Prospective, Randomized, Double-Blind, Placebo-Controlled Phase II Trial of Intravenous L-Citrulline (Turnobi) to Delay and Potentially Prevent the Need for Invasive Mechanical Ventilation for Acute Hypoxemic Respiratory Failure in Patients With COVID-19 (SARS-CoV-2) Illness
Phase: PHASE2
Conditions: Acute Hypoxemic Respiratory Failure
Sponsor: Asklepion Pharmaceuticals, LLC

Confidential 
 
 
 
Prospective, Randomized, Double-Blind, Placebo-Controlled Phase II Trial of Intravenous 
L-Citrulline to Delay and Potentially Prevent the Need for Invasive Mechanical Ventilation for Acute 
Hypoxemic Respiratory Failure in Patients with COVID-19 (SARS-CoV-2) Illness 
 
Protocol Number: 
CIT-COVID19-002-01 
 
Protocol Version/Date: 
Version 3.0, 11 June 2021 
IND Number: 
150762 
 
Study Drug: 
L-Citrulline 
 
Principal Investigators: 
Nikhil K. Meena, MD 
Associate Professor of Medicine and Pediatrics 
Director, Medical Intensive Care Unit 
Division of Pulmonary and Critical care Medicine 
Department of Medicine 
University of Arkansas for Medical Sciences 
4301 West Markham St, Slot 555 
Little Rock, AR 72205 
(501) 686-5526 
 
Michael P. Wilson, MD PhD 
Assistant Professor 
Department of Emergency Medicine 
University of Arkansas for Medical Sciences 
4301 West Markham Street, Slot #584 
Little Rock, Arkansas 722105-7199 
Phone: (217) 621-0393 
 
Principal Consultant: 
Frederick (Rick) E. Barr, MD MBA 
Chair of Pediatrics & Associate Dean for Child Health 
Robert H. Fiser, Jr MD Endowed Professor 
Pediatrician-In-Chief, Arkansas Children’s Hospital 
President (interim), Arkansas Children’s Research Institute 
Arkansas Children’s and the University of Arkansas for Medical 
Sciences 
One Children’s Way 
Little Rock, AR 72202 
(501) 364-5262 
 
Program Director: 
Gurdyal Kalsi, MD 
Chief Medical Officer 
Asklepion Pharmaceutical, LLC 
729 E. Pratt Street, Suite 360 
Baltimore MD 21020 
(410) 545-0494 

1 
Confidential 
 
 
Protocol Number: 
Protocol CIT-COVID19-002-01: Version 3.0 
Protocol Date: 
11 June 2021 
Title: Prospective, Randomized, Double-Blind, Placebo-Controlled Phase II Trial of Intravenous L-
Citrulline to Delay and Potentially Prevent the Need for Invasive Mechanical Ventilation 
for Acute Hypoxemic Respiratory Failure in Patients with COVID-19 (SARS-CoV-2) Illness 
 
 
SIGNATURES: 
 
11-Jun-2021 
 
  
 
Vincent Elliott, BS, Associate Director of Clinical Operations 
Asklepion Pharmaceuticals, LLC 
 
 
 
Gurdyal Kalsi MD, Chief Medical Officer 
Asklepion Pharmaceuticals, LLC 
Date 
 
 
 
 
 
 
 
 
11-Jun-2021 
 
Date 
 
 
 
 
 
 
 
 
15-Jun-2021 
 
  
 
Nikhil K Meena, MD, Principal Investigator, Associate Professor of 
Medicine and Pediatrics, Director, Medical Intensive Care Unit 
University of Arkansas for Medical Sciences 
Date 
 
 
 
These signatures constitute approval of this protocol and an assurance that this study will be 
conducted according to all requirements of this protocol. 

2 
Confidential 
 
 
Table of Contents 
1.0 
ABBREVIATIONS...................................................................................................................................... 4 
2.0 
STUDY SUMMARY ................................................................................................................................... 5 
3.0 INTRODUCTION ............................................................................................................................................... 8 
3.1 
The Urea/NO Cycle ...................................................................................................................................... 8 
3.2 
The Urea/NO Cycle and Oxidative Stress in Acute Hypoxemic Respiratory Failure ................................ 10 
3.3 
Citrulline Replacement ............................................................................................................................... 11 
3.4 
COVID-19 (SARS-CoV2) and Acute Hypoxemic Respiratory Failure Requiring Invasive Mechanical 
Ventilation ............................................................................................................................................................... 12 
3.4 Summary ........................................................................................................................................................... 12 
4.0 
OBJECTIVES ............................................................................................................................................ 13 
4.1 Primary Endpoints (objectives) .......................................................................................................................... 13 
4.2 Secondary Endpoints (objectives) ...................................................................................................................... 13 
5.0 
STUDY DESIGN ....................................................................................................................................... 13 
5.1 
Study Description ....................................................................................................................................... 13 
5.1.1 Randomization, Stratification, and Blinding ................................................................................................... 14 
5.1.2 Study Assessments.......................................................................................................................................... 14 
5.2 Treatment Arms and Study Drug Administration .............................................................................................. 15 
5.2.1 Citrulline Arm ................................................................................................................................................. 15 
5.2.2 
Placebo Arm ............................................................................................................................................... 15 
5.2.3 
Drug Interruptions ...................................................................................................................................... 15 
6.0 STUDY POPULATION AND ENROLLMENT .............................................................................................. 15 
6.1 
Participant Enrollment................................................................................................................................ 15 
6.2 
Inclusion Criteria ........................................................................................................................................ 16 
6.3 
Exclusion Criteria ....................................................................................................................................... 16 
6.4 
Informed Consent ....................................................................................................................................... 16 
6.5 
Excluded Medications ................................................................................................................................ 17 
6.6 
Concomitant Medications .......................................................................................................................... 17 
6.7 
Criteria for Discontinuation or Withdrawal ................................................................................................ 17 
6.8 
Procedures for Discontinuation or Withdrawal of a Participant ................................................................. 17 
7.0 
STUDY PLAN ........................................................................................................................................... 18 
7.1 
Definition of Study Procedures (see Appendix A: Schedule of Events)..................................................... 18 
7.1.1 
Demographics, Medical History, and Medication History ......................................................................... 18 
7.1.2 
Vital Signs Excluding Hemodynamics ....................................................................................................... 18 
7.1.3 
Hemodynamic Assessments ....................................................................................................................... 18 
7.1.3.1   Blood Pressure Measurements ................................................................................................................... 18 
7.1.4 
Mortality, Hospital, and ICU Length of Stay Assessment .......................................................................... 18 
7.1.5 
Clinical Laboratory Tests ........................................................................................................................... 19 
7.1.6 
Pregnancy ................................................................................................................................................... 19 
7.2 
Schedule of Observations and Procedures .................................................................................................. 19 
7.2.1 
Baseline Assessments ................................................................................................................................. 19 
7.2.2 
On Study (days 0-DC) Assessments ........................................................................................................... 19 
7.2.3 
Clinical Outcome Assessments .................................................................................................................. 20 
8.0 
PROTOCOL DEVIATIONS ...................................................................................................................... 20 
9.0 
ADVERSE EVENTS ................................................................................................................................. 20 
9.1 Adverse Events .................................................................................................................................................. 21 
9.2 Clinically Significant Laboratory Values ........................................................................................................... 21 
9.3 Pre-Existing Conditions ..................................................................................................................................... 21 
9.4 Serious Adverse Events ..................................................................................................................................... 22 
9.5 Adverse Event Collection Period ....................................................................................................................... 22 
10.0 
STATISTICAL METHODS ...................................................................................................................... 22 
10.1 
Analysis Populations .................................................................................................................................. 22 
10.2 
General Statistical Approach ...................................................................................................................... 22 
10.3 
Demographics and Baseline Characteristics ............................................................................................... 23 

3 
Confidential 
 
 
10.4 
Sample Size ................................................................................................................................................ 23 
10.5 
Primary Endpoints ...................................................................................................................................... 23 
10.6 
Secondary Safety Endpoints ...................................................................................................................... 24 
10.6.1    Secondary Clinical Endpoints .................................................................................................................... 24 
10.7 
Interim Analyses ........................................................................................................................................ 24 
11.0 
ETHICS ...................................................................................................................................................... 24 
11.1 
Ethical Conduct of the Trial ....................................................................................................................... 24 
11.2 
Institutional Review Board......................................................................................................................... 25 
11.3 
Participant Information, Informed Consent, and Participant Authorization ............................................... 25 
11.4 
Data and Safety Monitoring Plan ............................................................................................................... 25 
11.5 
Potential Risks ............................................................................................................................................ 26 
11.6 
Alternative Treatments ............................................................................................................................... 27 
11.7 
Potential Benefits ....................................................................................................................................... 27 
12.0 
DATA HANDLING AND RECORDKEEPING ....................................................................................... 28 
12.1 
Sources of Research Material ..................................................................................................................... 28 
12.2 
Participant Confidentiality ......................................................................................................................... 28 
12.3 
Case Report Forms ..................................................................................................................................... 28 
12.4 
Medrio Computerized Database ................................................................................................................. 28 
12.5 
Record Retention ........................................................................................................................................ 29 
13.0 
REFERENCES ........................................................................................................................................... 30 
14. Investigator Signature Page ............................................................................................................................... 32 
APPENDIX A: Schedule of Events ......................................................................................................................... 33 
APPENDIX B- Protocol Clarification Memo .............................................................................................................. 35 
APPENDIX C: Protocol Clarification Memo- Procedure Window Allowance ........................................................... 36 
15. 
Summary of Changes...................................................................................................................................... 38 

4 
Confidential 
 
 
1.0 
ABBREVIATIONS 
ALI 
Acute Lung Injury 
ARDS 
Acute Respiratory Distress Syndrome 
CPS1 
Carbamoyl-phosphate synthetase 1 
AL 
Arginine-succinate Lyase 
AS 
Arginine-succinate Synthase 
BMT 
Bone Marrow Transplant 
CFR 
Code of Federal Regulations 
CNS 
Central Nervous System 
CRF 
Case Report Form 
DC 
Discharge 
DIC 
Disseminated Intravascular Coagulopathy 
EBC 
Exhaled Breath Condensate 
ELISA 
Enzyme-linked Immunosorbent Assay 
eNOS 
Endothelial Nitric Oxide Synthase 
FiO2 
Fraction of Inspired Oxygen 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICF 
Informed Consent Form 
ICU 
Intensive Care Unit 
IL-1 
Interleukin-1 
IL-6 
Interleukin-6 
IL-8 
Interleukin-8 
iNOS 
Inducible Nitric Oxide Synthase 
IRB 
Institutional Review Board 
IV 
Intravenous 
L-NMMA 
NG-Monomethyl-L-Arginine 
MAP 
Mean arterial blood pressure 
mL 
Milliliter 
mm Hg 
Millimeter of Mercury 
NAC 
N-Acetyl Cysteine 
nNOS 
Neuronal Nitric Oxide Synthase 
NO 
Nitric Oxide 
NOS 
Nitric Oxide Synthase 
NOx 
Nitric Oxide Metabolites 
O2- 
Superoxide 
OTC 
Ornithine Transcarbamylase 
ROS 
Reactive Oxygen Species 
SAE 
Serious Adverse Event 
S/F 
SaO2 / FiO2 ratio 
SOFA 
Sepsis-related Organ Failure Assessment 
TNF 
Tumor Necrosis Factor 
TNF-α 
Tumor Necrosis Factor-alpha 
VDI 
Vasopressor Dependency Index 

5 
Confidential 
 
 
2.0 
STUDY SUMMARY 
Title: Prospective, Randomized, Double-Blind, Placebo-Controlled Phase II Trial of Intravenous L- 
Citrulline to Delay and Potentially Prevent the Need for Invasive Mechanical Ventilation for Acute 
Hypoxemic Respiratory Failure in Patients with COVID-19 (SARS-CoV2) Illness. 
 
