Conditions: Infant,Premature Bronchopulmonary Dysplasia Pulmonary Hypertension
Keywords: L-citrulline Nitric oxide
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 16
Healthy volunteers: no

Brief summary:
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects up to 35% of very low birth weight infants (VLBW \< 1500 g). Based on the current numbers of VLBW infants born annually in the U.S., between 5,000-10,000 neonates will develop BPD each year. It is estimated that 8-42% of infants with BPD will develop pulmonary hypertension (PH). Moreover, it has been known since the 1980's that echocardiographic evidence of PH in infants with BPD is associated with up to 40% mortality.

Treatment options to ameliorate PH in infants with BPD (BPD-PH) are limited. There have been no randomized clinical trials of any therapy in infants with BPD-PH. The standard care for the management of BPD-PH is to attempt to resolve the underlying lung disorder and the judicious use of oxygen as a potent pulmonary vasodilator. Using this management approach, which has not changed since the 1980's, the survival rates for infants with BPD-PH in the 2000's has been reported to be 64% at 6 months and 53% at 2 years after diagnosis of PH. The lack of improvement in outcomes for the past 3 decades has led to the widespread agreement that novel and effective therapies are desperately needed for infants with BPD-PH.

The goal is to develop oral L-citrulline clinically for the treatment of pediatric pulmonary hypertension associated with bronchopulmonary dysplasia (BPD-PH); before pursuing a large scale treatment trial, pharmacokinetic (PK) dose-finding, tolerability studies in patients at high risk of developing BPD-PH are warranted.

The hypothesis is that oral L-citrulline will be well tolerated, without significant adverse effects in infants at high risk of developing pulmonary hypertension (PH) associated with BPD. The investigators propose to first characterize the PK profile of oral L-citrulline in order to define an appropriate dose range and treatment interval for infants at high risk of developing BPD-PH. Then using the doses and intervals generated by the PK profile, with a maximum dose of 3 g/kg/d, the investigators propose to evaluate the tolerability and ability to achieve the target study drug level (100-150 micromolar) in babies treated for 72 hours with oral L-citrulline. These studies will provide the data needed to design a full-scale randomized multi-center trial to evaluate the efficacy of oral L-citrulline therapy to ameliorate BPD-PH in human infants, a patient population that has a desperate need of new therapies.

Detailed description:

Arms:
arm 1: Participants will be enrolled into the two groups, Group 1 (which will consist of 10 participants) and Group 2 (which will consist of 8 participants) in an alternating basis. Both Group 1 and Group 2 participants will receive a single, 150 mg/kg dose of oral L-citrulline. Population PKs will be done for both groups, at up to 3 time points.

Multiple interim time points and following completion of enrollment into Groups 1 and 2, data analysis will be done and results reviewed by the data safety monitoring board (DSMB). After the DSMB review is complete, enrollment into Group 3 will begin. arm 2: To evaluate the tolerability and ability to achieve target trough L-citrulline levels of 100-150 µM, an additional group of 18 infants (group 3) will be given oral L-citrulline doses at intervals over a total of 72 hours. If the participant is not nipple feeding, the dose will be delivered via the participant's indwelling gavage feeding tube. The dose and interval of L-citrulline will be based on results from the studies that assess pharmacokinetic parameters using a maximum daily dose of 3 g/kg/d. Blood draws for PKs will be done at baseline and prior to last dose of L-citrulline. Urine will be collected to measure nitric oxide metabolites.

Interventions: intervention 1: L-Citrulline
Intervention descriptions: intervention 1: The L-citrulline will be procured in powder form and will be solubilized in sterile water to achieve a concentration of 50 mg/ml. Therefore, 3 ml/kg of the solubilized L-citrulline (50 mg/ml) will be administered per dose for the single-dose groups and the dose administered to the steady-state group will be determined from results from the single-dose studies.

Protocol document:
Prot_SAP_000.pdf: 
Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
                          Page 1 of 22       
             
 
PROTOCOL 
 
Pharmacokinetics of oral L-citrulline in infants at high risk of developing pulmonary 
hypertension associated with Bronchopulmonary Dysplasia 
 
 
 
 
Principal Investigator: Candice D. Fike, M.D. 
Internal Staff and  Sub-Investigators: Kevin Watt, M.D. 
Alfred Balch MA, PhD 
Carrie Rau, RN 
Kathryn Woodbury, RN 
Trisha Marchant 
Kathie Coleman 
Anastasiya Mankouski, M.D. 
Matthew Douglass, M.D. 
 
 
External Sub-Investigators: 
Judy Aschner, M.D. 
Catherine Sherwin, M.D. 
Angela Birnbaum, Ph.D. 
 
 
 
 
 
US IND Number: 134349 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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This confidential document is the property of Dr. Candice D. Fike,. No unpublished information 
contained herein may be disclosed without prior written approval from Dr. Candice D. Fike. 
Access to this document must be restricted to relevant parties. 
 
