PEEP upward within 5 minutes if there are consistent measurements below the 
oxygenation target range 
12. Adjust FiO2 or PEEP downward within 30 minutes if there are consistent measurements above the 
oxygenation target range. 
13. There are no requirements for maintaining a specific PEEP to FiO2 ratio. The lower PEEP/higher FiO2 
table represents a consensus approach developed by ARDS Network investigators in 1995.  The higher 
PEEP/lower FiO2 table (ALVEOLI) yielded equivalent results in a randomized trial and would be 
acceptable and perhaps preferable in patients who appear to respond with a substantial increase in 
arterial oxygenation in the transition from lower to higher PEEP. 
 
 
 
 
 
 


 
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Lower PEEP/Higher FiO2 Treatment Group 
  
FiO2 
.30 .40 .40 
.50 
.50 
.60 
.70 
.70 
.70 
.80 
.90 
.90 
.90 
1.0 
PEEP 
5 
5 
8 
8 
10 
10 
10 
12 
14 
14 
14 
16 
18 
18-24 
 
Higher PEEP/Lower FiO2 Study Group 
 
FiO2 
.30 .30 .30 .30 .30 .40 .40 .50 
.50 
.50 – .80 .80 .90 
1.0 
1.0 
PEEP 
5 
8 
10 
12 
14 
14 
16 
16 
18 
20 
22 
22 
22 
24 
 
Note: Levels of PEEP in these FiO2/ PEEP tables represent levels set on the ventilator, not levels of 
total-PEEP, auto-PEEP, or intrinsic-PEEP. 
 
14. No specific rules for respiratory rate. It is recommended that the respiratory rate be increased 
in increments to a maximum set rate of 35 if pH < 7.30. 
15. No specific rules about I:E ratio.  It is recommended that duration of Inspiration be ≤ duration of 
Expiration. 
16. Bicarbonate is allowed (neither encouraged nor discouraged) if pH < 7.30. 
17. Changes in more than one ventilator setting driven by measurements of PaO2, pH, and Pplat may 
be performed simultaneously, if necessary. 
 
C.2. Weaning 
Commencement of Weaning (applicable to patients ventilated invasively or non-invasively) Patients 
will be assessed for the following weaning readiness criteria each day between 0600 and 1000. If a patient 
procedure, test, or other extenuating circumstance prevents assessment for these criteria between 06:00 and 
10:00, then the assessment and initiation of subsequent weaning procedures may be delayed for up to six 
hours. 
 
1. At least 12 hours since enrollment in the trial 
2. FiO2 ≤ 0.40 and PEEP ≤ 8 cm H2O or FiO2 ≤ 0.50 and PEEP = 5 cm H2O 
3. Values of both PEEP and FiO2 ≤ values from previous day 
4. Not receiving neuromuscular blocking agents and without neuromuscular blockade 
5. Patient exhibiting inspiratory efforts. If no efforts are evident at baseline, ventilator set rate will 
be decreased to 50% of baseline level for up to 5 minutes to detect inspiratory efforts. 
6. Systolic arterial pressure ≥ 90 mm Hg without vasopressor support (≤ 5 mcg/kg/min dopamine 
will not be considered a vasopressor) 
 
Spontaneous Breathing Trial Procedure and Assessment for Unassisted Breathing 
 
If criteria 1-6 above are met, then initiate a trial of up to 120 minutes of spontaneous breathing 
with FIO2 < 0.5 using any of the following approaches: 
1. Pressure support (PS) < 5 cm H2O, PEEP < 5 cm H2O 
2. CPAP < 5 cm H2O 
3. T-piece 
4. Tracheostomy collar (mask) 
 
The clinical team may decide to change mode during spontaneous breathing (PS = 5, CPAP, 


 
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tracheostomy mask, or T-piece) at any time during the spontaneous breathing trial. 
 