 
Objective: To evaluate safety and efficacy of a bolus loading dose and continuous intravenous infusion 
of L-Citrulline compared to placebo in patients hospitalized with COVID-19 infection (SARS-CoV-2). 
 
Hypothesis: Intravenous L-citrulline administration will safely restore the homeostasis of nitric oxide 
synthase by increasing both plasma citrulline and arginine levels. We also reason that restoration of 
citrulline/arginine balance through citrulline administration will safely re-establish homeostasis of NOS, 
lower oxidative stress, and reduce inflammation, thereby delaying and potentially preventing the need for 
invasive mechanical ventilation in patients hospitalized with COVID-19 infection (SARS-CoV-2). 
 
Study Design: 
Prospective, randomized, placebo-controlled, double-blind design. 
1. Enrollment: approximately 6-9 months 
2. Plasma citrulline and arginine levels at day 4 will represent the 
primary biochemical variable. 
3. Vasomotor stability, as measured using a vasopressor dependency 
index (VDI), through study day 10, will represent the primary safety 
variable. 
4. The length of time from the initiation of the treatment to an 
intubation event for invasive mechanical ventilation measured in 
hours will represent the primary clinical outcome. 
5. Patients will be followed until hospital discharge for mortality, ICU 
and hospital lengths of stay, and a requirement for invasive 
mechanical ventilation and non-invasive mechanical ventilation with 
high flow nasal cannula or BiPAP as additional clinical endpoints. A 
further comparison will be performed at D28 and D60 based on 
telephone follow up for those discharged earlier. 
6. Patients will exit the study early (prior to 10 days) if they recover 
and no longer require oxygen therapy. 
Treatment Arms: 
1. Patients will be randomized to IV Citrulline or placebo 
2. Patients will receive an initial bolus of 20 mg/kg (maximum 1500 
mg), followed by study infusion of 9 mg/kg per hour (maximum 
700mg) for up to 10 days. 
3. Patients, healthcare providers, and the sponsor will be blinded to 
which treatment the patient is receiving. The investigational 
pharmacy independent pharmacist will be unblinded to the treatment 
assignment and will deliver the correct infusion to the patient’s 
bedside. 
4. The time of initiation of the study drug infusion (either L-Citrulline 
or placebo will represent time zero. 
5. Intravenous Citrulline: Patients randomized the citrulline arm will 
receive an initial loading bolus of 20 mg/kg (maximum 1500 mg) 
followed by an infusion of 9 mg/kg per hour (maximum 700 mg) for 

6 
Confidential 
 
 
a maximum of 10 days. The citrulline solution will be prepared as a 
5% (50 mg/mL) isotonic solution in 5% dextrose water. 
6. Placebo: Patients randomized to placebo will receive equal volume 
bolus and study infusion of 5% dextrose water for a maximum of 10 
days. 
Sample Size/Statistical 
Considerations: 
1. The study will enroll up to 66 patients in a 1:1 ratio. 
2. Patients will be analyzed on an intention to treat basis. 
3. The primary outcome will be time to intubation event—from the 
initiation of the treatment until intubation for invasive mechanical 
ventilation—and will be compared using a log-rank test. 
4. Plasma citrulline, arginine, and nitric oxide levels will be measured 
at 2 and12 hours post-bolus and on days 2, 4, 6, 8, and 10 and 
compared using Linear mixed model or repeated measures ANOVA 
analysis. 
5. The vasopressor dependency index (VDI) will be measured for 
hemodynamics. Worst vasopressor dependency index score over the 
first 6 days will be compared using ANCOVA analysis with the 
baseline index as the covariate. 
6. SaO2/FiO2 or PaO2/FiO2 ratio will be utilized to measure the 
progression of ALI. Worst values from days 0, 2, 4, or 6, 8, or 10 
will be compared between groups using repeated measures 
ANCOVA with the baseline value as the covariate. 
Inclusion Criteria: 
1. Age 18-65 years inclusive. 
2. Clinical and laboratory evidence of COVID-19 (SARS-CoV2) 
infection with an acute hypoxemic respiratory illness requiring 
hospitalization and oxygen therapy. 
3. Admitted and transferred to floor without intubation. 
Exclusion Criteria: 
1. Subject unable to provide consent. 
2. Patient, surrogate, or physician not committed to full support. 
3. Malignant or other irreversible condition and estimated 28-day 
mortality ≥ 50% 
4. Moribund patient not expected to survive 48 hours (as defined by 
the primary medical team) from the start of study infusion 
5. End-stage Liver Disease as defined by Child-Pugh Score > 9 
6. Currently enrolled in, or having participated, in another study of an 
investigational compound within the last 30 days 
7. Pregnant female or female who is breastfeeding 
8. Allergy to L-citrulline or arginine or any citrulline- or arginine- 
containing product 
9. Patient not otherwise suitable for the study in the opinion of any of 
the investigators 
10. Requirement for intubation and invasive mechanical ventilation 
prior to study enrollment 

7 
Confidential 
 
 
Primary Clinical Efficacy 
Variables: 
The primary clinical efficacy variable is time to intubation event in 
hours from the start of study infusion. 
Primary efficacy variables will be evaluated comparing intravenous L- 
citrulline vs. placebo in separate analyses. 
Primary Biochemical 
Efficacy Variables: 
The primary biochemical efficacy variable is plasma levels of citrulline 
that are associated with primary and secondary clinical efficacy 
outcomes. It may serve as a surrogate marker for response. 
Primary Safety Variable: 
Primary safety variable is hemodynamic status, specifically worst 
vasopressor dependency index through day 10. 
Secondary Clinical 
Efficacy Variables: 
1. All-cause hospital mortality 
2. The requirement for intubation and invasive mechanical 
ventilation 
3. Length of non-invasive mechanical ventilation in hours 
4. Length of oxygen therapy in hours 
5. Length of ICU stay in hours 
6. Length of hospital stay in hours 
7. Difference in proportion of patients intubated 
8. Difference in proportion of patients intubated and on MV 
9. Duration of mechanical ventilation from consent & post- 
infusion 
Secondary Biochemical 
Efficacy Variables: 
1. Plasma levels of citrulline and arginine at 2 hours, 12 hours, and 
study days 2, 4, 6, 8, 10. 
2. Plasma NOx levels at 2 hours, 12 hours, and study days 2, 4, 6, 8, 
10. 
3. PK-PD analysis/modeling will enable dose proportionality (dose- 
response effect) determinations for a follow on confirmatory trial. 
Secondary Safety 
Variables: 
Incidence of reported adverse events to include D28 and D60 in-hospital 
or telephone follow up 
Safety Evaluations: 
In addition to vasopressor dependency index, the following laboratory 
values will be used for safety assessment at baseline and daily through 
study day 4 when available. 
1. Complete blood count with platelets 
2. Basic Metabolic profile, including renal function 
3. Liver function tests, including SGOT, SGPT, total bilirubin, 
and alkaline phosphatase 
Adverse events will be recorded prospectively, including incidences of 
hypotension, hepatitis (i.e., the elevation of liver enzymes), and injection 
site reactions. 

8 
Confidential 
 
 
 
 
 
 
 
3.0 INTRODUCTION 
 
3.1 
The Urea/NO Cycle 
 
A critical biochemical pathway to maintain metabolic homeostasis is the urea/nitric oxide (NO) cycle [1]. 
The urea/NO cycle is involved in the intermediary metabolism of the key amino acids citrulline and 
arginine, forming a critical bridge between urea/NO cycle activities in the liver and the nitric oxide 
synthase (NOS) system present in other tissues for cell signaling and immune functioning. Alterations in 
the urea cycle in the liver can lead to systemic effects, including oxidant stress and inflammation (Figure 
1). Kupffer cells, the largest fixed macrophage population in the body, have the capacity to react to 
various inflammatory insults, including infectious agents, and release inflammatory cytokines like TNFα, 
IL-1, and IL-8, into the circulation [2,3]. In addition, although the full urea cycle is limited to the liver, 
other organs, including the lung, contain many of the cytosolic enzymes, including nitric oxide synthase 
(NOS). The degree to which Kupffer cells and other macrophages, including pulmonary macrophages, 
deal with oxidant stress, and maintain metabolic homeostasis is closely related to NOS function and 
availability of arginine. NOS, in its natural coupled form, utilizes arginine as a substrate for producing 
nitric oxide (NO) and citrulline. However, when uncoupled, NOS preferentially produces the superoxide 
radical (O2-). This free radical reacts with NO to form peroxynitrite and other ROS, which stimulate 
monocytes to produce and release additional proinflammatory cytokines [4,5], further propagating and 
prolonging the inflammatory cascade (Figure 1). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The rate-limiting enzyme catalyzing the first committed step of ureagenesis in the urea/NO cycle, 
carbamoyl-phosphate synthetase 1 (CPS1) (Figure 2), is highly tissue specific, with function and 
production limited to the liver and small intestine. CPS 1 forms carbamoyl phosphate from ammonia, 
LIVER 
Urea/NO cycle 
Hepatic/Kupffer cells: 
Arginine inadequate 
Citrulline/Arginine 
homeostasis 
Uncoupled NOS 
Superoxide 
Oxidant stress 
Direct injury 
LUNG 
Partial Urea/NO Cycle 
TNFα, IL-1, etc (early) 
Amino acids 
Ammonia 
Proinflammatory cytokines Cytokine 
Arginine inadequate 
Oxidant 
Damage 
Uncoupled NOS 
Superoxide 
GI tract 
oxidant stress 
direct injury 
Urea 
Proinflammatory cytokines 
TNFa, IL-1, IL-8 (late) 
Other 
Tissues 
Kidney 
Figure 1 

9 
Confidential 
 
 
carbon dioxide, and ATP-derived phosphate, which is then joined to ornithine by ornithine 
transcarbamylase (OTC) to form citrulline. Citrulline is not found in any enzyme or structural protein, 
thus its principal function is its role in the urea cycle. The vast majority of both CPS1 and OTC are found 
in hepatic mitochondria. The remaining enzymes, arginine-succinate synthase (AS), arginine-succinate 
lyase (AL), and arginase are cytoplasmic enzymes with wide tissue distribution, including the lung [6]. 
Sequential actions of AS and AL convert citrulline to arginine, the only non-dietary source of arginine. 
The liver either takes up citrulline from GI production/absorption or manufactures it from ornithine and 
converts it to arginine, releasing the latter into the bloodstream. Ordinarily in the liver, urea is cleaved 
from arginine to regenerate ornithine. However, arginine exported from the liver also serves as a 
substrate for systemic NOS, heme proteins that catalyze oxidation of L-arginine to NO and L-citrulline. 
NOS is found in all cells in at least one of three isoforms: nNOS (neuronal, constitutive), eNOS 
(endothelial, constitutive), and iNOS (inducible). NOS substrate, arginine, is manufactured in the cell 
from citrulline or is supplied to the body by the liver or the kidney. Under normal physiological 
conditions, homeostasis of NOS activity is maintained by levels of both citrulline and arginine. 
 