TABLE OF CONTENTS 
List of abbreviations.................................................................................................................... 3 
1. Background and Introduction………………………….…………...……………...………....3 
1.1  
Introduction……………………………............………………………..............3 
1.2 
Nitric oxide metabolism………………………..…………….……………..…..4 
1.3  
Nitric oxide and Pulmonary Hypertension…...……...………..…………….......4 
1.4  
Citrulline and Nitric Oxide……………………………...….....……...…….…...5 
1.5       Oral L-citrulline supplementation and previous human and animal studies…....5 
1.6   
Hypothesis, what is being proposed, and value provided……...…………….....6 
2. Purpose and Objectives……………..………………………………....……………….…….7 
2.1 Specific Goals of this Proposal………………………………......….……………...7 
2.2 Objectives…………………..…………………….…………….……...…...…..…...7 
2.2.1 Primary Objectives………………………………...….…………..…........7 
2.2.2 Secondary Objective…………………………………..……………….….7 
3. Study Population……………… ………………………………………………………..……7 
 3.1 Age of Participants…………………………………………………...………..……7 
 3.2 Sample Size……………………………….…………...…………..……………..…7 
3.3 Inclusion Criteria…………………………………………….……………….…..….7 
3.4 Exclusion Criteria…………………………………………………………………....8 
4. Design…………………...…………………………………………………….…………..…..8 
5. Study Procedures………………………………………………………………………….......8 
5.1 Recruitment/Participant Identification Process……………………………….…..…8 
5.2 Informed Consent……………………………………………………………...….....8 
5.2.1 Description of location where consent will be obtained…………..............8 
5.2.2 Description of the consent process(es), including the timing of consent.....8 
5.3 Procedures…………………………………………….……………………………..9 
5.3.1 Single-dose pharmacokinetic studies …………………………………......9 
5.3.2 Steady-state pharmacokinetic studies…………………………………......9 
5.3.3 Blood samples………………………………………………………...…...9 
5.3.4 Urine samples……………………………………………………………...9 
5.3.5 Additional data………………………………………………………..…...9 
5.3.6 Participant monitoring………………………………………………..........9 
6. Statistical Methods, Data Analysis and Interpretation……………………………..….….. ..10 
 
6.1 Data and Safety Monitoring Board (DSMB)………………………………….…....11 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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7. Regulatory Ethics Compliance 
 
7.1 Investigator Responsibilities……………………………………………………......12 
 
7.2 IRB and FDA Submissions……………………………………………………...….13 
7.3 Informed Consent Form………………………………………………………….....13 
7.4 Adverse Event Reporting…………………………………………………..……...14  
7.6 Deviation Reporting…………………………………………………….………....15 
8.  Study Records Retention…………………………………………………………………...17 
9.  References……………………………………………………………………..……….…..17 
 
 
LIST OF ABBREVIATIONS 
ASL                           arginine succinate lyase 
ASS                           argininosuccinate synthetase 
BPD                           bronchopulmonary dysplasia 
eNOS                         endothelial nitric oxide synthase 
FDA  
           Food and Drug Administration 
ICF            
           Informed Consent Form 
iNO                            Inhaled nitric oxide 
iNOS                          inducible nitric oxide synthase 
IRB 
 
           Institutional Review Board 
MELAS                     mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes 
NO                             nitric oxide 
NOS                           nitric oxide synthase 
nNOS                         neuronal nitric oxide synthase 
PAECs                       Pulmonary arterial endothelial cells 
PH                              pulmonary hypertension 
VLBW                       very low birth weight 
 
1.0       Background and Introduction 
 
1.1  
Introduction 
 
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects up to 35% of very 
low birth weight infants (VLBW < 1500 g) (1, 2). Based on the current numbers of VLBW 
infants born annually in the U.S., between 5,000-10,000 neonates will develop BPD each year 
(3).  It is estimated that 8-42% of infants with BPD will develop pulmonary hypertension 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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(PH)(4-8). Moreover, it has been known since the 1980’s that echocardiographic evidence of 
PH in infants with BPD is associated with up to 40% mortality (9, 10).   
 
Treatment options to ameliorate PH in infants with BPD (BPD-PH) are limited. There have 
been no randomized clinical trials of any therapy in infants with BPD-PH.  The standard care 
for the management of BPD-PH is to attempt to resolve the underlying lung disorder and the 
judicious use of oxygen as a potent pulmonary vasodilator (11, 12). Using this management 
approach, which has not changed since the 1980’s, the survival rates for infants with BPD-PH 
in the 2000’s has been reported to be 64% at 6 months and 53% at 2 years after diagnosis of 
PH (13).  The lack of improvement in outcomes for the past 3 decades has led to the 
widespread agreement that novel and effective therapies are desperately needed for infants 
with BPD-PH.   
 
 
1.2 Nitric oxide metabolism 
 
Nitric oxide (NO) is a potent vasodilator synthesized from the amino acid substrate, L-
arginine, by one of three NO synthase (NOS) isoforms: neuronal NOS (nNOS), inducible NOS 
( iNOS), and endothelial NOS (eNOS).  L-citrulline provides an intracellular source for L-
arginine via a two-step biosynthetic pathway, involving the co-substrate, aspartate and the 
enzymes argininosuccinate synthetase (ASS) and arginine succinate lyase (ASL).  Through 
this recycling pathway, L-citrulline serves as both a substrate for arginine and as the end 
product when arginine is converted to NO by NOS.  Thus, L-citrulline provides an approach to 
deliver bioavailable L-arginine to synthesize NO.   A simplified diagram of the L-citrulline-L-
arginine-NO pathway is shown below:   
 
    
1.3Nitric oxide and Pulmonary Hypertension 
 
Evidence that impaired NO signaling plays a pivotal role in the pathobiology of PH in humans 
is now well established (14-17). There is evidence that inhaled NO (iNO) may be beneficial 
for the treatment of infants with BPD-PH.  In particular, iNO has been shown to function as an 
acute pulmonary vasodilator in infants with BPD-PH (18, 19).  There is also some data 
showing clinical improvement when iNO is used long term, for 8-90 days (20). Use of iNO is 
expensive and cumbersome especially when used long-term.  The responsiveness to iNO 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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suggests that other agents that improve NO bioavailability might have beneficial effects in 
infants with BPD-PH.  
 