Monitor for tolerance using the following: 
1. SpO2  ≥ 90% and / or PaO2  ≥ 60 mm Hg 
2. Mean spontaneous tidal volume ≥ 4 ml/kg PBW (if measured) 
3. Respiratory Rate ≤ 35 / min 
4. pH ≥ 7.30 (if measured) 
5. No respiratory distress (defined as 2 or more of the following): 
a. Heart rate ≥ 120% of the 0600 rate ( ≤ 5 min at > 120% may be tolerated) 
b. Marked use of accessory muscles  
c. Abdominal paradox 
d. Diaphoresis 
e. Marked subjective dyspnea 
 
If any of the goals a-e are not met, revert to previous ventilator settings or to PS greater than or equal to 
10 cm H2O with Positive End-expiratory Pressure and FiO2 = previous settings and reassess for weaning 
the next morning.  The patient will be reassessed for weaning (Section C2) the following day. 
 
Decision to remove ventilatory support: 
If tolerance criteria for spontaneous breathing trial (a-e above) are met for at least 30 minutes, the 
clinical team may decide to discontinue mechanical ventilation. However, the spontaneous breathing 
trial can continue for up to120 minutes if tolerance remains in question. 
 
C.3. Definition of Unassisted Breathing 
1.  Spontaneously breathing with face mask, nasal prong oxygen, or room air, OR 
2.  T-tube breathing, OR 
3.  Tracheostomy collar (mask) breathing, OR 
4.  CPAP ≤ 5 without PS or IMV assistance 
5.  Use of CPAP or BIPAP solely for sleep apnea management 
 
C.4. Definition of Extubation 
1.  Removal of an oral or nasotracheal tube 
2.  If a patient receives a tracheostomy, the time of extubation is defined as the time when the patient 
achieves unassisted breathing as defined in section C.3 
 
C.5. Completion of Ventilator Procedures 
 
Patients will be considered to have completed the study ventilator procedures if any of the following 
conditions occur: 
 
1.  Death 
2.  Hospital discharge 
3.  Alive 28 days after enrollment 
 
If a patient requires positive pressure ventilation after a period of unassisted breathing, the study 
ventilator procedures will resume unless the patient was discharged from the hospital or > 28 days 


 
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elapsed since enrollment. 
 
C.6. Removal from the Ventilator Management Protocol 
 
Patients may be removed from the 6 ml/kg PBW tidal volume ventilation requirement if they develop 
neurologic conditions where hypercapnia would be contraindicated (e.g., intracranial bleeding, GCS < 8, 
cerebral edema, mass effect [midline shift on CT scan], papilledema, intracranial pressure monitoring, 
fixed pupils). 
 
 


 
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46
APPENDIX D: FACTT LITE Conservative Fluid Management Approach 
If patient does not have a MAP > 60mmHg and has not been off vasopressors for > 12 hours, then patient does 
not meet criteria for any actions prescribed in the Fluid Management Approach.  Document as “Not Clinically 
Indicated”.   
 
This fluid protocol captures the primary positive outcome of the FACTT trial on increasing ventilator free 
days.  If clinically possible, for patients with a CVC, this protocol should be initiated within four hours of 
randomization in enrolled patients, and continued until UAB or study day 7, whichever occurs first. 
 
o 
Discontinue maintenance fluids. 
o 
Continue medications and nutrition. 
o 
Manage electrolytes and blood products per usual practice. 
o 
For shock, use any combination of fluid boluses# and vasopressor(s) to achieve MAP ≥ 60 mmHg as fast as possible.  Wean vasopressors as quickly as 
tolerated beginning four hours after blood pressure has stabilized. 
o 
Withhold diuretic therapy in renal failure § and until 12 hours after last fluid bolus or vasopressor given. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
§ Renal failure is defined as dialysis dependence, oliguria with serum creatinine > 3mg/dl, or oliguria with serum creatinine 0-3 with  
   urinary indices indicative of acute renal failure. 
# Recommended fluid bolus= 15 mL / kg crystalloid (round to nearest 250 mL) or 1 Unit packed red cells or 25 grams albumin 
*Recommended Furosemide dosing = begin with 20 mg bolus or 3 mg / hr infusion or last known effective dose.  Double each subsequent dose until goal achieved 
(oliguria reversal or intravascular pressure target) or maximum infusion rate of 24 mg / hr or 160 mg bolus reached.  Do not exceed 620 mg / day.  Also, if patient has 
heart failure, consider treatment with dobutamine. 
 
