A Phase 1 Dose Escalation Study of Continuous Intravenous L-citrulline During Sickle Cell Pain Crisis or Acute Chest Syndrome
Phase: PHASE1
Conditions: Sickle Cell Disease
Sponsor: University of Mississippi Medical Center

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A phase 1 dose escalation study of continuous intravenous 
L-citrulline during sickle cell pain crisis or acute chest syndrome 
 
 
Principal Investigator:  
 
Suvankar Majumdar MD 
 
 
 
 
 
Associate Professor 
 
 
 
 
 
Pediatric Hematology/Oncology 
 
 
 
 
 
 
 
Director, Comprehensive Pediatric Sickle Cell Program 
 
 
 
 
 
2500 North State Street 
 
 
 
 
 
Jackson, MS 39216 
 
 
 
 
 
Phone: 601 984 5220 
 
 
 
 
 
Fax: 601 815 5860 
 
 
 
 
 
e-mail: smajumdar@umc.edu 
 
 
Study Drug:  
 
 
IV L-citrulline 
 
Drug supplier:  
 
 
Asklepion 
 
Clinical Phase:  
 
 
Phase I 
 
Indication:   
 
 
Sickle Cell Disease 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
1.0 General Information 
1.1 Contact Information 
2.0 Study objectives 
2.1 Primary Objectives 
2.2 Secondary Objectives 
3.0 Hypothesis 
4.0 Study Overview 
4.1 Description of Design of Study 
5.0 Study Population 
5.1 Inclusion Criteria 
5.2 Exclusion Criteria 
6.0 Study Conduct 
6.1 Procedures 
6.2 Administration of Investigational Product 
6.3  Data collection at Enrollment and Follow-up 
7.0 Blood Sample Processing 
7.1 Citrulline Pharmacokinetic and Nitric Oxide Testing 
7.2 Collection and Processing of Blood Samples 
7.3 Amino Acid Analysis 
7.4 Nitric Oxide Assay 
7.5 Pharmacokinetic analysis 
8.0 Safety Assessments 
8.1 Clinical Parameters 
8.2 Laboratory Parameters 
9.0 Adverse Events/ Serious Adverse Events 
9.1 Adverse Event (AE) Definition 
9.2 Adverse Event Reporting  
9.3 Serious Adverse Event (SAE) Reporting 
10.0 Safety Monitoring Plan 
11.0 Data Handling and Record Keeping 
12.0 Institutional Review Board (IRB)/Ethics 
13.0 References 
 
 
 
 
 
 
 
 
 
 
 
 

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1.0 General Information 
 
1.1 Contact Information 
 
PRINCIPAL INVESTIGATOR 
Suvankar Majumdar MD 
Associate Professor 
Pediatric Hematology/Oncology  
 
Director, Comprehensive Pediatric Sickle Cell Program 
University of Mississippi Medical Center 
Phone: 601 984 5220 
Fax: 601 815 5860 
e-mail: smajumdar@umc.edu 
 
2.0 Study Objectives 
 
2.1 Primary Objectives 
 
2.1.1 To characterize the pharmacokinetic profile of continuous IV L-citrulline in   
patients > 6 years old during a sickle cell pain crisis or acute chest syndrome. 
 
2.1.2 To evaluate the safety profile of continuous IV L-citrulline in patients >6 
years during a sickle cell pain crisis or acute chest syndrome 
 
2.2 Secondary Objectives 
 
2.2.1 To determine the effect of continuous IV L-citrulline on NO and NO 
metabolites in individuals with sickle cell disease experiencing a sickle cell 
pain crisis or acute chest syndrome 
 
3 
Hypotheses 
 
3.1 Continuous IV L-citrulline will be well tolerated, without significant adverse 
effects in patients with sickle cell disease during a sickle cell pain crisis or acute chest 
syndrome 
 
3.2 
Continuous IV L-citrulline will increase NO and NO metabolites in patients 
with sickle cell disease receiving IV L-citrulline.  
 
4 Study Overview 
 
4.1 Description of Design of Study 
 
This study is a single center, prospective, open label phase 1 trial for subjects admitted 
for sickle cell pain crisis or acute chest syndrome. A total of 18 participants will be 

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recruited during their hospitalization at the University of Mississippi Medical Center 
(UMMC).  
 