Administering L-arginine, the amino acid substrate for NO synthesis, has been studied as a 
therapy to improve NO signaling and ameliorate PH.  We, and other investigators, have shown 
that limited bioavailability of L-arginine contributes to vascular dysfunction in PH (21-23).  
Unfortunately, results with L-arginine supplementation have not been consistent, with some 
studies showing no benefit from either acute (24) or prolonged (25) L-arginine 
supplementation in animals or humans with PH.  In addition, the feasibility and logic of 
chronic oral L-arginine supplementation are questionable, because the presence of arginase in 
gut bacteria, intestinal epithelial cells, and hepatocytes dictates that orally administered L-
arginine will be largely catabolized to ornithine and urea.  This catabolic loss of L-arginine 
necessitates the administration of massive L-arginine doses to achieve increases in circulating 
levels that are therapeutically effective (26).  These large doses are often poorly tolerated and 
patient compliance can be difficult to maintain (27).  Thus, alternative means of increasing NO 
production merit investigation.   
 
1.4  
Citrulline and Nitric Oxide 
 
Unlike arginine, L-citrulline is not subject to presystemic breakdown in the gut.  Instead, L-
citrulline is produced and released into the circulation by enterocytes.  Adult humans who 
receive oral L-citrulline achieve dramatic elevations in circulating L-citrulline, with minimal 
urinary losses (28-30).  The bioavailability of oral L-citrulline has also been demonstrated in 
newborn piglets (31).    
 
Pulmonary arterial endothelial cells (PAECs) express ASS, ASL, and eNOS and can, 
therefore, use L-citrulline to produce NO (32).  In studies with piglet PAECs, we showed that 
L-citrulline treatment increases NO production in PAECs cultured under both normoxic and 
hypoxic conditions (33).  Moreover, in our newborn piglet model of chronic hypoxia-induced 
pulmonary hypertension, L-citrulline supplementation was shown to increase plasma and 
tissue concentrations of citrulline, improve pulmonary vascular NO production, and ameliorate 
the development of pulmonary hypertension (31).   Thus, the presence of the enzymes needed 
to produce L-arginine-NO from L-citrulline in PAECs affords the opportunity to use L-
citrulline supplementation to increase NO bioavailability in the pulmonary vasculature and 
ameliorate pulmonary hypertension. Additional rationale to evaluate the impact on pulmonary 
hypertension from oral L-citrulline supplementation is provided by a recent study showing that 
plasma L-citrulline levels were lower in infants with BPD-PH than in infants with BPD and no 
PH (34).  
 
1.5       Oral L-citrulline supplementation and previous human and animal studies 
 
Long-term treatment options are needed for infants and children with chronic conditions, such 
as those with BPD.  Intravenous therapies are difficult to maintain and are fraught with 
complications when used long-term. Infants and children with certain types of urea cycle 
defects receive oral L-citrulline replacement therapy for decades and show no evidence of 
toxicity from its use (35, 36). A study recently performed in children, ages 4 to 16 years, with 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) 
syndrome reported 5 fold increases in plasma L-citrulline (increase from 21 to 156 µM) and 3 
fold increases in NO synthesis with the administration of oral L-citrulline (dose of 10 g/m2 
body surface area/day if weight >20 kg and 500 mg/kg/dose if weight < 20 kg) every 4 hours 
for 48 hours (37).  Of note, no adverse effects were found indicating that oral L-citrulline 
doses up to 3 g/kg/d (500 mg/kg/dose given every 4 hours) are safe and tolerated in infants and 
children (37).  However, the pharmacokinetics of oral L-citrulline has not been evaluated in 
infants or children.  
 
The role of oral L-citrulline supplementation has been under studied in infants and children 
with cardiovascular disease.  A pilot phase two clinical trial in children with sickle cell disease 
showed that  oral L-citrulline given twice daily in doses of 0.1 g/kg decreased vascular 
complications and improved the symptoms of well-being (38).  Supplementation with oral L-
citrulline using perioperative doses of 1.9 g/m2 (approximately 0.13 g/kg given every 12 hours 
for a total of 5 doses), was found to be well tolerated and safe in children, ages 0.3-29 months,  
undergoing cardiopulmonary bypass at risk for development of postoperative pulmonary 
hypertension (39).  Notably, postoperative pulmonary hypertension did not develop in those 
children who had naturally elevated levels of citrulline or who achieved plasma citrulline 
levels greater than 37 µM with the oral L-citrulline supplementation, reflecting an approx. 50-
100% increase from their basal L-citrulline levels.  In our newborn piglet model we found that 
the development of chronic hypoxia-induced pulmonary hypertension was ameliorated in 
piglets treated with an oral dosing strategy that achieved increases in trough plasma L-
citrulline that were 50-100% above baseline levels (total daily dosing of 0.26-0.52 g/kg/d) (31, 
40).  However, piglets treated with a higher oral L-citrulline dose strategy (total daily dosing 
of 1-1.5 g/kg/d) achieved a 3-5 fold increase in trough plasma L-citrulline and had a more 
marked reduction in chronic hypoxia-induced pulmonary hypertension than did piglets treated 
with the lower dose strategy (31).  These findings in our newborn pig model provide proof of 
concept to test the efficacy of using chronic oral L-citrulline to treat human infants at high risk 
of developing pulmonary hypertension in conditions that are associated with intermittent or 
persistent hypoxia, such as BPD.  In addition, our pre-clinical studies in newborn piglets and 
the clinical study in infants and children undergoing cardiac bypass surgery suggest targeting a 
circulating plasma L-citrulline trough that is at least 100%, and perhaps 3-5 fold, above basal 
levels.  Based on published plasma L-citrulline levels of 13-70 µM in infants and children 
(41), the dose and interval of L-citrulline treatment should target trough L-citrulline plasma 
levels of approx. 100-150 µM.   Moreover, total daily doses of 3 g/kg/d (500 mg/kg/dose given 
every 4 hours) were recently shown to be safe and tolerated in infants and children (37).         
 