Within the endothelial cell, the chemically bound caveolar HSP-90 pathway restricts NO production to 
arginine derived from HSP-90-bound citrulline. In addition, we have recently observed that in human 
vascular endothelial cells, extracellular citrulline and not arginine, is the effective precursor of NO 
production. These findings suggest that circulating levels of citrulline may be more predictive of NOS 
function than arginine levels. Citrulline serves as a negative feedback inhibitor on NOS and arginine, 
couples NOS to produce NO preferentially over O2-. 
 
There are clear links between a poorly functioning urea/NO cycle in the liver, altered balance of citrulline 
and arginine, decreased NO production in the lung and increased oxidant stress, which results in increased 
pulmonary vascular resistance, development of ALI and mortality. We also reason that restoration of 
citrulline/arginine balance through citrulline administration will safely re-establish homeostasis of NOS, 
lower oxidative stress, and reduce inflammation without resulting in overproduction of NO and 
precipitating hypotension. 

10 
Confidential 
 
 
 
 
 
HCO3 +NH4 + 2 ATP 
Hepatic 
Mitochondrion 
CPS - 1 
N-acetylglutamate 
Carbamyl 
phosphate 
 
 
Cytoplasm 
 
Aspartate 
 
OTC 
Citrulline 
Citrulline 
 
Ornithine 
AS 
NO 
NOS 
Argininosuccinate 
 
 
 
Urea 
Ornithine 
 
 
Arginase 
 
 
Arginine 
 
AL 
Fumarate 
 
 
 
3.2 
The Urea/NO Cycle and Oxidative Stress in Acute Hypoxemic Respiratory Failure 
 
Although the biochemical relationships and interlocking metabolic pathways in health and disease are 
complex, the urea cycle and NO metabolites play an integral role in the inflammatory cascade and 
oxidative stress reactions of many disease states. Homeostasis of redox signaling is contingent on many 
aspects which are complex, take place in a variety of tissues and depend on a balance of competing 
factors, including the amount and location of arginine, citrulline, NO and superoxide generation as well as 
whether or not these reactions are coupled [7]. As such, citrulline and arginine form a critical bridge 
between urea/NO cycle activities in the liver and the NOS system for cell signaling and immune 
functioning in the liver, lung, microvasculature, and elsewhere in the body. Intracellular pools of citrulline 
primarily and arginine determine NO synthesis and play a major role in oxidative stress. It has been 
shown in vitro that iNOS superoxide generation is decreased substantially with physiologic 
concentrations of arginine and is abolished completely in similarly designed nNOS systems [8,9]. 
 
Inducible NOS is upregulated during physiologic stress; however, if NOS substrate is insufficient then 
NOS can uncouple and switch from NO generation to production of damaging peroxynitrites. We 
hypothesized that NOS substrate levels are low in patients with COVID 19 (SARS-CoV2) and that low 
levels of the NOS substrate citrulline would be associated with end organ damage including acute 
respiratory syndrome. We further hypothesized that arginine levels would not be associated with acute 
respiratory syndrome because of substrate channeling from citrulline to NO. Furthermore, citrulline enters 
the cell with relatively little competition through neutral amino transport, while arginine uses dibasic 
amino acid transport, and we detect very little free arginine inside cells, findings that also favor a primary 
role for citrulline availability in regulation of NOS coupling. 

11 
Confidential 
 
 
 
In inflammatory conditions, ROS are produced within endothelial cells and neutrophils. These free 
radicals interact with endothelial cell membranes, especially in the lung, via lipid peroxidation, causing 
altered membrane fluidity and function. In acute respiratory failure and other inflammatory states, the 
urea cycle/NO system plays an integral role in this process. Intracellular deficiency of arginine and its 
precursor citrulline is common due to a combination of poor intake of substrate and reduced activity of 
CPS1, due to oxidation then proteolytic degradation. In addition, AS and AL are often co-induced along 
with inducible NOS (iNOS), especially in macrophages, making the citrulline-arginine cycle hyperactive 
in many inflamed tissues, including the lung in patients with severe infections. The resultant decreased 
production and increased consumption of citrulline and arginine results in dramatically decreased 
intracellular and plasma levels of both [10], which have been linked to abnormal liver function, lung 
injury, coma, and death [11-13]. The hepatic and systemic deficiency of arginine, like those seen in 
severely septic or BMT patients, contributes to the uncoupling of NOS. Uncoupled NOS preferentially 
generates O2- rather than NO [8,13-15] (Figure 2). Additionally, citrulline becomes a conditionally 
essential amino acid in these inflammatory states as levels fall below those needed to maintain negative 
feedback inhibition on NOS, resulting in hyperactive NOS and overproduction of both NO and O2-. 
Increased NO production results in vasomotor instability and contributes to the hypotension seen in septic 
shock. The subsequent reaction between NO and superoxide generates peroxynitrite [16], which 
contributes to vasomotor dysfunction [17,18] and may inactivate catecholamines [19]. Peroxynitrite also 
reacts with unsaturated fatty acids resulting in lipid peroxidation [20]. Lipid peroxidation of the 
endothelial cell membranes result in altered membrane structure and function which leads to capillary 
permeability and the syndrome of ALI. In addition, the resultant oxidative stress stimulates monocytes to 
release more pro-inflammatory cytokines, further potentiating the inflammatory cascade and subsequent 
end-organ damage, especially lung injury. 
 
 
3.3 
Citrulline Replacement 
 
As mentioned, citrulline’s main function appears to be related to the urea/NO cycle, and it may be a better 
molecule than arginine to support this function for several reasons. First, citrulline has free access to the 
mitochondrial matrix and facile entry into the urea cycle pathway. Specific dibasic amino acid 
transporters allow shuttling of ornithine and citrulline across the mitochondrial membrane [21]. Dietary 
manipulation of citrulline content clearly influences urea cycle intermediate levels. Addition of citrulline 
to the diet of arginine deficient rats supports a normal level of arginine in the blood, reduces plasma 
ammonia to normal, and eliminates excretion of orotic acid in the urine [22]. Recycling of citrulline to 
arginine is well documented in several species and in many tissues [23]. In a study of 30 patients with 
Reye’s syndrome, a disease whose characteristics are similar to sepsis with depression of CPS1 activity 
and markedly decreased plasma citrulline levels, provision of enteral citrulline (n=8) promoted more 
rapid lowering of plasma ammonia and among the sickest patients, showed a trend toward reduced 
mortality compared to untreated patients (n=14) (50% vs. 88%; P=0.075) (DeLong 1982). In healthy 
adults, patients undergoing chemotherapy for bone marrow transplant, and children undergoing 
congenital heart surgery, supplementation with citrulline (either oral or IV) supports a higher plasma level 
of arginine and is associated with decreased in vivo lipid peroxidation suggesting that citrulline is superior 
to arginine as a method of enhancing circulating arginine and reducing oxidative stress. 
 
In elegant studies of plasma arginine, citrulline, ornithine, and NO flux using stable isotopic tracers [24- 
30], Castillo and colleagues showed that: (1) Arginine-free intake does not significantly affect the rate of 
either in vivo arginine or citrulline biosynthesis, despite a decline in the plasma arginine flux and free 
arginine concentration. (2) Temporary arginine-free diet did not reduce total nitrate output (i.e. NO 
production was not reduced). Together, these suggest that a reduction in arginine does not limit citrulline 

12 
Confidential 
 
 
or NO production (3) Labeled citrulline leaves the plasma and is metabolized by a full turn of the urea 
cycle before reentering as arginine, (4) Oral administration of 15N-arginine results in a nearly five-fold 
greater recovery of tracer in 15NO3 than when an equimolar amount of tracer is given intravenously, 
documenting significant splanchnic production of NO. Administration of IV citrulline significantly 
bypasses the splanchnic circulation, thus limiting the splanchnic production of NO. (5) About 15% of the 
plasma arginine turnover is associated with urea formation; only 1.2% is associated with NO production. 
As arginase is upregulated in sepsis, the amount of arginine available as substrate for NOS is further 
reduced. Although this may decrease NO production somewhat (likely limited due to the low percentage 
of arginine converted to NO normally), it also alters homeostasis by uncoupling NOS and limiting the 
amount of NO available to scavenge free radicals. Citrulline is resistant to arginase and our data 
demonstrate that citrulline administration produces a more consistent plasma level of arginine than 
arginine administration itself. 
 
3.4 
COVID-19 (SARS-CoV2) and Acute Hypoxemic Respiratory Failure Requiring 
Invasive Mechanical Ventilation 
 
Although reports in the US literature on COVID-19 (SARS-CoV2) and critical illness is limited, a recent 
case series published in the New England Journal of Medicine detailed the clinical course of 24 critically 
ill adults with acute hypoxemic respiratory failure and confirmed COVID-19 infection (SARS-CoV2) in 
Seattle, Washington at 3 hospitals in the University of Washington health care system (106). The mean 
age was 64 +/- 18 years with a range of 23-97 years and 63% were male. Patients had symptoms an 
average of 7 days before hospital admission. Of the 24 patients, 18 or 75% required invasive mechanical 
ventilation but after exclusion of 3 patients who had “do not intubate” directives the percentage was to 
18/21 or 86% requiring invasive mechanical ventilation. The median duration of invasive mechanical 
ventilation was 10 days (IQR 7-12) and overall mortality was 50% in this critically ill population. 
 
3.4 Summary 
 
The body lives in a delicate balance of homeostasis. The urea/NO cycle plays a critical role in 
maintaining redox homeostasis and as such, also plays a role in regulating inflammation.  The 
biochemical relationships are complex and depend on inter-organ transfer, membrane transport, and 
intracellular compartmentation. However, data above demonstrate that citrulline, arginine, and NO are 
critical in maintaining this homeostasis through their regulation of NOS. Inflammation, especially from 
infection, results in decreased activity of CPS1 and increased activity of arginase, which decreases levels 
of both citrulline and arginine. These decreased levels result in dysregulated and uncoupled NOS, which 
drives both overexuberant NO production and formation of ROS. Both the NO production and ROS 
further exacerbate the inflammatory cascade, resulting in other organ dysfunctions, including acute lung 
injury. Both inflammation and oxidative stress have been shown to be driving forces for the development 
of ALI and regulated NOS function is vital to reducing both. Both plasma citrulline and arginine are 
deficient in sepsis and levels are inversely associated with development of ALI. Furthermore, citrulline 
replacement safely increases plasma levels of both citrulline and arginine in healthy volunteers, BMT 
patients, adults with sepsis, children with sickle cell disease, and children after congenital heart surgery. It 
seems highly likely that citrulline therapy in the setting of COVID-19 (SARS-CoV2) induced acute 
hypoxemic respiratory illness will safely increase citrulline and arginine levels and help re-establish NOS 
homeostasis, resulting in NO production in compartments that are more homeostatically appropriate so as 
to reduce pulmonary vascular resistance and enhance coupling of NOS to minimize superoxide 
production thus reducing free radical mediated ALI. 

13 
Confidential 
 
 
4.0 
OBJECTIVES 
 
4.1 Primary Endpoints (objectives) 
 
The primary objectives of the study are as follows: 
 
• 
The primary biochemical objective of this trial is to evaluate the effects of intravenous L- 
Citrulline on plasma levels of citrulline and arginine in patients admitted to the hospital with 
COVID-19 infection (SARS-CoV2) and acute hypoxemic respiratory symptoms requiring oxygen 
therapy. An association of plasma amino acid levels to clinical outcomes may serve as surrogate 
marker for response. 
 
• 
The primary safety objective is a beneficial effect of intravenous L-Citrulline on hemodynamics. 
• 
The primary clinical objective is to evaluate the difference in the length of time to an intubation 
event in hours from the start of study infusion between the study arms. 
 