5 Study Population 
 
5.2 Inclusion criteria: 
 
5.2.1 Sickle cell disease genotypes (HbSS, HbS/β-thalassemia, 
HbS/β+-thalassemia, HbSC)  
 
5.2.2 Patients with sickle cell disease aged 6 to 50 years old 
 
5.2.3 Presence of sickle cell pain crisis defined by the presence of 
pain requiring hospitalization and parental opioid therapy 
 
5.2.4 Presence of acute chest syndrome defined by the presence 
of a new CXR infiltrate and any one of the following 
respiratory symptoms of fever, shortness of breath, 
wheezing, chest pain, cough or new onset hypoxia.  
 
5.3 Exclusion criteria 
 
5.3.1 Presence of any other complication related to sickle cell 
disease 
requiring 
hospitalization 
such 
as 
splenic 
sequestration, hepatic sequestration, stroke, avascular 
necrosis of the hip/shoulder, acute priapism, etc.  
 
5.3.2 Severe anemia (hemoglobin < 5g/dL) 
 
5.3.3 History of red blood cell transfusion within the last 30 days 
 
5.3.4 Systemic steroid therapy within the last 48 hours 
 
5.3.5 Pregnant (as confirmed by a negative urine pregnancy test) 
or lactating female  
 
5.3.6 Alanine/aspartate transferase >2x upper limit of normal 
laboratory range for age.  
 
5.3.7 Subject has the following serum creatinine: 
5.3.7.1 
Age 6 to 13 years > 0.9 mg/dL 
5.3.7.2 
Age 14-17 years 1.0 mg/dL 
5.3.7.3 
Age 18 years >1.5mg/dL 
 
5.3.8 Patients with an inability to give consent will be excluded 

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6 Study Conduct 
 
6.2 Procedures - Whether participants participate in this study or not, they will 
still receive the standard of care for treatment of their sickle cell pain crisis or 
acute chest syndrome. 
 
6.2.1 Participant Enrollment and Screening 
 
Prior to enrollment, the investigator will: 
 
1. Determine initial eligibility prior to performing any study-specific 
procedures. 
 
2. Obtain signed informed consent from the patient participant before any 
study-specific procedures are performed. 
 
3. Review patient eligibility. 
 
4. Assign the potential participant a unique enrollment number, beginning 
with 005.  If a participant withdraws from participation in the study, then 
his/her enrollment code cannot be reused. 
 
 
Once the consent form is signed and the participant is enrolled into the study, the 
investigator will: 
 
5. Collect demographics, medical history and concomitant medications. 
 
6. Obtain vital signs (weight, height, blood pressure, heart rate, 
temperature, respiratory rate) and complete a physical examination. 
 
7. Obtain a blood sample for clinical chemistry, liver function tests and 
hematology assessments (if standard of care labs are obtained within 
48, those labs can be used for screening). 
 
8. Collect a urine sample in females of childbearing potential for a urine 
pregnancy test. 
 
9. The study drug will be administered within 72 hours of admission to the 
hospital for the sickle cell pain crisis or acute chest syndrome 
 
 
6.2.2 Initiation of Investigational Product 

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Once the eligibility of the participant is confirmed, the investigator will: 
 
1. Establish intravenous access for drug infusion and blood sample 
collection. 
 
2. Apply a blood pressure cuff for continuous cardiopulmonary monitor 
 
3. Collect about 2 teaspoon of blood immediately prior to the drug 
infusion for a baseline level of citrulline (trough level), and for NO and 
NO metabolites.  
 
4. Administer intravenous citrulline at a bolus dose of 20 mg/kg over 5 
minutes and then continuous at a rate of 7 mg/kg/hour for the next 23 
hours. 
 
5. Collect about 2 teaspoon of blood at each of the following time-points: 
10 minutes (peak level), 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 
6 hours, 8 hours, 12 hours, 24 hours and 48 hours after start of the IV 
citrulline infusion. 
 