Pharmacokinetic dose-finding, tolerability studies of oral L-citrulline in infants at high risk of 
developing BPD-PH are needed to determine appropriate dosing strategies (dosing ranges and 
treatment intervals) before use in large scale efficacy treatment trials for BPD-PH.  
 
Of note, the first phase of this study has now been completed and the data reveals basal plasma 
L-citrulline levels of 9-36 µM, with a mean of 28 +/- 8 µM for infants in this study (infants at 
high risk of developing BPD-PH).  Therefore, for infants at high risk of developing BPD-PH, 
the appropriate plasma L-citrulline trough to target is a level that is 50-100% above basal 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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levels of approx. 9-36 µM.  Therefore, the trough L-citrulline plasma level to target for 
patients in this study has been revised from approx. 100-150 µM to approx. 50-80 µM.   
 
1.6       Hypothesis, what is being proposed, and value provided 
 
The hypothesis is that oral L-citrulline will be well tolerated, without significant adverse 
effects in infants at high risk of developing pulmonary hypertension (PH) associated with 
BPD.  We propose to first characterize the PK profile of oral L-citrulline in order to define an 
appropriate dose range and treatment interval for infants at high risk of developing BPD-PH.  
Then using the doses and intervals generated by the PK profile, with a maximum dose of 3 
g/kg/d (37), we propose to evaluate the tolerability and ability to achieve the target trough L-
citrulline plasma level of approx.50-80 µM in patients at high risk of developing BPD-PH 
treated for 72 hours with oral L-citrulline.  These studies will provide the data needed to 
design a full-scale randomized multi-center trial to evaluate the efficacy of oral L-citrulline 
therapy to ameliorate BPD-PH in human infants, a patient population that has a desperate need 
of new therapies. 
 
 
 
2.          Purpose and Objectives 
 
 2.1        Specific Goals of this Proposal: 
The goal is to develop oral L-citrulline clinically for the treatment of pediatric pulmonary 
hypertension associated with bronchopulmonary dysplasia (BPD-PH); before pursuing a large 
scale treatment trial, pharmacokinetic dose-finding, tolerability studies in patients at high risk 
of developing BPD-PH are warranted. 
 2.2         Objectives: 
      2.2.1       Primary Objectives 
• 
To characterize the pharmacokinetic profile of oral L-citrulline in infants at high risk of 
developing BPD-PH in order to define an appropriate dose range (dose and treatment 
interval that achieves trough L-citrulline levels of approx. 50-80 µM) 
• 
To evaluate the tolerability and ability to achieve the target L-citrulline plasma level of 
approx.50-80  µM in patients at high risk of developing BPD-PH treated for 72 hours 
with oral L-citrulline 
 
      2.2.2        Secondary Objective 
• To assess the effect of L-citrulline administration on urinary nitric oxide metabolite 
(nitrite/nitrate) levels in patients at high risk of developing BPD-PH 
 
3.          Study Population 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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3.1        Age of Participants:  
      32 +/- 1 week postmenstrual age 
 
3.2        Sample Size: 
 28 participants: 10* participants for pharmacokinetic profile characterization and 18 
participants for evaluating the tolerability and ability to achieve target trough plasma 
levels 
 
• Group 1: 5 participants 
• Group 2:   5 participants 
• Group 3: 18 participants 
* Note that the total participant size for Groups 1 and 2 has been reduced from the originally 
proposed total of 18 participants to 10 participants.  This change was made based on the single 
patient PK analysis on the 10 enrolled participants.  This analysis was assessed to be sufficient 
to perform the population PK modeling needed to design the next phase of the study. Both the 
DSMB and the FDA approved the change in participant size.     
3.3      Inclusion Criteria: 
 
1. Infants born prematurely at <28 weeks gestation requiring invasive (mechanical 
ventilation) or non-invasive positive pressure support (nasal continuous positive 
airway pressure, high flow nasal cannula >1 lpm) at 32 +/- 1 weeks postmenstrual 
age 
2. Tolerating at least one-half of full volume oral/gavage tube feedings (using 120 
ml/kg/d as full volume oral/gavage tube feedings) 
3. The continuous need for some form of respiratory support (supplemental oxygen 
and/or flow, or mechanical ventilation) for the prior 14 days 
4. Hemoglobin  ≥ 10 mg/dL 
 
 
3.4       Exclusion Criteria: 
 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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1. Known major fetal anomaly or chromosomal aneupleudy 
2. Clinical evidence of congenital heart disease (except patent ductus arteriosus, atrial 
septal defect, or ventricular septal defect [PDA, ASD, VSD]) 
3. Urine output < 1 ml/kg/hr 
4. History of or known to have liver failure 
5. History of or known to have necrotizing enterocolitis 
6. History of or known to have significant feeding intolerance beyond the first week of 
life  
7. Presence of any acute illness defined by fever >100.4F, vomiting, or diarrhea 
8. Hemoglobin < 10 mg/dL 
 
9. NICU cases determined to be futile (anticipated death prior to hospital discharge) 
10. Multiple births 
 
4.         Design  
 
Study to Assess Pharmacokinetics of L-Citrulline in infants at high risk of developing 
pulmonary hypertension associated with Bronchopulmonary Dysplasia   
 
 
5.         Study Procedures 
  
5.1     Recruitment/Participant Identification Process: 
Participants will be recruited while they are hospitalized at the Neonatal Intensive Care      
Nursery either at Primary Children's Hospital or University of Utah Hospital or 
Intermountain Medical Center Hospital. The study team will review patient medical 
records to determine eligibility. 
 