 
 
CVP 
(recommend) 
 
 
 
PAOP 
(optional) 
 
MAP > 60 mm Hg AND off vasopressors for > 12 hours 
Average urine output < 0.5 
ml/kg/hr 
Average urine output > 0.5 
ml/kg/hr 
>8 
> 12 
 
Furosemide* 
Reassess in 1 hour 
Furosemide* 
Reassess in 4 hours 
4-8 
8-12 
Give fluid bolus as fast as possible# 
Reassess in 1 hour 
 
< 4 
< 8 
No intervention 
Reassess in 4 hours 


 
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47
APPENDIX E: Time Events Schedule 
 
Assessments 
Hour 
0 
Q 24 hrs 1st 
7 days or in 
ICU 
Hour 
48 
Hour 
96 
Hour 
168 
Day 
28 
Day 
60 
VS: BP, HR, MAP, RR, Temp., O2 sats, 
CVP, Glasgow Coma Scale 
X 
 
X 
X 
X 
Body Weight 
A 
 
A 
A 
Suspected or known site of sepsis 
A 
 
A 
A 
A 
A 
A 
I/Os Total and Urine only 
 
X 
 
 
 
 
 
Assessment of Acute/Chronic Renal 
Failure and Use of Dialysis 
X 
 
X 
X 
X 
X 
Calculate SOFA Score (post study by 
biostatistician) 
X 
 
X 
X 
X 
Calculate VE40 Score (post study by 
biostatistician) 
A 
 
A 
A 
A 
Calculate Oxygenation Index (post study 
by biostatistician) 
A 
 
A 
A 
A 
 
Ventilator Data: 
Tv, FiO2, PEEP, Plateau Pressure, Peak 
Inspiratory Pressure, Mean Airway 
Pressure, Minute Ventilation 
A 
 
A 
A 
A 
Labs: 
Arterial Blood Gasses, Na+, K+, BUN, 
Cr, WBC, Hgb, Hct, Platelets, PT/INR 
X 
 
X 
X 
X 
Bilirubin Total 
X 
 
X 
X 
X 
Vasopressors or Inotropes: 
Epi, Nor-epi, Phenylephrine, 
Vasopressin, Dopamine 
A 
 
A 
A 
A 
Concomitant Medications: 
Methylprednisone, Hydrocortisone 
X 
 
X 
X 
X 
AE/SAE Assessments 
X 
 
X 
X 
X 
Blood for Biomarkers 
X 
 
X 
X 
X 
Ventilator Free Days  
 
X 
All Cause Mortality  
 
X 
ICU Free Days 
 
X 
Hospital Free Days 
 
X 
Glucose Monitoring 
I 
I 
I 
I 
I 
 
 
 
 
 
 
 
 
 
 
 
 
X = Required 
I= As Needed (See Section 3.3.2) 
A=When available 
 
 


 
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APPENDIX F:  Adverse Events 
  
Procedures for Reporting Adverse Events 
 
Assuring patient safety is an essential component of this protocol.  Each participating investigator has 
primary responsibility for the safety of the individual participants under his or her care.  The Principal 
Investigator will evaluate all adverse events.  The Study Coordinator must view patient records for possible 
adverse events throughout the study period.  
 