6. Vital signs will be collected prior to each blood collection.  
 
7. Monitor the participant for any adverse events. 
 
6.2.3 24 hour Post-Infusion Follow-Up 
 
At the end of 24 hours (+/- 3 hours) hours from start of infusion, the investigator 
will: 
 
1. Perform a physical examination. 
 
2. Evaluate the participant for any adverse events. 
 
3. Obtain blood sample for complete count, liver and renal function tests 
(if standard of care labs are performed, then those laboratory results 
will be used).  
 
At the end of 48 hours (+/- 3 hours) hours from start of infusion, the investigator 
will: 
 
1. Perform a physical examination. 
 
2. Evaluate the participant for any adverse events. 
 

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3. Obtain blood sample for complete count, liver and renal function tests 
(if standard of care labs are performed, then those laboratory results 
will be used).  
 
6.2.4 15 Day Post-Infusion Follow-Up 
 
The following assessment will be completed 15 days (+/- 3 days) following the 
discontinuation of IV citrulline. This assessment may be completed via telephone 
contact with the subject and will not require an office visit. 
 
1. Adverse event reporting.  
 
6.2.5  30 Day Post-Infusion Follow-Up 
 
The following assessment will be completed 30 days (+/- 3 days) following the 
discontinuation of IV citrulline. 
 
2. Adverse event reporting.  
 
3. Obtain blood samples for complete blood count, liver and renal 
function tests 
 
 
6.3 Administration of Investigational Product   
 
6.3.1 Administration Regimens 
 
This study will include 2 cohorts each consisting of 7 participants for a total of 14 
study participants.  The second cohort will receive an escalated or reduced dose 
of the continuous study medication until a target peak goal citrulline 
concentration of 100 µmol/L is achieved.  Each cohort will receive a single IV 
dose of L-citrulline administered as a single bolus infusion over a 5 minute period 
and then a continuous infusion for 23 hours. 
 
Cohort 1 will receive 20 mg/kg of study medication and then a continuous rate of 
7 mg/kg/hour for 23 hours.   After pharmacokinetic data and safety profile 
(toxicity) is analyzed for cohort 1, the dosage for cohort 2 will change as follows: 
 
1. Higher continuous dose: IV citrulline 20 mg/kg bolus infusion followed by 9 
mg/kg/hour for 23 hours OR 
 
2. Lower continuous dose: IV citrulline 20 mg/kg bolus infusion followed by 5 
mg/kg/hour for 23 hours  
 
7.2.2. Study Drug Accountability, Storage and Mode of Administration 
 

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The manufacturer of citrulline will supply the study drug in sufficient quantities to 
complete the study. The study drug will be stored at ambient room temperature. 
The pharmacist will be asked to maintain a temperature log to document storage 
temperature conditions. The pharmacist will prepare and label all study drugs, and 
all labeling will meet standard requirements. L-citrulline will be supplied from the 
pharmacy as open label vials, and administered as a 50 mg/mL (5%) isotonic 
solution, with distilled water as a suspending agent. 
 
6.4 Data Collection at Enrollment and Follow-up 
 
For this study, participant data will be collected by paper source documents.  Every 
effort should be made to collect all required data, including blood samples and to 
complete all assessments required for each visit as detailed in the study plan. 
 
Clinical information obtained as part of standard clinical care before the informed 
consent is signed may be used as part of the screening and evaluation process. 
 
The investigator will ensure the data are recorded on the CRFs as specified in the study 
protocol.  He will ensure the accuracy, completeness, and timeliness of the data 
recorded. 
 
 
 
7 Blood Sample Processing 
 
7.2 Citrulline Pharmacokinetic and NOx metabolite sampling 
 
Plasma sampling for PK studies and NOx metabolites will be collected. As noted 
above, baseline PK and NOx testing will be performed just prior to giving the bolus 
(trough levels), 10 minutes after the bolus (peak level), and then 30 minutes, 1 hour, 
2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours  and 24 hours after the bolus 
infusion.   
 
 
7.3 Collection and Processing of Blood Samples 
 
Ten ml of venous blood will be collected in collection tubes containing citrate or 
EDTA. Samples will be kept on ice and processed within 3 hours after collection. 
Specimens will be centrifuged at 2000 rpm for 5 min and plasma collected and 
stored at –80ºC. Samples will be shipped by overnight courier to Marshall Summar 
MD, Center for Genetic Medicine Research at the Children’s National Medical 
Center for amino acid testing, and Paresh Ray, PhD at Jackson State University for 
nitric oxide testing. 
 