5.2       Informed Consent: 
5.2.1  Description of location(s) where consent will be obtained:  
University of Utah Hospital Neonatal Intensive Care Nursery  
Primary Children’s Hospital Neonatal Intensive Care Nursery 
Intermountain Medical Center Neonatal Intensive Care Nursery 
 
5.2.2  Description of the consent process(es), including the timing of consent:  
Parents/guardians with children fitting the eligibility criteria will be approached by the 
PI or study coordinator and introduced to the study.  During that time the study 
procedures and participant involvement will be thoroughly discussed, questions 
encouraged, and full understanding of the protocol ensured.  Parents/guardians will be 
encouraged to take the consent form home to discuss with others before making any 
decision to sign the consent form.  Consent from the parent/guardian and assent if 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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applicable will be obtained before any L-citrulline wil be administered and before any 
sample will be collected.   
 
5.3 Procedures: 
 
Consent from the parent/guardian will be obtained. 
 
The study drug, L-citrulline, will be provided by Asklepion Pharmaceuticals.  
 
5.3.1  Single-dose pharmacokinetic studies:  Participants will be enrolled into the two 
groups, Group 1 (which will consist of 5 participants) and Group 2 (which will consist 
of 5 participants) in an alternating basis. Both Group 1 and Group 2 participants will 
receive a single, 150 mg/kg dose of oral L-citrulline. The L-citrulline will be provided 
by Asklepion Pharmaceuticals.  The L-citrulline will be procured in powder form and 
will be solubilized in sterile water to achieve a concentration of 50 mg/ml. Therefore, 3 
ml/kg of the solubilized L-citrulline (50 mg/ml) will be administered to the participant.  
  
To assess pharmacokinetic parameters, the study team will collect three, 0.3-1.0 ml 
blood draws (whole blood) from infants who receive the single dose of 150 mg/kg oral 
L-citrulline. Blood sampling volumes will be limited to a maximum of 1 ml/kg total 
over a 24-48 hour period. If the participant is not nipple feeding, the dose will be 
delivered via the participant’s indwelling gavage feeding tube.   
• For Group 1: one blood draw will be pre-dose and 2 blood draws will be post-dose. 
The pre-dose blood draw will occur 24-48 hours prior to administering the dose of 
oral L-citrulline and the two post-dose draws will occur at 1 hours (+/- 10 minutes) 
and 2.5 hours (+/- 10 minutes) post-dose.   
• For group 2: one blood draw will be pre-dose and 2 blood draws will be post-dose. 
The pre-dose blood draw will occur 24-48 hours prior to administering the dose of 
oral L-citrulline and the two post-dose draws will occur at 15 minutes (+/- 10 
minutes) and 3 hours (+/- 10 minutes) post-dose 
These sampling times are designed to estimate the population PK parameters of a 
typical infant using scaling and population PK analysis of available oral PK profiles in 
adults (28).   
The single dose pharmacokinetic studies have been completed.  PK data from 10 
subjects (5 in group 1 and 5 in group 2) were analyzed and assessed as being sufficient 
to provide the information needed to design the next phase, the steady state 
pharmacokinetic studies.   
 
5.3.2      Steady-state pharmacokinetic studies:  
To evaluate the tolerability and ability to achieve target trough L-citrulline levels of 
50-80 µM, an additional group of 18 infants (group 3) will be given oral L-citrulline 
doses at intervals over a total of 72 hours. The L-citrulline will be provided by 
Asklepion Pharmaceuticals.  If the participant is not nipple feeding, the dose will be 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
Project: L-citrulline   
                                                  Date: 23 Feb 2022                             
Status: FINAL 
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delivered via the participant’s indwelling gavage feeding tube.   The dose and interval 
of L-citrulline are based on results from the studies that assessed pharmacokinetic 
parameters using a maximum daily dose of 3 g/kg/d. The choice of the maximum 
daily dose is based on a recent study that showed good tolerability and reported no 
adverse effects in children with mitochondrial encephalomyopathy, lactic acidosis and 
stroke-like episodes (MELAS syndrome) treated with oral l-citrulline at a dose of 3 
g/kg/d (500 mg/kg/dose given every 4 hours) (37).   
 
Based on results from the single dose pharmacokinetic studies, the infants in group 3 
will be given oral L-citrulline doses of 60 mg/kg every 6 hours +/- 30 minutes (4 times 
a day) for 72 hours (total of 12 doses).  This is a total daily dose of 240 mg/kg/day.   
 
The L-citrulline will be provided by Asklepion Pharmaceuticals.  The L-citrulline will 
be procured in powder form and will be solubilized in sterile water to achieve a 
concentration of 50 mg/ml.  Therefore, 1.2 ml/kg of solubilized L-citrulline (50 
mg/ml) will be administered to the participant every 6 hours +/- 30 minutes (4 times a 
day) for 72 hours.  This is a total daily volume of 4.8 ml/kg/day.   
 