4.2 Secondary Endpoints (objectives) 
The secondary objectives of this study are as follows: 
• 
To evaluate the safety of intravenous L-Citrulline compared to placebo as measured by incidence 
of reported adverse events. 
• 
To evaluate the effect of intravenous L-Citrulline compared to placebo as measured by the total 
length of all mechanical ventilation, including non-invasive modalities such as high flow nasal 
cannula and BiPAP and oxygen therapy. 
• 
To evaluate the effect of intravenous L-Citrulline compared to placebo on Hospital all-cause 
mortality 
• 
To evaluate the effect of intravenous L-Citrulline compared to placebo on lengths of ICU and 
hospital stay 
• 
To evaluate overall difference in intubation rates 
• 
To evaluate overall duration of mechanical ventilation from consent and post-infusion 
 
5.0 
STUDY DESIGN 
 
5.1 
Study Description 
 
This is a randomized, double-blind, placebo-controlled, phase II study to evaluate the safety and efficacy 
of intravenous L-Citrulline in reducing the length of time from the start of study infusion to an intubation 
event in hospitalized acutely ill patients with an acute hypoxemic respiratory illness due to COVID-19 
infection (SARS-CoV2). Patient’s age of 18 years old and less than 65 years old inclusive with COVID- 
19 infection (SARS-CoV2) will be included in the study. 
 
Only subjects hospitalized at UAMS and requiring oxygen for an acute hypoxemic respiratory illness due 
to COVID-19 (SARS-CoV2) will be included in this study. Study drug administration will occur 
exclusively in the hospital. 

14 
Confidential 
 
 
In addition, subjects will receive standard supportive care for acute hypoxemic respiratory illness and 
failure. 
 
Up to 66 participants ages 18 years to 65 years, inclusive, who meet all the inclusion criteria and none of 
the exclusion criteria will be randomized into the study over approximately 6-9 months. 
 
The study consists of an initial Study Drug bolus followed by a continuous infusion for a maximum of 10 
days. The safety and efficacy will be evaluated through hemodynamic monitoring, laboratory assessment, 
oxygenation status, and biomarker measurements through study day 10.  Adverse events will be 
monitored through study day 10.  Clinical outcomes, including all-cause mortality, ICU length of stay, 
and hospital length of stay will be assessed through hospital discharge with a telephone follow up at D28 
and D60, if discharged earlier 
 
5.1.1 Randomization, Stratification, and Blinding 
Participants will be randomly assigned to one of two study groups. Randomization assignments will be 
determined prior to initiation of the study using a random size permuted block design. Randomization 
will be stratified by age group with participants divided into 2 age groups of 18-49 years and 50-65 years. 
After obtaining informed consent, the signed informed consent document will be faxed to the 
investigational pharmacy. Upon receipt, the investigational pharmacy will begin preparation of the study 
material infusion. Assignment will be communicated electronically using Medrio randomization system. 
Once the study drug material is prepared, it will be sent to the bedside for administration via the bedside 
nurse under the oversight of one of the site investigators and/or study coordinator. After baseline 
hemodynamic assessments, participants will be randomized to either placebo or citrulline in a 1:1 fashion 
stratified by age group. The participants, nurses, physicians, study coordinators and investigators will 
remain blinded to whether participants are receiving placebo or L-citrulline throughout the study. The 
investigational pharmacy will be unblinded and will be responsible for maintaining the blinding of 
investigators, participants, nurses, and primary medical team. The investigational pharmacy will deliver 
the correct infusion(s) to the patient’s bedside every 24 hours. The placebo and study drug solutions will 
be identical appearing on delivery to the ICU where the drug will be administered. 
 
5.1.2 Study Assessments 
The assessment of efficacy will include collection of plasma for analysis of citrulline and arginine levels 
at day 1 hours 0, 2, 12, and the alternating morning of study days the infusion is running for a maximum 
of 10 days, i.e., on days 2, 4, 6, 8 and 10. A patient will exit the study early if they have been weaned off 
all oxygen support before 10 days. Additional efficacy measures will include clinical outcomes, including 
progression of acute lung injury and lengths of hospital and ICU stay. 
 
The assessment of safety will include collection of hemodynamic measurements at least every 4 hours on 
a hospital unit and--if a patient is critically ill in an ICU--hourly while the study material is infusing and 
for 12 hours after completion of the infusion in each patient. Hemodynamic measurements will be 
converted to a vasopressor dependency index for analysis if a patient becomes critically ill. Adverse 
event reporting will also serve as a means of safety analysis. Additional safety measures will be 
biochemical and hematologic laboratory measurements. The biochemical laboratory measurements, 
including liver function tests (ALT, AST, bilirubin, alkaline phosphatase), kidney function tests, and 
electrolytes, along with hematologic laboratory measurements, including complete blood count and 
platelet count, will be collected when available for any study day in order to evaluate for adverse events. 

15 
Confidential 
 
 
5.2 Treatment Arms and Study Drug Administration 
All study material infusions (including the initial boluses) will be administered intravenously through a 
dedicated peripheral line or a dedicated port of a central line using a volumetric or syringe pump. The 
infusion will be restarted as soon as possible after any stoppages and will continue through day 10 unless 
the patient exits the study early. Regardless of amount of time the infusion is stopped, time missed during 
any stoppage will not be added to the end of the infusion. 
 
 
5.2.1 Citrulline Arm 
Patients randomized to citrulline will receive an initial intravenous bolus of 20 mg/kg (to a maximum of 
1500 mg) L-citrulline over 10 minutes. The study solution will be prepared as a 5% isotonic solution (50 
mg/mL) in 5% dextrose water. Immediately after the initial bolus, a continuous intravenous infusion of 
L-citrulline at 9 mg/kg (max 700 mg) per hour will be administered through a dedicated intravenous line 
or port of a multi-lumen catheter. 
 
5.2.2 Placebo Arm 
Patients randomized to placebo arm will receive an infusion of 5% dextrose water matched for volume 
and color to the citrulline infusion. The placebo infusion will consist of an initial iv bolus (up to 30 mL) 
over 10 minutes followed by a continuous infusion of 5% dextrose water (about 15 mL/hr). The initial 
bolus and subsequent infusion will be administered through a dedicated intravenous line or port of a 
multi-lumen catheter. 
 
5.2.3 Drug Interruptions 
Interruptions of study infusions should be avoided. As the study drug poses no known risk during 
procedures, infusions should be continued during bedside procedures. If the study infusion must be 
interrupted, it should be restarted at the same infusion rate as soon as possible. In cases where the 
infusion was interrupted, the infusion window will be restarted as soon as possible but will not be 
extended beyond the end of study day 10. Any study infusion interruptions will be recorded, including 
the reason for the interruption. 
 
In the case of a significant safety concern related to the drug administration, the study drug should be 
discontinued until the situation is evaluated by the safety officer. Since there is no specific antidote for L- 
citrulline, in almost all cases simply discontinuing the study drug is appropriate. Breaking the blind will 
not generally provide increased safety. The study medication blind shall not be broken unless information 
concerning the study medication is clearly necessary for the medical treatment of the participant. The 
decision to unblind study personnel for an adverse event or other circumstance will be determined by the 
safety officer in consultation with the investigators. If unblinding occurs, the investigator will notify the 
IRB within 24 hours. The date, time, and reason that the blind was broken will be provided and 
documented in the CRF. 
 
6.0 STUDY POPULATION AND ENROLLMENT 
6.1 Participant Enrollment 
Up to 66 participants will be enrolled. The study is expected to accrue over about 6-9 months. 
Participants will be recruited from the University of Arkansas for Medical Sciences Hospital. Additional 

16 
Confidential 
 
 
sites may be added at the discretion of the investigators and the sponsor. Participants who withdraw or 
prematurely discontinue study material infusion will be included in an intention to treat analysis. Safety 
data will continue to be collected up to hospital discharge for any participants who are withdrawn from 
the study. 
 
6.2 Inclusion Criteria 
Participant eligibility is determined based on the selection of criteria detailed below: 
 
1. Age 18-65 years. 
2. Clinical evidence of COVID-19 (SARS-CoV2) infection, defined as a positive COVID-19 
laboratory test plus evidence of an acute hypoxemic respiratory illness requiring oxygen. 
3. Admitted and transferred to floor without intubation. 
 
 
6.3 Exclusion Criteria: 
Patients meeting any of the following criteria will not be eligible for participation in the study: 
 
1. No consent/inability to obtain consent 
2. Patient, surrogate, or physician not committed to full support 
3. Malignant or other irreversible condition and estimated 28-day mortality ≥ 50% 
4. Moribund patient not expected to survive 48 hours (as defined by primary medical team) 
from start of study infusion 
5. End-stage Liver Disease as defined by Child-Pugh Score > 9 
6. Currently enrolled in, or participated in another study of an investigational compound within 
the last 30 days 
7. Pregnant female, or female who is breast feeding 
8. Allergy to L-citrulline or arginine or any citrulline- or arginine-containing product 
9. Patient not otherwise suitable for the study in the opinion of any of the investigators 
10. Requirement for intubation and invasive mechanical ventilation before study enrollment 
 
6.4 
Informed Consent 
 
Study personnel (either coordinator or investigators) will identify potential candidates for enrollment 
utilizing the inclusion and exclusion criteria per protocol. Once a potential participant is identified, the 
study coordinator or principal investigator will meet with the patient and/or legally authorized 
representative and will describe the proposed study protocol in lay terminology. Prior to performing any 
study procedures, an IRB-approved informed consent form (ICF) must be signed and dated by each study 
participant or legally authorized surrogate representative and the study investigator or person informing 
the subject and obtaining the consent. Prior to taking part in the study, the participant or his or her legal 
representative should receive a copy of the signed and dated ICF. This procedure will be noted in the 
medical record. ICF will include PK sample retention for any exploratory analysis of certain 
intermediates of special interest to support further understanding of the role of citrulline. 
 
A special vulnerable population, specifically cognitively impaired adults, will be eligible for enrollment 
in this proposed study. Although not all enrolled participants will be cognitively impaired, many could 
be. In cases where the potential participant is cognitively impaired, surrogate consent will be obtained 
from the participant’s legally authorized representative according to state law. In these cases, the 

17 
Confidential 
 
 
participant will be “re-consented” at the earliest opportunity and given the option of continuing or 
stopping their participation. 
 
All revised informed consent forms must be reviewed in the same manner and signed by any active 
participants or their legal representatives. The date that the informed consent is obtained will be 
documented in both the source documents and the case report form (CRF). 
 
A subject number will be assigned to each participant after the ICF is signed. Numbers will be unique 3- 
digit numbers, assigned in ascending order starting with 001. 
 
 
6.5 Excluded Medications 
As interactions between citrulline and other medications are not known, no specific medications will be 
excluded. Patients should not receive exogenous citrulline or arginine (either intravenously or enterally) 
while participating in this study. Additionally, patients enrolled in another study of an investigational 
medicine will be excluded from participating. 
 
6.6 Concomitant Medications 
All medications administered as part of standard supportive care for acute hypoxemic respiratory failure 
will be allowed, including antibiotics, replacement dosages of corticosteroids, catecholamines and other 
vasopressors, and insulin for glucose control. 
 
6.7 Criteria for Discontinuation or Withdrawal 
The primary reason for treatment discontinuation will be noted in the case report form using the following 
categories: 
 
a. Adverse Event: The participant has experienced an adverse event that the investigator 
believes requires early termination because continued participation imposes an 
unnecessary risk to the participant’s health. 
 
b. Major Protocol Deviation: the enrolled participant failed to meet protocol entry criteria 
or did not adhere to protocol requirements, excluding prolonged interruption of study 
drug. 
 
c. Voluntary Withdrawal: The participant or legally authorized representative wishes to 
withdraw from the study. The reason for withdrawal, if provided, will also be 
documented in the CRF. 
 
d. Other: This category includes participants withdrawn for occlusion of an IV or loss of IV 
access. 
 