AE/SAEs that meet the definition of reportable events (refer to Appendix F Table 1: Anticipated AEs) or as 
determined by the investigator, will be followed from the time of consent through study hour 168 until resolved, 
resolved with sequelae, unresolvable, or death. 
 SAEs will be collected over the same time period as stated above for AEs.  However, any AEs/SAEs assessed 
as related to study participation (e.g. Disease under study) will be recorded from the time of consenting up to 
and including study hour 168, discharge from the hospital, withdrawal, lost to follow up, or death.   
 Events that do not meet the definition of an AE include: 
• Any clinically significant abnormal laboratory findings or other abnormal safety assessments that are 
associated with the underlying disease, unless judged by the investigator to be more severe than 
expected for the subject’s condition. 
• The disease/disorder being studied, or expected progression, signs, or symptoms of the disease/disorder 
being studied, unless more severe than expected for the subject’s condition. 
• Medical or surgical procedure (e.g., endoscopy, appendectomy); the condition that leads to the 
procedure is an AE. 
• Situations where an untoward medical occurrence did not occur (social and/or convenience admission to 
a hospital). 
• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the 
start of the study that do not worsen; 
• "Lack of efficacy" or "failure of expected pharmacological action" would not be reported as an AE or 
SAE.  However, the signs and symptoms and/or clinical sequelae resulting from lack of efficacy will be 
reported if they meet the definition of an AE or SAE. 
 If an event does not meet the definition of an AE per Section 11, then it cannot be an SAE even if serious 
conditions are met (e.g., hospitalization for signs/symptoms of the disease under study, death due to progression 
of disease, etc). 
  
SAE Follow-up language: 
All AEs and SAEs will be followed until resolution, until the condition stabilizes, until the event is otherwise 
explained, or until the subject is lost to follow-up. 


 
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The investigator is obligated to perform or arrange for the conduct of supplemental measurements and/or 
evaluations as may be indicated or as requested by the Lead Site to explain as fully as possible the nature and/or 
causality of the AE or SAE.   The investigator is obligated to assist.  This may include additional laboratory 
tests or procedures, or consultation with other health care professionals.  If a subject dies during participation in 
the study or during a recognized follow-up period, the investigator will provide the Lead Site with a copy of any 
autopsy reports. 
 New or updated information will be recorded on the SAE report form.  The investigator will submit any 
updated SAE form to the Lead Site within the designated reporting time frames. 
 Once the investigator determines that an event meets the protocol definition of an SAE, the SAE will be 
reported to the Lead Site within 24 hours.  Any follow-up information on a previously reported SAE will also 
be reported to the Lead Site within 24 hours. 
 The investigator will always provide an assessment of causality at the time of the initial report as described in 
Section 11. 
 The primary mechanism for reporting SAEs to the Lead Site will be through REDCap and facsimile with an 
accompanying email.  If the electronic system is unavailable for greater than 24 hours, the site will fax and 
email.  Then the site will enter the serious adverse event data into REDCap as soon as it becomes available. 
 After the study is completed at a given site, REDCap will be taken off-line to prevent the entry of new data or 
changes to existing data.  If a site receives a report of a new SAE from a study participant or receives updated 
data on a previously reported SAE after REDCap has been taken off-line, the site can report this information on 
a paper SAE form via email. 
 Facsimile transmission of the SAE form is the preferred method to transmit this information to the Lead Site 
for SAE receipt.  In rare circumstances and in the absence of facsimile equipment, notification by telephone is 
acceptable, with a copy of the SAE data collection tool sent by overnight mail.  Initial notification via the 
telephone does not replace the need for the investigator to complete and sign the SAE data collection tool within 
the designated reporting time frames. 
 
1. Serious, Expected, AND Study-Related Adverse Events: adverse events occurring from the time of 
informed consent through study hour 168 or until discharged from the hospital, withdrawal from the 
study, or death will not be considered ‘reportable’ Adverse Events (AE)/Serious Adverse Events 
(SAE) unless the Investigator has a reasonable doubt regarding the relatedness of the event to the 
investigational product (refer to Table: Anticipated AE/SAEs for the SEPSIS ALI Study Population). 
 