For group 3, the study will collect two, 0.3-1.0 ml blood draws (whole blood) and two, 
1 ml urine samples.  Blood sampling volumes will be limited to a maximum of 1 
ml/kg total over a 24 hour period. One blood draw will occur between 10 minutes and 
6 hours prior to administering the first dose of oral L-citrulline and the second blood 
draw will occur 10 to 30 minutes prior to administering the last dose of oral L-
citrulline.  These sampling times are designed to assess baseline and trough L-
citrulline plasma levels.  One urine sample will be collected on the day prior to 
administering the first dose of oral L-citrulline and the second urine sample will be 
collected between 4 and 8 hours following the last dose of oral L-citrulline. These 
urine collection times are designed to assess baseline levels of nitric oxide metabolites 
(nitrite/nitrate) in the urine and assess whether there is an increase in levels of nitric 
oxide metabolites in the urine in response to 72 hours of L-citrulline dosing.   
 
5.3.3     Blood Samples 
Blood samples will be processed  and the plasma harvested into labeled tubes which 
are stored in a designated freezer section at -80 degrees C, where they will be 
collected by study personnel.   For each of the blood samples we will test for plasma 
citrulline and arginine levels.  Analysis will be done by ARUP.   
 
5.3.4     Urine samples 
Each 1 ml urine samples will be aliquoted into 2 separate tubes (0.5 ml urine per 
tube), then stored in a designated freezer section at -80 degrees C, where they will be 
collected by study personnel.  One of the aliquoted urine samples will be tested for 
NO metabolites (nitrites/nitrates) and the second aliquoted urine sample will be tested 
for Cr (to normalize the sample).  Urine nitrite/nitrate analysis will be done in Dr. 
Fike’s laboratory.  Urine Cr analysis will be done by ARUP.      
 
5.3.5     Additional data 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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Additional data collected will include demographics; vital signs including weight, 
length, heart rate, respiratory rate, temperature, and blood pressure; dietary/nutrition 
information; concomitant medications.  For the Group 1 and Group 2 participants 
(single dose PK studies), the blood pressure will be monitored at least 3 times after the 
participant receives the study drug.  For the Group 3 participants (steady state PK 
studies), the blood pressure will be monitored at least 4 times daily for 4 days (72 
hours when the participant is receiving the study drug and 12 hours after last dose). 
The heart rate, respiratory rate, temperature, and blood pressure will be monitored for 
12 hours following the last dose of L-citrulline.   
 
5.3.6     Participant monitoring 
All participants will be monitored for clinical evidence of necrotizing enterocolitis, 
gastrointestinal bleeding, sepsis, feeding intolerance (including vomiting and 
diarrhea), electrolyte disturbances, hemodynamics, alteration of hematological 
parameters and death.  Any electrolytes, hematologic laboratories, and eye 
examinations that are ordered as part of standard of care by the participants clinical 
care team will be monitored.  Hypotensive subjects (defined as a decrease in mean 
arterial blood pressure of more than 25% baseline) will be treated with 
pharmacological/volume supplementation interventions at the discretion of the 
attending physician.  Treatment unresponsive hypotensive subjects will be withdrawn 
from the study.  Subjects who develop significant feeding intolerance will be 
withdrawn from the study.  Infants who are made NPO for a time period long enough 
that they miss any doses of L-citrulline will be withdrawn from the study. 
 
 
 
6.        Statistical Methods, Data Analysis and Interpretation 
 
After the first 3 participants are enrolled in the single-dose PK study (3 participants enrolled in 
either Group 1 or Group 2), and after the first 3 participants are enrolled in the steady-state 
pharmacokinetic study (3 participants enrolled in Group 3), an initial PK analysis will be 
performed and the data will be reviewed by the DSMB to assess for safety to continue the 
single dose PK study and to continue the steady-state pharmacokinetic study.  
 
Throughout the study, participants who are withdrawn from treatment due to adverse events 
will be replaced for PK analysis.   
 
Data collected from this study will be examined to ensure that all participants in the analysis 
meet inclusion criteria. Summary statistical analysis will be conducted to describe participant 
demographics, citrulline dosing, and participant PK profiles. Outliers in the measured citrulline 
levels that may influence the estimation of PK parameters will be evaluated. A population 
model will be developed to describe the citrulline PK and population variability.  
 
The non-linear mixed effect modeling software NONMEM® version 7.3.0 (ICON 
Development Solutions, Ellicott City, MD, USA) will be used to develop models. Perl-speaks-