 
8.3 Amino Acid Analysis  

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Briefly, deproteinated plasma samples will be subjected to cation exchange 
chromatography using a 4-component pH and ionic strength graded lithium citrate 
buffer system on a Beckmann 7300 amino acid analyzer (Beckmann, Palo Alto, 
CA).  Arginine and citrulline will be the main focus as measurable products of urea 
cycle function, but will measure all urea cycle intermediates. In addition, the level of 
total essential amino acids (TEEA) to control for overall nutrition and metabolic state 
will be calculated. Analysis of amino acids will be completed as previously 
described. 
 
8.4 Nitric Oxide Assay  
 
NO will be measured by chemiluminescence using a Sievers 280 nitric oxide 
analyzer (NOA). Samples are mixed one part sample with two parts of cold ethanol 
at 00C for 30 minutes. Following centrifugation at 14000 RPM for 5 min, samples 
are injected into the NOA. This method relies on catalytic reduction of NOx by 
exposure to warm vanadium hydrochloride. Liberated NO is driven by nitrogen gas 
into an ozone chamber. Light released by NO/O3- interaction is captured by a 
photomultiplier tube and relayed to an analytical software program. A standard 
curve using sodium nitrite is required.  
 
8.5 Pharmacokinetic analysis 
 
Data obtained for each patient will be fit to a single-compartment pharmacokinetic 
model.  The appearance of citrulline in plasma is described by a zero-order process 
(rate of citrulline appearance, Rapp) to account for endogenous production, whereas 
the removal of citrulline is determined by a first-order process (constant of citrulline 
removal, krem). It is assumed that the values of all parameters will remain constant 
for each patient during the course of plasma sampling. Origin  (OriginLab 
Corporation, Northhampton, MA) will be used to solve for the pharmacokinetic 
parameters using a weighted, least squares procedure to obtain simultaneous 
values for Rapp, Co (plasma concentration at time zero), krem, and AUC (area under 
the curve).The volume of distribution (Vd)  will be calculated from the total dose/Co 
and the half-life from 0.693/krem. Clearance will be calculated from krem multiplied by 
the Vd. 
 
8 Safety Assessments 
 
Safety assessments will be collected as part of this bolus and continuous dose infusion, 
pharmacokinetic study.  Safety will be assessed throughout the infusion in all subjects, and 
monitored through 30 days after the end of the infusion. Subject safety will be assessed by 
monitoring adverse events (see section 10 for full detail), clinical laboratory tests, vital 
signs and physical examinations.  
 
8.2 Clinical Parameters 
 
Vital Signs 

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Vital signs (temperature, respiratory rate, blood pressure and heart rate) 
will be collected prior to the initiation of study drug and prior to each blood 
sample collection at 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 
hours, 6 hours, 8, 12 hours and 24 hours after the infusion. 
 
 
Physical Examination 
 
A complete physical examination will be performed during the screening 
period.  If the drug infusion occurs on a different day than the screening 
procedures are performed, then an abbreviated physical examination will 
be completed prior to the drug infusion.  A follow up complete physical 
examination will be performed at 24 hours after the end of the infusion. 
 
 
8.3 Laboratory Parameters 
 
Laboratory parameters will be evaluated at screening and then at 24 hours at 
completion of infusion and then at 30 days after the infusion to include the 
following: 
 
 Complete Blood Count (white blood cell count, hemoglobin, MCV, platelet 
count) 
 Renal Function (Sodium, potassium, blood urea nitrogen (BUN), HCO3, 
Creatinine)  
 Liver Function (Alkaline phosphatase, aspartate transferase, alanine 
transferase, and total bilirubin).  
 
9 Adverse/Serious Adverse Events 
 
9.2 Adverse Event (AE) Definition 
 
An adverse event (AE) is any untoward medical occurrence in a subject 
administered a pharmaceutical product that does not necessarily have a causal 
relationship with this treatment. AE may be reported by the subject, discovered 
by investigator questioning, or detected through physical examination, laboratory 
test, or other means.  
 