2.  Serious, Unexpected, AND Study-Related  Adverse Events: Investigators will report all serious, 
unexpected, AND study-related adverse events from the time of informed consent through study hour 
168, to the Clinical Coordinating Center within 24 hours by email.  The local Institutional Review 
Board must also be notified in a timely manner.  The investigator will then submit a detailed written 
report to the Clinical Coordinating Center and the local Institutional Review Board no later than 5 
calendar days after the investigator discovers the event. 
 
3.  Definitions of Adverse Events 


 
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a.  A serious adverse event is any event that is fatal or immediately life threatening, is permanently 
disabling, or severely incapacitating, or requires or prolongs inpatient hospitalization.  
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious adverse events when, based upon appropriate 
medical judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above. Life-threatening means that 
the patient was, in the view of the investigator, at immediate risk of death from the reaction as 
it occurred. This definition does not include a reaction that, had it occurred in a more serious 
form, might have caused death.  Assessment of the cause of the event has no bearing on the 
assessment of the event’s severity. 
b.  An unexpected event is any experience not identified by the type, severity, or frequency in 
the current study protocol or an event that is unexpected in the course of treatment for ALI 
or ARDS. 
c.  Adverse events will be considered to be study-related if the event follows a reasonable 
temporal sequence from a study procedure and could readily have been produced by the 
study procedure. 
d.  Organ failures or death related to ALI or ARDS or the patient’s underlying condition that are 
systematically captured by the protocol should not be reported as adverse events unless they 
are considered to be study related. 
 
4.   Assigning Causality 
a.  The investigator is obligated to assess the relationship between investigational product and 
the occurrence of each AE/SAE.  A "reasonable possibility" is meant to convey that there are 
facts/evidence or arguments to suggest a causal relationship, rather than a relationship cannot 
be ruled out.  The investigator will use clinical judgment to determine the relationship.  
Alternative causes, such as natural history of the underlying diseases, concomitant therapy, 
other risk factors, and the sequential relationship of the event to the investigational product 
will be considered and investigated.  The investigator will also consult the Investigator 
Brochure (IB) and/or Product Information, for marketed products, in the determination of 
his/her assessment. 
b.  There may be situations when an SAE has occurred and the investigator has minimal 
information to include in the initial report to VCU.  However, it is very important that the 
investigator always make an assessment of causality for every event prior to the initial 
transmission of the SAE data to VCU.  The investigator may change his/her opinion of 
causality in light of follow-up information, amending the SAE report form.   
• Unrelated:  
o Event occurred before dosing;  
o Event or concomitant illness due to factors other than drug or study procedure; 
• Possibly:  
o Reasonable sequential relationship with study procedure or drug treatment;  
o Event could be explained by patient’s clinical state or other factors 
• Probably:  
o Reasonable sequential relationship with study procedure or drug treatment;  
o Likely to be a known reaction to study agent or chemical group, or predicted by 
known pharmacology;  
o Event cannot easily be explained by patient’s clinical state or other factors. 
• Definitely:  
o Distinct sequential relationship with study procedure or drug treatment;  


 
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51
o Known reaction to study agent or chemical group, or predicted by known 
pharmacology;  
o Event cannot be explained by patient’s clinical state or other factors. 
 
 
ANTICIPATED AE/SAEs for the SEPSIS ALI Study Population 
The following is a list of anticipated Diseases/Illnesses associated with the SEPSIS ALI study population and are expected 
to occur in the study population, independent of investigation product exposure; will be captured as part of the study 
inclusion processes and/or study assessments and as such will not be considered ‘reportable’ Adverse Events (AE)/Serious 
Adverse Events (SAE) unless the Investigator has a reasonable doubt regarding the relatedness of the event to the 
investigational product, from the time of informed consent through Study hour 168 or until discharged from the hospital, 
withdrawn from the study or death.  
AE/SAEs that meet the definition of reportable events or as determined by the investigator, will be followed from the time 
of consent through study hour 168 until resolved, resolved with sequelae or considered unresolvable. 
NOTE: this list is meant to be as comprehensive as possible for expected events for the Disease under study and may not 
include all events.  The Investigator responsibilities for reporting of AE/SAEs still apply, regardless of the table below 
and should be reported if the Investigator has a reasonable doubt regarding the relatedness of the event to the 
investigational product. 
Table 1: 
Expected Disease(s) Under Study 
Associated Symptoms and Labs 
Infection  
 