6.8 Procedures for Discontinuation or Withdrawal of a Participant 
If a participant is withdrawn from treatment and/or the study because of a clinically significant serious 
adverse event (SAE), the investigator will evaluate the urgency of the event. For urgent situations, the 
investigator will proceed with immediate treatment of the SAE and the participant’s condition according 

18 
Confidential 
 
 
to standard of care. The IRB will be notified of any clinically significant, unexpected, related SAE 
leading to the participant’s withdrawal from the study and/or required treatment. 
 
The investigator may terminate participation of a subject for safety if he or she believes that continued 
participation in the study would put the participant’s health at unacceptable risk. In addition, a participant 
or legally authorized representative may discontinue his or her participation without giving a reason at 
any time during the study. 
 
7.0 
STUDY PLAN 
 
7.1 
Definition of Study Procedures (see Appendix A: Schedule of Events) 
7.1.1 Demographics, Medical History, and Medication History 
Demographics will include the age, gender, ethnicity, and race as described by the participant or 
participant’s legally authorized representative. A complete medical history will include a review of all 
major body systems. All concurrent medical conditions will be recorded. Medication history will include 
all medications taken or administered within 72 hours of study drug administration. 
 
7.1.2 Vital Signs Excluding Hemodynamics 
Vital signs will be collected and recorded on the CRFs. For this study, vital signs will include 
temperature, heart rate (pulse), and respiratory rate. Blood pressure will be considered a hemodynamic 
assessment in this study. 
 
7.1.3 Hemodynamic Assessments 
Blood pressure measurements will represent a major component of the hemodynamic assessments. 
Vasopressor dosages in critically ill patients will also form a significant component of these assessments. 
Vasopressor Dependency Index (VDI) will be used as the measure of hemodynamic assessment. VDI is a 
continuous variable calculated by dividing vasopressor index (summation of all pressors) by MAP. VDI 
will be calculated at baseline (immediately before start of the infusion), again 2 hours after completion of 
the bolus, and every 4 hours during the infusion. Any cases where the VDI increases by more than 20% 
from baseline will result in interruption of the study infusion and review by the safety officer in an 
expedited fashion. 
 
7.1.3.1 Blood Pressure Measurements 
Blood pressure measurements will be taken from an arterial catheter if available and sphygmomanometer 
if an arterial catheter is not available. If a participant has an arterial catheter in place, the MAP measured 
by both the arterial catheter and the sphygmomanometer will be documented in case the arterial line is 
discontinued and not replaced during the study. 
 
7.1.4 Mortality, Hospital, and ICU Length of Stay Assessment 
Participant survival will be assessed at hospital discharge. ICU length of stay will be the total number of 
days the participant spends in the ICU from time of enrollment to hospital discharge. Re-admissions to 
the ICU during the initial hospital stay will be added to the initial ICU length of stay to determine a total 
number of ICU days. Hospital length of stay will be calculated as the number of days from enrollment to 

19 
Confidential 
 
 
hospital discharge. Once the patient is discharged from the hospital (to any disposition including home, 
home with help, rehab, or another hospital) or dies, hospital days will cease. Subsequent re-admissions to 
the hospital or ICU after hospital discharge will not be added to the hospital or ICU lengths of stay, 
respectively. 
 
7.1.5 Clinical Laboratory Tests 
Hematology and serum chemistries, including basic metabolic panels, liver function testing, complete 
blood counts, and platelet levels will be performed as part of the participant’s standard medical care at 
clinical care laboratories at the treating site. Five milliliters of blood for special laboratory measurements, 
including serum citrulline and arginine levels will be collected at each of the scheduled time points (see 
Appendix A) separated into plasma and cellular components and frozen at –70oC. These samples will be 
thawed, and the variables measured, all at the same time, after all enrolled participants have completed 
the study. Citrulline and arginine will be measured using a Beckman 7300 Amino Acid Analyzer or 
equivalent. Safety labs, including complete blood count, complete platelet count, basic metabolic panel, 
and liver function tests, will be performed as part of the participant’s standard medical care at clinical care 
laboratories at the treating site. 
 
Five milliliters of blood will be collected in an EDTA anti-coagulated tube at hours 0, 2, 12, and every 2 
days until extubation for a maximum of 10 days- i.e. days 1 (hours 0, 2, 12) then on days 2, 4, 6, 8, and 
10. Plasma will be centrifuged and the supernatant and cellular portions will be frozen at –70oC. 
 
7.1.6 Pregnancy 
Female participants of childbearing potential must have a negative urine or serum HCG pregnancy test to 
be eligible for the study. 
 
7.2 
Schedule of Observations and Procedures 
The schedule for all study related procedures and evaluations is summarized in Appendix A. 
 
7.2.1 Baseline Assessments 
Eligibility of participants will be assessed based on the inclusion and exclusion criteria. Prior to initiation 
of study drug infusion (defined as study time=0 hours), baseline safety assessments, including complete 
blood and platelet counts, measures of renal and liver function, and metabolic panel, will be collected 
from the medical record. Measurements within 36 hours prior to starting study infusion will be 
considered appropriate for baseline measurements. In cases where more than one measurement is 
available in that 36 hours, the measurements closest to the start of study infusion will be used. Blood for 
baseline plasma citrulline and arginine will be drawn as close to time=0 as possible. Baseline 
hemodynamic measurements (i.e. MAP and vasopressor dosage) will be taken within 15 minutes prior to 
initiation of study material. 
 
7.2.2 On Study (days 0-DC) Assessments 
Study drug administration should begin as soon as possible after confirming the participant meets the 
eligibility criteria. Time=0 hours is defined as the time when study infusion is initiated. All times are 
calculated in relation to the time study drug was initiated at Hour 0. The study infusion will last for a 
maximum of 10 days. 

20 
Confidential 
 
 
MAP will be measured per hospital unit or ICU protocol during the entirety of the infusion. MAP and if 
required vasopressor dosages will be recorded in the CRF 2 and 12 hours after start of study infusion and 
as close as possible to 8AM on study days one through 10. VDI will also be calculated for these 
timepoints. 
 
Blood samples for plasma citrulline, arginine measurements will be collected as close to 8AM as possible 
on study days 2, 4, 6, 8, and 10, after Hours 0, 2, 12 are completed. All of these specimens will be 
centrifuged, and the supernatant and cellular portions frozen at –70oC for subsequent batch analysis of all 
specimens once all participants have completed the study. 
 
Patients will discontinue study drug on Day 10. Patients will continue to have all procedures in the 
schedule of procedures except for screening procedures, study drug, and blood draw for plasma citrulline. 
 
Patients who complete study visit day 11 will continue to be followed from day 12 to discharge. From 
day 12 to discharge the following values will continue to be collected during this time are vital signs, 
hospital status, ICU status, and non-invasive and invasive ventilator settings. A further follow up will 
occur on D28 and D60 as a telephone visit, if discharged earlier. 
 
Concomitant medications and adverse event monitoring will be performed and reported in CRFs. 
See the Schedule of Procedures in Appendix A for further details. 
7.2.3 Clinical Outcome Assessments 
ICU length of stay will be calculated in days from time = 0 to the earlier of ICU discharge or death. Any 
re-admissions to the ICU during the hospital stay will add days to ICU length of stay. Hospital length of 
stay will be calculated in days from time = 0 until the earlier of hospital discharge or death. A follow up 
at D28 and D60 will further evaluate the stated clinical outcomes. 
 
Concomitant medications and adverse events will continue to be monitored and recorded in the CRF for 
the duration of the participant’s hospital stay. 
 
See the Schedule of Procedures in Appendix A for further details. 
 
8.0 
PROTOCOL DEVIATIONS 
Protocol deviations will be recorded and reported to the local IRB. If the protocol deviation has placed the 
participant at increased risk, it will be reported to the safety officer and the IRB within 24 hours. If the 
protocol deviation has resulted in no increased risk, the event will be reported to the IRB at continuing 
review. 
 
9.0 
ADVERSE EVENTS 
Investigators will determine daily if any clinical adverse experiences occur during the period from 
enrollment to 48 hours after study drug termination. Most data normally collected as part of a clinical 
study to guarantee patient safety and determine efficacy is obtained as part of normal clinical care of 
patients in the hospital or ICU. Examples include arterial blood gases, electrolytes, liver function tests, 
hemograms, and chest radiographs. All available clinical data, including radiographs, laboratory values, 
vital signs, exam findings, and clinical impressions from the primary team, will be monitored for adverse 
events. The investigator will evaluate any changes in laboratory values and physical signs and will 

21 
Confidential 
 
 
determine if the change is clinically important and different from what is expected during treatment of 
patients with acute hypoxemic respiratory failure. If clinically important and unexpected adverse 
experiences occur, they will be recorded on the adverse event case report form. Investigators will report 
all serious and unexpected adverse events to the safety officer and the local IRB in a timely manner. All 
adverse events that are deemed non-serious and or not unexpected will be reported to the IRB annually at 
continuing review. 
 
Patients will be closely monitored in a hospital or ICU setting. This will allow for prompt treatment of 
any adverse event that is suspected to be related to study drug, in which case the safety monitor will be 
notified and will evaluate the situation. In such an instance study drug infusion will be withheld until the 
event is evaluated and a decision has been made whether the infusion will be restarted or permanently 
discontinued. 
 
9.1 Adverse Events 
An adverse event is defined as any untoward medical occurrence in a patient or clinical investigation 
participant administered a pharmaceutical product that does not necessarily have to have a causal 
relationship with this treatment. An adverse event therefore can be any unfavorable and unintended sign 
(e.g., abnormal laboratory finding), symptom, or disease temporally associated with the use of an 
investigational product, whether or not the incident is considered to be related to the investigational 
product. Incidences of hypotension, hepatitis (i.e. elevation of liver enzymes), and injection site reactions 
will be systematically collected and will be considered adverse events. 
 
9.2 Clinically Significant Laboratory Values 
Any change in laboratory values will be evaluated. If a laboratory abnormality is new from baseline and 
thought to be clinically significant, it is considered a laboratory adverse event. However, if the laboratory 
value abnormality is consistent with a current diagnosis, it will not necessarily be considered an adverse 
event. Changes in laboratory values will only be considered adverse events if they are judged to be 
clinically significant (if some action or intervention is required or if the investigator judges the change to 
be beyond the range of normal fluctuation). Due to most of the urea cycle enzymes being hepatically 
located, elevation of liver enzymes will be systematically collected. 
 
9.3 Pre-Existing Conditions 
Pre-existing conditions that are present before the start of the study infusion will be considered concurrent 
medical conditions and should NOT be recorded as adverse events. However, if the subject experiences a 
worsening or complication of such a concurrent condition, the worsening or complication may be 
recorded as an adverse event at the discretion of the investigator. 
 