At each evaluation, subject will be interviewed to elicit potential adverse reactions 
from the drug. The occurrence of an AE will be based on changes in the subject’s 
physical examination, laboratory results, and/or signs and symptoms. All AEs 
(except Grade 1 and 2 laboratory abnormalities that do not require an 
intervention), regardless of causal relationship, will be recorded in the case 
reporting form (CRF) and source documentation. The Investigator will determine 
the intensity of any AE according to the NCI Common Terminology Criteria for 

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Adverse Events (CTCAE) Version 4.03 (see http://ctep.info.nih.gov) and their 
causal relationship. 
 
In this phase 1 trial, a participant with at least a grade 2 level of toxicity will be 
considered as dose-limiting for IV citrulline, and will be used as the stopping rule 
for testing further cohorts of participants.  
 
 
 
10.2  Adverse Event Reporting 
 
AE will be monitored and reported on a CRF that includes the description of the 
event, onset date, stop date, and outcome. The subjects will be instructed to 
inform the study personnel or clinical staff of any AEs experienced during the 
trial. In addition, subject safety will be assessed by monitoring clinical laboratory 
tests, vital signs and physical examinations. Any subject who has an AE will be 
evaluated by the investigator and will be treated accordingly. The investigator or 
designated study physicians will review each event and assess its relationship to 
the drug treatment (unrelated, possibly related, or probably related) based on the 
following definitions: 
 
10.2.1. Serious Adverse Events (SAEs) 
 
An AE or suspected AE is considered serious if, in the view the Investigator, it 
results in any of the following outcome 
 
1. Death; 
 
2. A life-threatening adverse event; 
 
3. Inpatient hospitalization or prolongation of existing hospitalization, 
excluding hospitalization for VOC required for inclusion into the study; 
 
4. A persistent or significant disability/incapacity; 
 
5. Congenital anomaly/birth defect; or 
 
6. Other events that, based upon appropriate medical judgment, may 
jeopardize the subject and may require medical or surgical intervention to 
prevent one of the outcomes listed above. 
 
10.2.2. Unexpected Adverse Event 
 
An AE is considered unexpected if it is not described in the Investigator’s 
Brochure or if it is of greater frequency and/or severity than mentioned in the 
Investigator’s Brochure. Unexpected, as used in this definition, refers to an 
adverse drug event that has not been previously observed rather than from the 

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perspective of such event not being anticipated from the pharmacological 
properties of the pharmaceutical product. 
 
10.2.3. Relationship 
 
The Investigator will assess the relationship of the event (not-related, unlikely-
related, possibly related, probably related, or definitely related). The relationship 
of an adverse event will be assessed using the following definitions: 
 
Not Related: Evidence exists that the adverse event definitely has an etiology 
other than the investigational agent, such as a pre-existing condition or 
underlying disease, a concurrent illness, or concomitant medication, and does 
not meet any of the criteria above. 
 
Unlikely: The causality of the AE is supported by enough evidence to reasonably 
suggest that the event could more likely be attributed to something other than the 
investigational product such as the underlying disease or an intercurrent illness 
or injury. 
 
Possibly Related: A temporal relationship exists between the event’s onset and 
investigational agent administration. Although the event may appear unlikely to 
be related to the investigational agent, a relationship cannot be ruled out with 
certainty, and/or the event cannot be readily explained by the subject’s clinical 
state or concomitant treatment. 
 
Probably Related: A temporal relationship exists between the event’s onset and 
investigational agent administration, and the event appears with some degree of 
certainty to be related to investigational agent, based on the investigational 
agent’s known therapeutic and pharmacologic actions. The event cannot be 
readily explained by the subject’s clinical state or concomitant treatment. If the 
investigational agent is discontinued or the dose is reduced the event abates or 
resolves. 
 
Definitely Related: Strong evidence exists that the investigational agent caused 
the adverse event. There is a temporal relationship between the event’s onset 
and 
investigational 
agent 
administration, 
and 
strong 
therapeutic 
and 
pharmacologic evidence that the investigational agent caused the event. The 
subject’s clinical state and concomitant treatment have been ruled out as causes. 
If the investigational agent is discontinued or the dose is reduced the event 
abates or resolves, but it reappears upon rechallenge. 
 