(e.g., thorax, urinary tract, abdomen, skin, sinuses, bacterial meningitis, central 
venous catheters, and central nervous system, see Appendix A) 
 
** Use of antibiotics at time of consent (provided the antibiotics are not for 
prophylaxis) is considered evidence of suspected infection. Examples of 
prophylactic antibiotics include: pre-surgical incision, antibiotic for the 
prevention of pneumocystis jiroveci (aka carinii), herpes simplex, 
cytomegalovirus, and latent mycobacterial disease. 
Acute Lung Injury (ALI) 
Fever:  >38ºC (any route) or hypothermia: <36ºC (core temp only),  
Tachycardia: heart rate > 90 beats/min or receiving medications that slow heart 
rate or paced rhythm,  
Leukocytosis:  >12,000 WBC/µL or leukopenia:  <4,000 WBC/µL or >10% 
band forms.   
Respiratory rate > 20 breaths per minute or  
PaCO2 < 32 or invasive mechanical ventilation. 
Adult Respiratory Distress 
Syndrome (ARDS) 
Lung injury of acute onset, within 1 week of an apparent clinical insult and 
with progression of respiratory symptoms; 
Bilateral opacities on chest imaging not explained by other pulmonary 
pathology (e.g. pleural effusions, lung collapse, or nodules); 
Respiratory failure not explained by heart failure or volume overload; 
Decreased arterial PaO2/FiO2 ratio ≤ 300 mm Hg; 
Minimum PEEP of 5 cmH2O  
Systemic Inflammatory 
Responses (SIRS) 
Fever > 38°C (any route) or hypothermia: < 36°C (core temp. only) 


 
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52
Tachycardia: heart rate > 90 beats/min or receiving medications that slow heart 
rate or paced rhythm 
Respiratory Rate > 20 breaths per minute or PaC02 < 32 or invasive 
mechanical ventilation 
Leukocytosis: > 12,000 WBC /µL or leukopenia:  <4,000 WBC/µL or >10% 
band forms 
 
Sepsis associated System Organ 
Failure (SOF) 
The current trial will be enrolling 
patients with sepsis associated acute 
lung injury. We therefore expect 
that many of these patients will 
have some degree of organ 
dysfunction. 
 