The following are clinical outcomes of an acute hypoxemic respiratory illness progressing to critical 
illness due to COVID-19 infection (SARS-CoV2), and as such, will not be considered as adverse events: 
 
1. Respiratory: decreased SaO2/FiO2 or PaO2/FiO2, mechanical ventilation, acute lung 
injury or acute respiratory distress syndrome (ARDS), or respiratory failure 
2. Hepatic: hepatic injury or liver dysfunction; increased bilirubin 
3. Renal: renal failure, need for dialysis, increased creatinine 
4. Coagulation: disseminated intravascular coagulation (DIC), thrombocytopenia 
5. Central Nervous System (CNS): acute mental status changes (not associated with 
localizing neurological signs) 

22 
Confidential 
 
 
6. Vital Signs: tachypnea, hypothermia, hyperthermia, tachycardia 
 
Cardiovascular function will be systematically collected as both MAP and vasopressor dosages. VDI will 
be calculated at baseline (immediately before start of the infusion), again 60 minutes after completion of 
the bolus, and every 4 hours during the infusion. Any cases where the VDI increases by more than 33% 
from baseline will be considered an adverse event and submitted for review by the safety officer in an 
expedited fashion. 
 
9.4 Serious Adverse Events 
A serious adverse event (SAE) is defined as any untoward medical occurrence that meets any of the 
following criteria: 
 
a. Results in death 
b. Is life-threatening (defined as an event in which the participant was at risk of death at the 
time of the event and NOT an event that hypothetically might have caused death if it had 
been more severe) 
c. Requires inpatient hospitalization 
d. Prolongs an existing hospitalization 
e. Results in persistent or significant disability or incapacity 
f. Results in a congenital anomaly or birth defect 
g. Important medical event that requires an intervention to prevent any of a-f above. 
 
h. SAE qualifying as a SUSAR (serious unexpected suspect adverse reaction) will be 
subject to expedited reporting as per safety plan. 
 
9.5 Adverse Event Collection Period 
Collection of adverse events will commence from the time that the participant is first administered the 
study infusion (study hour =0). Routine collection of adverse events will continue until day 10. Any 
adverse events spontaneously reported to the investigator or observed by the investigator after day 10 will 
also be collected and recorded on the CRF. 
 
10.0 STATISTICAL METHODS 
10.1 Analysis Populations 
The intent-to-treat (ITT) population will be used for all efficacy and safety analyses. This will consist of 
all randomized patients irrespective of whether the patient received study drug or the patient’s compliance 
with the study protocol, in the treatment group assigned by the randomization system. 
 
10.2 General Statistical Approach 
Before database lock, a statistical analysis plan (SAP) will be issued as a separate document, providing 
full detailed methods for the analyses outlined below. Multiplicity adjustment approach will be described 
as per industry guidance for multiple comparisons across all the endpoints to eliminate Type 1 errors. 
Sensitivity analysis will be applied to handle missing data and sources of uncertainty. The protocol study 
team may revise the plan during the study to accommodate clinical trial protocol amendments and to 
make changes to adapt to unexpected issues in study execution and data that affect planned analyses. The 
protocol team will conduct all statistical analyses following the statistical principles for clinical trials as 
specified in International Council on Harmonization Topic E9. Any deviations from the planned analyses 

23 
Confidential 
 
 
will be described and justified in the final integrated clinical study report. The protocol study team will 
present overall and treatment specific data and summary tables. 
 
All statistical tests will be two-sided. All summary tables for quantitative measures will display means, 
standard deviations, median and range, as well as number of missing data (if relevant). All summary 
tables for categorical variables will display counts, percentages, and number of missing data (if relevant). 
 
10.3 Demographics and Baseline Characteristics 
Demographics and baseline characteristics will include age, gender, race, comorbidities, baseline vital 
signs and laboratories. Descriptive statistics, including mean and standard deviation, median, intra- 
quartile ranges, minimum and maximum, and the number and percent of subjects in specified categories 
will be used to summarize the demographic and baseline variables for the two study arms. These will be 
compared between groups using independent two-sample test or Wilcoxon rank-sum test, as appropriate, 
for continuous variables and Fisher’s Exact Test for categorical variables. All statistical analyses will be 
done using SAS statistical software. 
 
10.4 Sample Size 
This study will accrue approximately 66 patients in a 1:1 fashion to account for 10% attrition. This 
sample size was determined on the basis of an expected time to intubation event in the placebo group 
derived from historical data on critically ill patients with COVID-19 infection (SARS-CoV-2) of 3 days 
(72 hours) +/- 3 days (72 hours). An acceptable effect size in the intravenous citrulline group would be 
delay in time to an intubation event of at least 6.5 days (156 hours). Thus, a two-sided log-rank test with 
an overall sample size of 60 patients achieves 81% power at a 0.05 significance level to detect a hazard 
ratio of 0.46 when the placebo group time to intubation is 3 days. The study is expected to last 12 months, 
of which patient accrual will occur in the first 6-9 months. The accrual pattern across time is uniform. 
 
In terms of mortality, in patients who progress to acute hypoxemic respiratory failure requiring ICU care, 
the mortality is 50%. With a sample size of 60 patients, the study will have 80% power to detect a 
difference between the group proportions of -33%. The mortality in the intravenous citrulline group is 
assumed to be 50% under the null hypothesis and 17% under the alternative hypothesis. Again, the 
mortality rate for the placebo group is assumed to be 50%. The test statistic used is the two-sided z-test 
with pooled variance with a 0.05 significance level. 
 
10.5 Primary Endpoints 
Time to an intubation event in hours from the start of the study drug infusion or overall duration of 
mechanical ventilation will represent the primary clinical endpoint. The number of patients starting 
intubation over time will be displayed in the form of survival curves using the Kaplan-Meier method and 
analyzed using a two-sided log-rank test. Statistical significance will be claimed if the computed p-value 
is equal to or less than 0.05. 
 
Citrulline and arginine plasma levels will represent the primary biochemical endpoint of this study. A 5 
mL sample of blood will be obtained for amino acid analysis at hour 0 before study medication is 
administered, 2 and 12 hours after the bolus and as close as possible to 8 AM on days 2, 4, 6, 8, and 10. 
Samples will be collected in citrated tubes and placed on ice until they can be centrifuged. Plasma and 
cellular components will be frozen at –70 oC until batch analysis. Concentrations of citrulline, arginine, 
and ornithine will be measured in the plasma samples using cation-exchange chromatography with a 
Beckman 7300 amino acid analyzer or equivalent. The analysis team will provide the means and standard 

24 
Confidential 
 
 
deviations for citrulline, arginine, and ornithine plasma levels separately for each treatment group. A 
generalized linear mixed model (GLMM) with appropriate link function (i.e., identity link for continuous 
outcome) will be used to compare these plasma amino levels across the two intervention groups. The 
model will examine how the treatment means differ (i.e., main treatment effect), how treatment means 
change over time (i.e., main time effect), and how differences between treatment means change over time 
(i.e., treatment-by-time effect). 
 
The primary safety outcome will be worst VDI any time after start of infusion. VDI is a continuous 
variable calculated by dividing vasopressor index (summation of all pressors) by MAP. VDI will be 
calculated at baseline (immediately before start of the infusion), 2 hours after completion of the bolus, and 
every 4 hours during the infusion. Worst VDI will be compared between groups using ANCOVA analysis 
with the baseline VDI as the covariate. 
 
10.6 Secondary Safety Endpoints 
Although VDI will represent the primary outcome for determining safety, other measures will also be 
employed. 
 
10.6.1 Secondary Clinical Endpoints 
All-cause, hospital mortality, defined by whether the patient was discharged from the hospital alive or 
deceased, will be analyzed via two statistical methods. The proportion of patients who have died by 
hospital discharge will be analyzed using a Chi-square analysis or Fisher’s Exact Test, if needed. In 
addition, time to death will also be analyzed using a log-rank method of a Kaplan-Meier analysis. ICU 
length of stay (LOS) will be defined in days as the time from study enrollment to the day ICU discharge 
orders are written, regardless of when the patient physically leaves the ICU. Comparison will be made 
between the two groups for total ICU length of stay which will include any days of ICU re-admission 
prior to hospital discharge. Hospital length of stay will be defined as time from study enrollment to first 
hospital discharge. We will consider both ICU LOS and hospital LOS as count measure and has the 
potential to follow a skewed distribution. Initially, we will assess the distributional assumption. We will 
use a generalized linear model (GLM) to compare the expected ICU LOS and hospital LOS between the 
two treatment groups (placebo and intravenous citrulline). Specifically, we will use a negative binomial 
distribution and log-link to account for potential over-dispersion. We will report point estimates for the 
group mean difference along with a 95% confidence interval. Overall duration of mechanical ventilation 
will be modeled with zero-inflated Poisson or negative binomial method using consent and post-infusion 
start time for two separate analysis. 
 
10.7 Interim Analyses 
No formal interim analyses for efficacy will be done in this phase 2 study. A safety analysis looking at 
VDI and hypotension will be undertaken after the first 20 patients have completed enrollment to ensure 
that citrulline administration is not causing hypotension. 
 
 
11.0 
ETHICS 
11.1 
Ethical Conduct of the Trial 
This study will be conducted with the highest respect for individual participants according to the protocol, 
the World Medical Association Declaration of Helsinki, the Guideline for Good Clinical Practice, and the 

25 
Confidential 
 
 
Belmont Report. Furthermore, the study will be conducted in accordance with the Common Rule (45 
CFR 46), as guided by the Department of Health and Human Services via the Office of Human Research 
Protections. 
 
11.2 
Institutional Review Board 
The investigator and sponsor will submit all relevant documents to the IRB for the protocol’s review and 
approval. The study, protocol, informed consent form, investigator’s brochure, and any proposed 
advertising or recruitment materials must be approved by the local IRB prior to commencing the study. 
Written approval by the IRB of the protocol and participant informed consent document must be obtained 
prior to enrollment of any participants. If any member of the IRB has direct participation in this trial, he 
or she must refrain from any IRB discussions or votes pertaining to this study. 
 
Sites must adhere to all requirements stipulated by their respective IRBs. This may include notification to 
the IRB regarding: protocol amendments, updates to the informed consent document, recruitment 
materials, local safety reporting requirements, and closure of the study. 
 
11.3 
Participant Information, Informed Consent, and Participant Authorization 
All consent for participation in this study must occur via written consent documents, which should 
contain the elements of informed consent as required by 45 CFR 46 and the Declaration of Helsinki. The 
informed consent document will describe the planned and permitted uses, transfers, and disclosures of the 
participant’s personal and personal health information for purposes of conducting the study. 
 
The investigator is responsible for the preparation, content, and submission to the IRB approval of the 
informed consent form. The informed consent form and any participant information sheet must be 
approved by the IRB prior to use. 
 
The participant, or his or her legally authorized representative, must be presented with an informed 
consent document written in their native language and given ample opportunity to: 1) inquire about 
details of the study, and 2) decide whether or not to participate in the study. If the participant or legally 
authorized representative agrees to participate in the study, the informed consent form must be signed and 
dated by the participant or legally authorized representative prior to the participant being enrolled in the 
study. The investigator must document the date the ICF is signed in the participant’s medical record. The 
investigator must also sign and date the informed consent form at the time of consent and prior to the 
participant being entered into the study. 
 
Once signed by both the participant and investigator, a copy of the informed consent document will be 
given to the participant (or legally authorized representative). The original informed consent document 
will be stored in the investigator’s site file. All revised informed consent forms must be reviewed and 
signed in the same manner as the original informed consent. The date the revised consent was obtained 
should be recorded in the participant’s medical record and the participant should receive a copy of the 
revised ICF. If surrogate consent was utilized for initial consent, the participant should be “re-consented” 
by signing the original ICF when he or she regains the capacity to consent. The participant should date 
this “reconsent” with the actual date of reconsenting and not with the date of the original consent signing 
by the legally authorized representative. 
 