10.2.4. Severity of Adverse Events 
Severity or intensity of adverse events will be graded using the National Cancer 
Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), 
Version 4.03, and reported as indicated on the CRF.  
 

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10.3. Serious Adverse Event Reporting 
 
10.3.1. Time-Frame for Reporting 
 
Under 21 CFR 312.32(c), the Principal Investigator will notify the FDA within 7 calendar 
days an event that meets all three definitions of a suspected adverse reaction, serious 
adverse reaction and unexpected adverse reaction via a secure e-mail account with the 
FDA. The Principal Investigator will evaluate the available information and decide 
whether there is a reasonable possibility that the drug causes the adverse event, and 
therefore, that the event meets the definition of suspected adverse reaction. In addition, 
the investigator will identify in each IND safety report all IND safety reports previously 
submitted to the FDA concerning a similar suspected adverse reaction in light of 
previous, similar reports of any other relevant information. As per 21 CFR part 312, the 
investigator will conduct ongoing safety evaluations, including a periodic review and 
analyses of the entire safety database, not only for IND safety reporting purposes, but 
also to update investigator brochures, protocols, and consent forms with new safety 
information.  
 
10.3.2 Follow-Up of Adverse Events 
 
Any SAE or AE assessed as possibly, probably, or definitely related after last dose of 
study treatment will be followed until either resolution of the event or determination by 
the Investigator that the event has become stable or irreversible. This follow-up 
guidance also applies to possibly, probably, or definitely related SAE that occur 7 days 
after the last dose of study drug administration. The status of all other continuing AEs 
will be documented as of 7 days after last dose of IV citrulline. Follow-up data 
concerning reported SAE must also be reported to the FDA. 
 
 
10.3.3. Regulatory Reporting 
 
Reporting of SAEs by the Investigator to the IRB will be done in accordance with the 
standard operating procedures and policies of the IRB. Adequate documentation will be 
maintained showing that the IRB was properly notified. 
 
 
 
11. Safety Monitoring Plan 
 
A Data Safety Monitoring Board (DSMB) will be comprised of 3 University of Mississippi 
Medical Center physicians who are not related to the study.  Every third patient will be 
reviewed by the DSMB to ensure the safety of the study participants. 
 
12. Data Handling and Record Keeping 

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In compliance with ICH guidelines, the investigator will maintain copies of all source 
documents that support data collected from each patient and all study documents as 
specified in ICH Section 8, Essential Documents for the Conduct of a Clinical Trial. The 
investigator will take measures to prevent accidental or premature destruction of these 
documents.  
All data containing protected health information will be collected and stored in a locked 
cabinet located in the Children’s Cancer Clinic. In addition, all electronic data in for the 
form of Microsoft excel spreadsheet, word documents etc. will be stored in a password 
protected encrypted computer. The study coordinator will file all the material. Any 
information that is shared with other study personnel via e-mail will be de-identified in 
order to protect the participants’ health information.  
 
13. Institutional Review Board (IRB)/Ethics 
The protocol complies with the principles of the Declaration of Helsinki, 1964. In 
addition, the protocol complies with the laws and regulations of the state of Mississippi.  
The collection and processing of personal data from patients enrolled in the this study 
will be limited to that data which is necessary to investigate the efficacy, safety, quality, 
and utility of the investigational study medicine used in this study.  
The data will be collected and processed with adequate precautions to ensure 
confidentiality and compliance with applicable data privacy protection laws and 
regulations.  
Only approval by the Institutional Review Board (IRB), explicit consent for the 
enrollment in this research study, and processing of personal data will be obtained from 
the participating patient before collection of data.  
The patient has the right to request through the investigator access to his/her personal 
data and the right to request rectification of any data that is not correct or complete. 
Reasonable steps should be taken to respond to such a request, taking into 
consideration the nature of the request, the conditions of the study, and the applicable 
laws and regulations.  
Data will be kept confidential and if published in a scientific journal, will be done without 
identification of the individual patients.  
 
14. Reference List 
 
 
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