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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NONMEM (PsN) 4.4.0, Pirana 2.9.0, and R packages (version 3.2.1) will be used to facilitate, 
evaluate, and visualize the model development.  
The model development process would include two major steps: base model structure 
evaluation and covariate screening. The development of a base structural model will include 
the evaluation of compartment numbers, baseline effects, absorption and elimination kinetics. 
The stochastic models describing between-subject variability in PK parameters and intra-
subject variability in observations will be also examined.  
Potential covariates that are predictive of citrulline PK variability will be screened using the 
stepwise covariate model (scm) building tool of PsN. The continuous covariates to be 
examined will include, but not be limited to, body weight, length, postnatal age, gestational 
age, etc. Categorical covariates to be examined will include, but not be limited to, sex, race, 
ethnicity, and concomitant medication (yes/no). The predefined parameter-covariate 
association for continuous covariates will be included as a power relation, while categorical 
covariates will be linearly included. Both stepwise forward selection and backward elimination 
will be incorporated for covariate searching. Changes in OFV will be considered significant at 
p<0.05 (χ2-distribution, 1 df, ΔOFV > 3.84) during forward selection, and p<0.01 (1 df, ΔOFV 
>6.63) during backward elimination. In addition to the aforementioned statistical criteria, the 
following standard diagnostic plots will also be used to visually assess the final covariate 
model fit: observed concentrations versus population- and individual-predicted concentrations, 
conditional weighted residuals versus time and population-predicted concentrations. To assess 
the model stability, a nonparametric bootstrap analysis will be conducted. The visual predictive 
check (VPC) will be performed to further evaluate the final model performance. 
Once the final population PK model is determined, an optimal dosing plan will be developed 
using the model to achieve target plasma trough concentrations of 50-80 µM in patients at high 
risk of developing BPD-PH. Statistical comparison between actual trough concentrations and 
model predicted concentrations will be conducted. Model prediction capability will be 
evaluated and refinement will be performed. 
The final population PK model has been determined and the optimal dosing plan has been 
developed to target plasma trough concentrations of 50-80 µM (trough plasma concentrations 
that are based on data from the single dose PK study and that are appropriate for infants in this 
study).  The optimal dosing plan is to give oral L-citrulline doses of 60 mg/kg every 6 hours 
+/- 30 minutes (4 times a day) for 72 hours (total of 12 doses).  This is a total daily dose of 240 
mg/kg/day.   
6.1     Data and Safety Monitoring Board 
This study will use a Data and Safety Monitoring Board (DSMB). The plan is to perform 
an initial PK analysis after the first 3 participants are enrolled in the single dose PK arm 
and also after the first 3 participants are enrolled in the steady-state pharmacokinetic arm.  
The data will be reviewed by the DSMB to assess for safety to continue the single-dose 
pharmacokinetic study and to continue the steady-state pharmacokinetic study.   
 


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7.      Protection of Human Subjects 
 
 
7.1     Investigator Responsibilities 
The investigator is responsible for ensuring that the study is performed in accordance 
with the protocol, current ICH guidelines on Good Clinical Practice (GCP), and 
applicable regulatory and country-specific requirements. 
 
Good Clinical Practice is an international ethical and scientific quality standard for 
designing, conducting, recording, and reporting studies that involve the participation of 
human subjects. 
 
Compliance with this standard provides public assurance that the rights, safety, and well-
being of study subjects are protected, consistent with the principles that originated in the 
Declaration of Helsinki, and that the study data are credible. 
 
7.2     Institutional Review Board and FDA Submissions 
 
The protocol, informed consent form(s), recruitment materials, and all participant 
materials will be submitted to the IRB for review and approval.  Approval of both the 
protocol and the consent form must be obtained before any participant is enrolled. Any 
amendment to the protocol will require review and approval by the IRB before the 
changes are implemented to the study. All changes to the consent form will be IRB 
approved; a determination will be made regarding whether previously consented 
participants need to be re-consented. 
 
During the study, the investigator will send the following documents and updates to the 
FDA for their review and the IRB for their review and approval, where appropriate: 
• Protocol amendments (excluding the ones that are purely administrative, with no 
consequences for subjects, data, or study conduct). 
• Revision(s) to ICF and any other written materials to be provided to subjects. 
• Summaries of the status of the study at intervals stipulated in guidelines of the IRB 
and the FDA (at least annually). 
• Reports of AEs that are serious, unlisted/unexpected, and associated with the study 
drug. 
• New information that may adversely affect the safety of the subjects or the conduct of 
the study. 
• Deviations from or changes to the protocol to eliminate immediate hazards to the 
subjects. 
• Report of deaths of subjects under the investigator’s care. 
• Notification if a new investigator is responsible for the study at the site. 
• At least once a year, the IRB will be asked to review and reapprove this study, where 
required.  An annual report will be submitted to the FDA.   
• At the end of the study, the investigator will notify the IRB and the FDA about the 
study completion  


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7.3     Informed Consent Form 
 
Written permission from parents or legal guardians will be required for participation. 
Parental consent will also be required. After determining that a subject is eligible, the site 
investigator or designee will approach the parent or legal guardian to oﬀer participation 
in the study. The parent or legal guardian will be informed about the objectives of the 
study and the potential risks and beneﬁts of participation. If the parent or legal guardian 
refuses consent to participate OR refuses permission for their infant to participate, then 
all clinical management for the infant will be provided by the clinical staﬀ in accordance 
with institutional practice and judgment. 
 
7.3.1   Child Assent 
Subjects who are eligible for this study are under 6 months of age and assent is not 
applicable. 
 
7.4       Adverse Event Reporting 
 
7.4.1  Assessment of Safety 
 
Safety assessments will consist of monitoring and reporting adverse events (AEs) and 
serious adverse events (SAEs) per protocol for 48 hours after the last study drug dose. 
This includes all events of death, and any study speciﬁc issue of concern. 
 
7.4.2  Adverse Events 
 
Investigators must report all potential unanticipated problems and events to the IRB. 
Unanticipated problems (UPs) are defined as any incident, experience or outcome that 
meets all of the following criteria: 
• 
Unexpected (unforeseen by the researcher or the research participant) in terms of 
nature, severity, or frequency, given the research procedures and the subject population 
being studied; 
 
and 
• 
Related or probably related to participation in the research, or if the event or 
problem probably or definitely affects the safety, rights and welfare of current 
participants; 
 
and 
• 
Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic or social harm) than was previously known 
or recognized. 


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The Investigator must consider each problem, event, or new information and decide 
whether or not it represents a UP, using the criteria above.  
In fatal or life-threatening serious adverse events, either unexpected and unrelated, or 
suspected to be related to the study, notification will be sent to the DSMB Chair, the IRB, 
and NHLBI program office within 7 calendar days of the PI becoming aware of the event.   
 
In non-fatal, non-life-threatening serious adverse reactions either unexpected and 
unrelated, or suspected to be related to the study, notification will be sent to the DSMB 
Chair, the IRB, and NHLBI program office within 14 calendar days of the PI learning of 
the event.   
 