11.4 
Data and Safety Monitoring Plan 

26 
Confidential 
 
 
The Principal Investigator, along with the Chief Medical Officer at Asklepion (safety officer) will be 
responsible for overseeing the safety of this trial. There is no formal data safety monitoring board planned 
for this trial. Laboratory and clinical data will be reviewed daily for adverse events by the safety officer. 
In addition, the safety officer will be available at any time for questions from the co-investigators at 
UAMS, who will be monitoring the patients continuously for potential adverse events. There will be no 
interim analyses of the data for efficacy with the exception of an initial independent PK evaluation 
(separate from study team) of first 2 subjects for all the time points to ensure steady state concentration is 
being attained The safety officer will be available to review serious adverse events in a timely manner. 
Furthermore, in cases of serious adverse events or significant hypotension, the safety officer can request 
unblinding of the treatment arm and/or an urgent amino acid analysis to better guide decision-making. 
The safety officer will have the ability to pause the trial in agreement with the co-investigators to enable 
investigation of possible safety issues and/or suggest changes to the design of the study to abrogate any 
safety issues, including discontinuation of the initial bolus should it result in unacceptable hypotension. 
 
11.5 
Potential Risks 
All patients will reside in a hospital unit or intensive care unit environment, where close monitoring can be 
achieved and rapid response to any potential adverse event, including hypotension, will occur. Should a 
patient improve and be weaned off all oxygen support prior to completion of the study drug infusion, the 
study drug will be stopped but data will continue to be collected. Patients will be monitored by the bedside 
or ICU nurse for the duration of the infusion per standard unit protocol. The investigator and/or coordinator 
will be available by pager 24 hours a day 7 days a week to answer any questions that the bedside nurse has. 
 
All patients will have blood drawn for research purposes. The risks of drawing blood are uncommon and 
may include bleeding and bruising. Commonly, having blood drawn is painful, and rarely can lead to 
infection at the site of the blood draw. It is anticipated that most of the blood obtained for research purposes 
will be obtained through existing intravenous peripheral, central, or arterial catheters since these patients 
routinely have such catheters while in the intensive care units. The amount of blood drawn is minimal and 
represents a small percentage of the amount of blood taken during a standard hospital or ICU stay and will 
not represent a significant risk to the patient. 
 
The risks of citrulline are theoretical. In previous studies enrolling hundreds of humans, no serious 
adverse events have been attributed to either oral or intravenous citrulline. Citrulline may increase NO 
production and exacerbate hypotension. Arginine, a product of citrulline via the urea cycle, has been 
reported to exacerbate hypotension. The blood pressure of every participant in this study will be 
monitored closely throughout the study and especially during the infusion. As these patients will be cared 
for in a hospital unit or ICU, blood pressure measurements will be taken per hospital unit or ICU 
protocol. Hypotension, as measured by vasopressor dependency index (VDI) represents an a priori 
endpoint of the study.  VDI will be calculated 2 hours after the bolus and daily.  Any worsening of VDI 
by 33% or more will prompt review by the safety monitor. Although it is likely to reduce organ 
dysfunction by attenuating inflammation and oxidative stress, daily labs will be monitored for adverse 
events on organ functioning. The citrulline will be administered in a small volume of 5% dextrose water 
(30 mL bolus followed by ~15 mL/hr). This small amount of dextrose should not dramatically increase 
blood sugar levels in these patients and as such, should pose minimal risk. Furthermore, any event which 
the investigator or primary team is concerned may be related to study drug administration will be reported 
as an adverse event. Participants will be monitored for any signs of allergic reaction, including new 
wheezing, itching and/or rash. Blood pressure, which might fall during severe allergic reactions such as 
anaphylaxis, will be closely monitored in the hospital or ICU setting with treatment available as needed. 

27 
Confidential 
 
 
The placebo will be a small volume of 5% dextrose water (30 mL bolus followed by ~15 mL/hr). 
Although administration of dextrose in theory could raise blood sugar levels, the small volume will result 
in little dextrose being administered which should not significantly increase blood sugar levels in these 
patients and as such, should pose minimal risk. In addition, blood sugar levels are routinely measured 
and monitored closely as part of routine care in intensive care unit patients, especially those with severe 
sepsis. 
 
Another potential risk to participants is the breach of confidentiality including information from their 
medical record. There are many safeguards in place to prevent the release of information from this study. 
All data for this study will be stored in a password-protected, HIPAA security compliant computer 
database.  Initially it will be recorded on case report forms which will be stored in a locked office that 
only the study personnel have access to. This is especially true since some genetic tests will be run 
utilizing these samples. All samples obtained for this study will be assigned a code which will not identify 
the subject. The only key to the code will be in the password-protected computer database. Dr. Meena, 
and the study co-investigator will have access to the code and information that identifies a participant as a 
being in this study. The results of analyses run on those samples will not be reported to the participant or 
primary study team. No one else, including relatives, doctors, employers, or insurance companies will be 
allowed to view the test results. The codes linking the patients with their data will be destroyed after all 
participants have completed the study and the data has been through quality assurance processes. 
Informed consent documents will not bear the study identification number so that once the code is 
destroyed; there will be no possible way the results could be traced back to the individual. 
 
11.6 
Alternative Treatments 
There are no alternative treatments. Patients who are participating in another interventional study will be 
ineligible for this study. Participants will be told they have the option to choose not to participate in this 
study. The primary team will manage the participants’ clinical condition according to their best clinical 
judgment. Any potential treatments for acute hypoxemic respiratory illness, including low tidal volume 
ventilation in patients requiring mechanical ventilation will be allowable, determined by the primary 
medical team, and not restricted by participating in this study. The blinded study drug material will be 
added to the treatment course that the primary team considers appropriate. 
 
11.7 
Potential Benefits 
The potential benefits to science and mankind that may result from this study include the knowledge that 
we will obtain regarding the effects of citrulline and the urea/NO cycle on inflammation and oxidative 
stress in an acute hypoxemic respiratory illness. In addition, we will also gain valuable information 
regarding the safety of IV citrulline in COVID-19 (SARS-CoV2) infected patients. Since the urea/NO 
cycle and NOS play an integral role in redox homeostasis, administration of citrulline may decrease 
oxidative stress and improve organ function, especially pulmonary function, even potentially mitigating 
acute lung injury in these patients. The patients randomized to the placebo arm will not directly benefit 
from participating in this study but will provide vital information for the interpretation of the study results 
and a valuable contribution to medical science. 
 
These benefits are reasonable in relation to the relatively few known risks of L-citrulline. L-citrulline, by 
re-establishing urea/NO cycle homeostasis and reducing oxidant stress, may improve clinical outcomes, 
including mitigation of organ dysfunction, particularly lung injury. These improved outcomes are 
desirable by all patients seeking medical attention. L-citrulline has been used in humans previously and 
has been shown to be safe. 

28 
Confidential 
 
 
 
12.0 
DATA HANDLING AND RECORDKEEPING 
12.1 
Sources of Research Material 
All materials will be collected and recorded for research purposes. The data collected during this 
investigation will be obtained from multiple sources, including medical records, observational data, and 
serum samples. One of the main sources of data will be chart abstraction utilizing the patient’s medical 
record. The progress notes and bedside nursing notes and daily charting will also be used to collect data. 
Interviews with patients and/or patients’ family will be conducted both in person and over the phone on 
occasion. The patients will have serum samples collected at the specified timepoints for specialized 
laboratory analyses. All the remaining data will be derived from the interactions with the patient in the 
form of the nurses’ and physicians’ examinations and interviews. 
 
12.2 
Participant Confidentiality 
The investigators affirm and uphold the principle of the participant’s right to protection against invasion 
of privacy. Throughout this study, a participant’s source data will only be linked to the clinical study 
database or documentation via a unique study identification number. As permitted per HIPAA 
regulations, limited participant attributes such as sex, age, or gender may be used to verify the accuracy of 
the participant’s unique identification number. Copies of any participant source documents provided to 
entities outside of the University of Arkansas for Medical Sciences (including the FDA, IRB, or NIH) 
will have all personally identifiable information removed (e.g., participant name, address, date of birth) 
 
The research coordinator, principal investigator, or co-investigators will collect data and record it on case 
report forms designed specifically for the study. The data will then be entered in a password protected, 
privacy-secured, computer database. The data collection forms will be kept in a locked cabinet in a 
locked office at each site. Only the study investigators and study coordinators will have access to the 
locked cabinet. All samples obtained for this study will be assigned a code which will not identify the 
subject. The only key to the code will be in the password-protected computer database. The Principal 
Investigator will be responsible for maintaining confidentiality of data and codes. Only study 
investigators and the study coordinator will have access to the code and information that identifies a 
volunteer as a being in this study. The results of analyses run on those samples will not be reported to the 
participant or primary team. The codes linking the patients with their data will be destroyed after all 
participants have completed the study and the data has been through quality assurance processes. 
 
12.3 
Case Report Forms 
CRFs will be completed for each participant randomly assigned to study medication or placebo. CRFs 
will be completed in legible black ink and will be written in English. Corrections will be made via a 
single line strikeout of the incorrect information and writing in the revisions. All corrections must be 
initialed and dated. The completed original CRFs are the sole property of site where enrolled. 
 
12.4 
Medrio Computerized Database 
Medrio EDC will be used as the data capture tool. It is a secure, cloud-based application that provides 1) 
intuitive navigation for validated data entry; 2) detailed audit trails for tracking data manipulation (e.g. 
old/new/deletion, approvals, locking); 3) reporting modules and on-demand data extraction capabilities; 
and 4) tools for importing data from external sources. 

29 
Confidential 
 
 
Data within Medrio is siloed and independently structured to ensure that a study and its users can access 
only their own study. Thus, only study personnel for this project with appropriate permissions will be 
allowed to access the database. Authorized users can input data into Medrio from anywhere in the world 
with secure web authentication and data logging. 
 
12.5 
Record Retention 
The investigator will keep all study-specific documents, an identification log of all participating patients, 
source worksheets, original signed and dated informed consent documents in a locked and secure office or 
storage space until at least three years after the completion of the study. In addition, the secure Medrio 
database will be maintained for at least three years after completion of the study. The data will be housed 
with the sponsor from the Medrio EDC. 