 
Sepsis associated hypotension (systolic blood pressure (SBP) < 90 mm Hg or 
an SBP decrease > 40 mm Hg unexplained by other causes or use of 
vasopressors for blood pressure support (epinephrine, norepinephrine, dopamine 
=/> 5mcg, phenylephrine, vasopressin); 
Arterial hypoxemia (PaO2/FiO2 < 300) or supplemental O2 > 6LPM. 
Lactate > upper limits of normal laboratory results 
Urine output < 0.5 ml/kg/hour for > two hours despite adequate fluid 
resuscitation 
Platelet count < 100,000 per mcL 
Coagulopathy (INR > 1.5) 
Bilirubin > 2 mg/dL 
Glasgow Coma Scale < 11 or a positive CAM ICU score 
Biomarkers of Inflammation, 
Vascular Injury and Alveolar 
epithelial injury 
Increases in C-reactive protein   (CRP), procalcitonin (PCT), thrombomodulin 
(TM) alveolar epithelial injury (Receptor for Advanced Glycation Products) 
Lactic Acidosis 
Patients with sepsis are at high risk of metabolic acidosis (including lactic 
acidosis). To prevent the possibility of metabolic acidosis due to drug 
administration, the study drug is formulated to a neutral pH of 7.4. Therefore, we 
do not anticipate the need for additional monitoring of acid/base balance beyond 
standard-of-care provided at each institution. Any observed abnormalities will be 
evaluated according to standard-of-care practice and documented in the research 
record. 
Use of Dialysis 
In the presence of Acute or Chronic renal failure. 
Septic Shock 
Fluid management during shock will not be prescribed per study protocol. In 
subjects who are not in shock, a conservative fluid management approach will be 
administered, if possible.  (refer to Appendix D, Table 5). 
Use of Ventilator 
As per protocol (refer to Section 5.5 and Appendix C) 
Plasma Ascorbate levels 
Septic patients exhibit subnormal plasma ascorbate levels. Phase I studies 
performed at Virginia Commonwealth University (VCU) show mean ascorbate 
levels of 17.5 µM (normal human ascorbate levels 60 to 70 µM).  The day 2 – 7 
plasma ascorbate levels are expected to be between 500 to 1000 µM. 
Death 
Organ failures or death related to ALI or ARDS or the patient’s underlying 
condition that are systematically captured by the protocol should not be reported 
as adverse events unless they are considered to be study related. (refer to 
Appendix F). 
NOTE: All AE/SAEs considered “Reportable” will be captured on the adverse event log and reported to the 
sponsor. 
 
 
 
 


 
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53
APPENDIX G: Modified SOFA Score Calculator 
 
Value 
0 
1 
2 
3 
4 
Score 
Respiratory 
  
PaO2/FiO2 
>400 
≤400 
≤300 
≤200 
with 
respiratory 
support 
≤100 
with 
respiratory 
support 
  
Coagulation 
  
Platelets 
>150 
≤150 
≤100 
≤50 
≤20 
  
GI 
  
T Bilirubin 
<1.2 
1.2-1.9 2.0-5.9 
6.0-11.9 
≥12 
  
Cardio- 
Vascular 
  
No 
Hypo- 
tension 
MAP 
<70 
Dopa ≤5 
PE <100 
Dopa > 5 
Epi ≤ 0.1 
NE ≤ 0.1 
PE 100-
300 
  
Dopa>15 
Epi>0.1 
NE>0.1 
PE>300 
VP>0.01 
  
Neuro 
  
GCS 
15 
13-14 
10-12 
6-9 
<6 
  
Serum 
Creatinine 
  
-OR- 
  
Urine  
Output 
<1.2 
1.2-1.9 2.0-3.4 
3.5-4.9 
  
<500cc/ 
day 
≥5.0 
  
<200cc/ 
day 
  
  
Total 
 
 
  
1. For patients on supplemental oxygen, add 0.03 to the room air FiO2 (0.21) for each liter of nasal cannula 
oxygen (e.g. 2 liters = FiO2 of 0.27).  Face mask FiO2 is whatever amount is being delivered (e.g. 40% 
face mask = FiO2 of 0.40).  For non-rebreather face masks, use FiO2 of 0.99. 
2. Doses of dopamine (Dopa), epinephrine (Epi), norepinephrine (NE) are in micrograms/kg/min; phenylephrine 
(PE) is micrograms/min; vasopressin (VP) is U/min. Vasopressors must have been administered for at least one 
hour. 
 
 


 
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54
APPENDIX H: P/F from S/F  Calculator 
 
 
 
To calculate FiO2 for a non-intubated patient: 
 
.21+(3.5 x per liter O2) = FiO2 
 
Calculate PO2/FiO2 by finding SpO2 along the top row and calculated FiO2 along the left vertical axis and finding the 
intersection of the two. 
 
SpO2/FiO2 ratio should only be analyzed if the SpO2 is < 97%.  At values of 97% or greater there is no 
longer an interpretable relationship between SpO2/FiO2 and PaO2/FiO2 
 
 


 
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55
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