In both cases, the DSMB chair will review the event, determine if a full DSMB meeting 
should be held, and will respond to the PI and NHLBI program office with 
recommendations within 3 business days of notification.   
 
Unanticipated problems that are not a serious adverse event (SAE) will be reported to the 
IRB, DSMB, and NHLBI within 14 days of the PI becoming aware of the problem.   
 
If UP Criteria Are Not Met: 
1. Document in the research record how the problem/event/information does not meet 
the UP Criteria.  
2. Summarize the problem/event/information in the next Continuing Review 
application. 
Examples of Potential Unanticipated Problems 
• 
A breach of confidentiality or privacy that involves real or potential risk (e.g. 
unauthorized use of disclosure of protected health information.)  
• 
Data and safety monitoring reports that indicate that frequency or magnitude of 
harms or benefits may be different than initially presented to the IRB  
• 
A publication that shows that the risks or potential benefits or the research may be 
different than initially presented to the IRB  
• 
Change in FDA labeling or withdrawal from marketing of a drug, device, or 
biologic used in a research protocol  
• 
Complaints from participants or others involved in the research that indicate 
unexpected risks or cannot be resolved by the research team  
• 
Warning or determination letters issued by any funding agency or regulatory body 
including Office of Human Research Protections (OHRP), Department of Health and 
Human Services (DHHS), Food and Drug Administration (FDA) 
 


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7.4.3   Mandatory FDA Safety Reporting  
 
Initial reporting: 
 
The IND application sponsor investigator must report any suspected adverse reaction or 
adverse reaction to study treatment that is both serious and unexpected as soon as 
possible but no later than within 15 calendar days following the sponsor’s initial receipt 
of the information 
Unexpected fatal or life-threatening suspected adverse reactions represent especially 
important safety information and must be reported to FDA as soon as possible but no 
later than 7 calendar days following the sponsor’s initial receipt of the information. 
• 
Follow-up reporting: Any relevant additional information obtained by the 
sponsor that pertains to a previously submitted IND safety report must be submitted as a 
Follow-up IND Safety Report. These reports should be submitted without delay, as soon 
as the information is available but no later than 15 calendar days after the sponsor 
receives the information. 
7.4.4     Deviation Reporting 
A deviation is ANY departure from the defined procedures and treatment plans as 
outlined in the protocol version or application version submitted and previously approved 
by the Institutional Review Board (IRB). Deviations have the potential to place 
participants at risk and can also undermine the scientific integrity of the study thus 
jeopardizing the justification for the research.  
Deviations are unplanned and/or unintentional events. Any changes in the IRB-approved 
research protocol should not be initiated without submission of an amendment for IRB 
review and approval.  If such changed are initiated before IRB approval for the change is 
given, a deviation has occurred.  
Criteria for Reporting Deviations 
The University of Utah IRB requires researchers to submit deviations that meet one or 
more of the following criteria:  
• 
Intended to eliminate apparent immediate hazard to a research participant 
(such as changing the dose of a medication due to possible toxicity);  
or 
• 
Caused possible harm to participants or others, or places them at increased risk 
of harm - including physical, psychological, economic, or social harm, such as a breach 
of confidentiality;  
or 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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• 
Possible serious or continued non-compliance (such as a deviation that has 
happened previously and is now being repeated).  
o Serious non-compliance is an act or omission to act that resulted in significant 
harm (physical, psychological, safety, or privacy) or significantly increased the 
possibility of harm to the rights and welfare of research participants.  
o Continuing non-compliance is a pattern of repeated actions or omissions to act 
that suggests a future likelihood of reoccurrence and that indicates a deficiency in 
the ability or willingness to comply with Federal regulations, VHA Handbooks or 
the policy, requirements, and determinations of the IRB governing human subject 
research.  
Some deviations pose no conceivable threat to participant safety or scientific integrity. 
For example, when the subject misses a clinic visit and the only available re-schedule 
date is outside the study visit window but no study procedures or medication doses are 
missed. In this case, the subject may not incur possible harm from a missed dose or 
missed procedures meant to maintain or evaluate the subject's safety and welfare.  As 
such, reporting is left to the discretion of the PI within the context of the guidelines 
above. Though a deviation may not pose a conceivable threat or possible harm, it may 
represent possible continuing non-compliance.  
The Investigator must consider each deviation and decide whether or not it meets the 
reporting criteria above. If the reporting criteria are met, the report must be submitted 
promptly, within 10 working days from the time the investigator learns of the deviation.  
 
If criteria are not met,  
a. Document in the research record how the deviation does not meet the reporting 
criteria. 
b. Log the deviation such that it can be evaluated in the future if other deviations occur. 
Any deviations that do not require a change in protocol or consent documents will be submitted 
to the FDA in the annual report.  
 
8.0     Study Records Retention  
 
Study documents will be retained for a minimum of 2 years after the end of the study.  
 
 9.        References 
 
 
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and neonatal morbidity among infants 501 to 1500 grams from 2000 to 2009. Pediatrics 
2012; 129: 1019-1026. 


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2. Stoll BJ, Hansen NI, Bell EF, S.Shankaran, Laptook AR, Walsh MC, Hale EC, Newman NS, 
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Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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297: 506-511. 


Principal Investigator: Candice D. Fike, M.D.                                                                                       Version:v16 
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41. Goossens L, Bouvry M, Vanhaesebrouck P, Wuyts B, Maele GV, Robberecht E. Citrulline 
levels in a paediatric age group:  does measurement on dried spots have additional value? 
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