30 
Confidential 
 
 
13.0 
REFERENCES 
1. Moncada S, Higgs A (1993). The L-arginine-nitric oxide pathway. N Engl J Med. 329(27):2002- 
2012. 
2. Matuschak GM (1994). Liver-lung interactions in critical illness. New Horizons. 2:488-04. 
3. Matuschak GM, Mattingly ME, Tredway TL, Lechner AJ (1994). Liver-lung interactions during 
E. coli endotoxemia. TNF-alpha: leukotriene axis. Am J of Resp Crit Care Med. 149:41-9. 
4. Blackwell TS, Christman JW, Hagan T, et al (2000). Oxidative stress and NF-kappaB activation: 
correlation in patients following allogeneic bone marrow transplantation. Antioxidants & Redox 
Signalling. 2(1):93-102. 
5. Koay A, Christman JW, Segal BH, et al (2001). Impaired pulmonary NF-6B activation in 
response to lipopolysaccharide in NADPH oxidase-deficient mice. Infection and Immunity. 
69:5991-5996. 
6. Cynober L (1994). Can arginine and ornithine support gut functions? Gut. Suppl 1:S42-45. 
7. Dasgupta T, Hebbel RP, Kaul DK (2006). Protective effect of arginine on oxidative stress in 
transgenic sickle mouse models. Free Radic Biol Med. 41(12):1771-1780. 
8. Xia Y, Tsai AL, Berka V, et al (1998). Superoxide generation from endothelial nitric-oxide 
synthase. A Ca++/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol 
Chem. 273:25804-25808. 
9. Kaul DK, Liu XD, Fabry ME, et al (2000). Impaired nitric oxide-mediated vasodilation in 
transgenic sickle mouse. Am J Physiol Heart Circ Physiol. 278(6):H1799-H1806. 
10. Magarik J, West N, Bernard G, Wheeler A, Christman B, Cunningham G, Canter J, Bossert F, 
Barr R, Summar M, Ware LB. Low levels of the nitric oxide (NO) precursor citrulline are 
associated with acute respiratory distress syndrome (ARDS) in patients with severe sepsis. Proc 
Am Thorac Soc. 177:A117, 2008. 
11. Sprung CL, Cerra FB, Freund FR, et al (1991). Amino acid alterations and encephalopathy in the 
sepsis syndrome. Crit Care Med. 19:753-757. 
12. Legras A, Labarthe F, Maillot F, et al (2002). Late diagnosis of ornithine transcarbamylase 
defect in three related female patients: polymorphic presentations. Crit Care Med. 30(1):241- 
244. 
13. Wiesinger H (2001). Arginine metabolism and the synthesis of nitric oxide in the nervous 
system. Prog Neurobiol. 64:365-391. 
14. Pou S, Pou WS, Bredt DS (1992). Generation of superoxide by purified brain nitric oxide 
synthase. J Biol Chem. 267:24173-24176. 
15. Stuehr D, Pou S, Rosen GM (2001). Oxygen reduction by nitric-oxide synthases. J Biol Chem. 
276:14533-14536. 
16. Beckman JS, Beckkman TW, Chen J, et al (1990). Apparent hydroxyl radical production by 
peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl 
Acad Sci USA. 87:1620-1624. 
17. Salvemini D, Ischiropoulos H, Cuzzocrea S (2003). Roles of nitric oxide and superoxide in 
inflammation. Methods Mol Biol. 225:291-303. 
18. Cuzzocrea S, Mazzon E, Di Paola R, et al (2006). A role for nitric oxide-mediated peroxynitrite 
formation in a model of endotoxin-induced shock. J Pharmacol Exp Ther. 9(1):73-81. 
19. Macarthur H, Westfall TC, Riley DP, et al (2000). Inactivation of catecholamines by superoxide 
gives new insights on the pathogenesis of septic shock. Proc Natl Acad Sci USA. 97(17):9753- 
9758. 
20. Hogg N, Darley-Usmar VM, Wilson MT, et al (1993). The oxidation of alpha-tocopherol in 
human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide. 
FEBS Lett. 326:199-203. 

31 
Confidential 
 
 
21. Cheung CE, Cohen NS, Raijman L (1989). Channeling of urea cycle intermediates in situ in 
permeabilized hepatocytes. J Biol Chem. 264:4038-4044. 
22. Hartman WJ, Torre PM, Prior RL (1994). Dietary citrulline but not ornithine counteracts dietary 
arginine deficiency in rats by increasing splanchnic release of citrulline. J Nutr. 124:1950-60. 
23. Shuttleworth CW, Conlon SB, Sanders KM (1997). Regulation of citrulline recycling in nitric 
oxide-dependent neurotransmission in the murine proximal colon. Br J Pharm. 120:707-713. 
24. Castillo L, Chapman TE, Sanchez M, et al (1993a). Plasma arginine and citrulline kinetics in 
adults given adequate and arginine-free diets. Proc Natl Acad Sci USA. 90:7749-7753. 
25. Castillo L, Chapman TE, Yu YM, et al (1993b). Dietary arginine uptake by the splanchnic region 
in adult humans. Am J Phys. 265:E532-E539. 
26. Castillo L, deRojas TC, Chapman TE, et al (1993c). Splanchnic metabolism of dietary arginine 
in relation to nitric oxide synthesis in normal adult man. Proc Natl Acad Sci USA. 90:193-197. 
27. Castillo L, Sanchez, Chapman TE, et al (1994a). The plasma flux and oxidation rate of ornithine 
adaptively decline with restricted arginine intake. Proc Natl Acad Sci USA. 91:6393-6397. 
28. Castillo L, Ajami A, Branch S, et al (1994b). Plasma arginine kinetics in adult man: response to 
an arginine-free diet. Metabol Clin Exp. 43:114-122. 
29. Castillo L, Sanchez M, Vogt J, et al (1995). Plasma arginine, citrulline, and ornithine kinetics in 
adults, with observations on nitric oxide synthesis. Am J Phys. 268:E360-E367. 
30. Castillo L, Beaumier L, Ajami AM, et al (1996). Whole body nitric oxide synthesis in healthy 
men determined from [15N] arginine-to-[15N]citrulline labeling. Proc Natl Acad Sci USA. 
93:11460. 
31. Bhatraju PK, Ghassemieh, BJ, Nichols M, et al (2020). COVID-19 in Critically Ill patients in the 
Seattle Region- Case Series. New England Journal of Medicine epub March 2020: 1-11. 

Confidential 
 
 
14. Investigator Signature Page 
Protocol Title: 
Prospective, Randomized, Double-Blind, Placebo-Controlled Phase II Trial 
of Intravenous L-Citrulline to Delay and Potentially Prevent the Need for 
Invasive Mechanical Ventilation for Acute Hypoxemic Respiratory Failure 
in Patients with COVID-19 (SARS-CoV-2) Illness 
 
Protocol Number: 
CIT-COVID19-002-01 Version 3.0 Dated 11 June 2021 
 
 
 
Confidentiality and cGCP Compliance Statement 
 
I, the undersigned, have reviewed this protocol, including appendices and I will conduct the study as 
described in compliance with this protocol, GCP, and relevant ICH guidelines. 
 
Once the protocol has been approved by the IRB/IEC, I will not modify this protocol without obtaining 
prior approval of the Asklepion Pharmaceuticals, LLC and of the IRB/IEC. I will submit the protocol 
modifications and/or any ICF modifications to Asklepion Pharmaceuticals, LLC and IRB/IEC, and 
approval will be obtained before any modifications are implemented. 
 
I understand that all information obtained during the conduct of the study regarding the subjects’ state of 
health will be regarded as confidential. No subjects’ names will be disclosed. All subjects will be 
identified by assigned numbers on all Case Report Forms, laboratory samples or source documents 
forwarded to the sponsor. Clinical information may be reviewed by the sponsor or its agents or regulatory 
agencies. Agreement must be obtained from the subject before disclosure of subject information to a third 
party. 
 
Information developed in this clinical study may be disclosed by Asklepion, to other clinical 
investigators, regulatory agencies, or other health authority or government agencies as required. 
 
 
 
 
 
 
Investigator Signature 
Date 
 
15-Jun-2021 
 
Nikhill K. Meena, MD 
 
Printed Name 
 
UAMS 
 
Institution 
 
 
32 

Confidential 
 
 
 
APPENDIX A: Schedule of Events 
 
 
 
Measurement 
Day 0 
(baseline) 
Day 1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
6 
Day 
7 
Day 
8 
Day 
9 
Day 
10 
Day 
11 
Day 
12-DC 
Day 28 e 
Follow-up 
Day 60 e 
Follow-up 
Informed Consent 
X 
Demographics- Hx 
& Px 
X 
Eligibility 
Assessment 
X 
Pregnancy Test 
Xc 
Study Drug 
Administration 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Mean Arterial 
Pressure 
Xa,b,d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vasopressor 
Dosages 
Xa,b,d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Basic Metabolic 
Panel 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
CBC 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
LFTs 
Ab 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
D-dimer blood 
draw 
Xa,b 
X 
X 
X 
X 
X 
X 
CRP 
Xa,b 
X 
X 
X 
X 
X 
X 
5 ml blood for 
citrulline etc. 
Xa,b 
X 
X 
X 
X 
X 
SaO2/ FiO2 ratio 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Pa02 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ABG 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
Hospital status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ICU status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Non-invasive 
ventilation status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Invasive ventilation 
status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ConMed Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
33 

Confidential 
 
 
 
 
 
 
Adverse Event 
Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
a= measurement should be recorded or performed at hours 0, 2 and 12 
b= Day 1 hour 0 samples/procedure must be collected/recorded before study drug is administered. 
c= only perform if patient is of childbearing potential 
d= must be collected 15 minutes prior to administration of study medication 
e= Day 28 and Day 60 are telephone follow ups 
X= Study Procedure A= Standard of Care 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
34 

APPENDIX B- Protocol Clarification Memo 
Confidential 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
35 

36 
Confidential 
 
 
APPENDIX C: Protocol Clarification Memo- Procedure Window Allowance 
 
 
 
 

37 
Confidential 
 
 
 
 
 
 

38 
Confidential 
 
 
15. 
Summary of Changes: 
 
SECTION 
DESCRIPTION of CHANGES 
1.0 
Protocol Version Date 
2.0 STUDY SUMMARY 
Study Design-D28 and D60 telephone follow up 
2.0 STUDY SUMMARY 
Treatment Arms-added independent pharmacist 
2.0 STUDY SUMMARY 
Sample Size/Statistical Considerations-added PaO2 
2.0 STUDY SUMMARY 
Primary Biochemical Efficacy Variables-added surrogate marker info 
2.0 STUDY SUMMARY 
Secondary Biochemical Efficacy Variables-added dose proportionality info 
4.1 Primary Endpoints (objectives) 
Primary Endpoints Objectives-added association of plasma amino acids 
5.1 Study Description 
Study Description-added telephone follow up for D28 and D60 
6.4 Informed Consent 
Informed Consent-added PK Sample explanatory analysis 
7.1.5 Clinical Laboratory Tests 
Clinical Laboratory Tests-added timepoints 
7.2.2 On Study (days 0-DC) 
Assessments 
On Study Assessments-added timepoints 
10.2 General Statistical Approach 
Clinical Outcomes-added D28 and D60 
9.3 Pre-Existing Conditions 
Pre-Existing Conditions-added Sa02/Fi02 
10.2 General Statistical Approach 
General Statistical Approach-added multiplicity adjustment approach 
10.4Sample Size 
Data and Safety Monitoring Plan-added independent PK evaluation info 
APPENDIX A: Schedule of Events 
Schedule of Events-added footnotes to D28 and D60 
APPENDIX A: Schedule of Events 
Schedule of Events-added Pa02 
APPENDIX A: Schedule of Events 
Schedule of Events-added D-dimer test and CRP 
APPENDIX A: Schedule of Events 
Schedule of Events-added Conmed and AE review 
 
 
Changes from Protocol Version 2.0 dated 08 September 2021 to Version 3.0 Dated 11 June 2021 
Section 
Description of Changes 
1.0 
Protocol Version and date were updated to reflect Version 3.0 dated 11 June 2021 
and added the IND number. 
Table of Contents 
The table of contents was updated to reflect the addition of Appendix B and C. 
2.0 Study Summary 
Updated Section “Sample Size/Statistical Considerations:” to reflect that up to 66 
participants can be enrolled. 
5.1 Study Description 
The wording is updated to reflect. Up to 66 participants can be enrolled. 
6.1 Participant Enrollment 
The wording is updated to reflect up to 66 participants can be enrolled. 
10.4 Sample Size 
The wording is updated to reflect up to 66 participants can be enrolled. The 
wording was also updated to account for the additional patients in the analysis 
14.0 Investigator Signature Page 
Updated the protocol version and date. 
APPENDIX 
Protocol Clarification Memo: Requirement of Arterial Blood Gas (ABG) 
Timepoint was added (APPENDIX B), and the Protocol Clarification Memo- 
Procedure Window Allowance was added (APPENDIX C)