Clinical trial NCT01576003
Conditions: Bloodstream Infections Short Bowel Syndrome Development Nutrition Biological Markers
Keywords: Short Bowel Syndrome Necrotizing Entercolitis (NEC) Atresia Parenteral Nutrition Biological Markers Growth
Sponsor: Children's Hospital Medical Center, Cincinnati
Start date: 2012-04-01; Completion date: 2015-06-01
Enrollment: 10 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this research study is to evaluate the effects (good and bad) of supplementation with Glutamine to that of a placebo (L-alanine), on your child and their Short Bowel Syndrome. Researchers are doing this study to see if the addition of Glutamine to oral/tube feeding (nutrition therapy) will work better by preventing bloodstream infections, improving growth, and/or changing the make-up of bacteria in your child's intestine. Glutamine is approved by the FDA for use in adults with Short Bowel Syndrome. In this study, the investigators will be assessing how well Glutamine affects Short Bowel Syndrome in children.

Detailed description:
This is a double-blind, randomized placebo-controlled pilot study to investigate the efficacy of enteral glutamine (GLN) supplementation in 36 infants, ≤ 12 months of age with parenteral nutrition (PN)-dependent short bowel syndrome (SBS) due to massive small bowel resection for NEC and/or atresia on improving weaning of PN and preventing infections. The investigators intend to evaluate the effect of enteral feeding and GLN supplementation on the gut bacteria. The investigators will also recruit 12 age-matched controls to evaluate the normal gut bacteria.

Arms: arm 1: Infants randomized to the Glutamine group will receive L-Glutamine (GLN) administered enterally at a dose of 0.6g/kg body weight/day (0.3g/kg/dose) in 2 divided daily doses for 6 months. GLN will be dissolved in water, breast milk or formula and administered to the subject orally or through their enterostomy tube approximately every 12 hours (twice a day). arm 2: Infants randomized to the placebo group will receive L-alanine (ALA) administered enterally at a dose of 0.6g/kg body weight/day in 2 divided doses (0.3g/kg/day twice a day) for 6 months. ALA will be dissolved in water, breast milk or formula and administered to the subject orally or through their enterostomy tube approximately every 12 hours (twice a day). arm 3: Healthy age-matched infants (n=12) will have serial stools collected on 4 occasions, each separated by 60 days.
Interventions: intervention 1: Glutamine intervention 2: L-alanine
Intervention descriptions: intervention 1: 0.3 g/kg, taken orally/feeding tube every 12 hours/twice a day for 180 days (6 months) intervention 2: 0.3 g/kg, taken orally/feeding tube every 12 hours/twice a day for 180 day (6 months)

Protocol documents:
Prot_SAP_000.pdf: 
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Cincinnati Children’s Hospital Medical Center 
Research Protocol 
 
 
 
Title:  
 
Efficacy of Enteral Glutamine in Reducing Bloodstream Infections in SBS 
Infants 
Investigator:   
Conrad R. Cole, MD 
 
Co-Investigators:  Samuel Kocoshis MD, Michael Helmrath MD, Andrew South MD 
 
 
I.  Abstract 
Short bowel syndrome (SBS) following massive bowel resection for various conditions, 
especially necrotizing entercolitis (NEC), is a devastating complication that affects neonates 
especially those delivered < 36 weeks gestational age. These children are especially susceptible 
to recurrent blood stream infections (BSI), repeated hospital admissions and subsequent poor 
growth. All of these contribute to a very high burden on the primary caretakers and the health 
care system. Organisms associated with recurrent BSI in these children are predominantly gut 
related. However, there is still a lack of understanding of the intestinal microbial ecology of 
these children and whether it has a role in BSI, enteral feeds and ultimately growth. Glutamine 
(GLN) supplementation has been shown to decrease mucosal damage, lower rates of BSI and 
associated sepsis and improve positive nitrogen balance in adults with SBS. In children, there is 
currently insufficient data to determine whether enteral GLN supplementation improves clinical 
outcomes in young infants with significant gastrointestinal resection leading to SBS. The overall 
purpose of this pilot clinical study is to obtain needed preliminary data on the efficacy of enteral 
GLN supplementation in children with SBS as a result of significant intestinal resection due to 
NEC, omphalocele, gastroschisis and intestinal atresia in the context of a rigorous, double-blind 
exploratory clinical trial with the following specific aims: Specific Aim 1: To perform a double-
blind, randomized, placebo controlled, 6-months pilot study of enteral GLN treatment in PN-
dependent SBS children due to NEC, omphalocele, gastroschisis and/or atresia evaluating the 
efficacy of enteral GLN supplementation to decrease BSI and serial serum concentrations of 
TNF-α, IL1- β, IL-6 and IL-8; Specific Aim 2: To assess the efficacy of 6 months of therapy 
with enteral GLN as a method to improve somatic growth parameters (length velocity, weight 
velocity, head circumference and mid arm circumference) in Aim 1 subjects and the impact of 
BSI on growth parameters; Specific Aim 3: To comprehensively assess the gut lumenal 
microbiome (using state-of-the art molecular methods) in Aim 1 pediatric SBS patients versus 
non-SBS, age-matched controls, and, in the SBS patients to correlate major bacterial populations 
present in the lumen with BSI due to these microbes and to explore whether the gut lumenal 
microbiome is altered by enteral GLN treatment. This proposal will facilitate the development of 
a multicenter, multidisciplinary team from Cincinnati Children’s Hospital Medical Center, 
University of Michigan and University of Colorado to assess new methods for decreasing BSI 
and improving growth as well as to gain insights into the intestinal microbial ecology of SBS 
children.   
 
 


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II. Purpose of Study 
 
This is a double-blind, randomized placebo-controlled pilot study to investigate the efficacy of 
enteral glutamine (GLN) supplementation in 36 infants, ≤ 12 months of age with parenteral 
nutrition (PN)-dependent short bowel syndrome (SBS) due to massive small bowel resection for 
NEC, omphalocele, gastroschisis and/or atresia on improving weaning of PN and preventing 
infections. We intend to evaluate the effect of enteral feeding and GLN supplementation on the 
gut bacteria. We will also recruit 12 age-matched controls to evaluate the normal gut bacteria. 
Investigators at the University of Michigan and Cincinnati Children’s Hospital Medical Center 
will recruit the potential subjects (18 patients and 6 controls will be recruited at each site). We 
will estimate residual small and large bowel length and the presence, or absence of the ileocecal 
valve and colon by review of surgical records and postoperative intestinal barium studies in the 
subjects. Estimates of bowel length for gestational age have been previously published1,2. Data 
on parenteral or enteral GLN supplementation (L-GLN or the dipeptide Alanyl-GLN) in infants 
have not shown consistent effects, potentially because these studies were under powered; the 
dose of GLN was suboptimal or other factors. 
 
III. Background 
 
Significance 
Pediatric SBS is characterized by insufficient bowel length causing inability to maintain protein, 
energy, fluid, electrolyte or micronutrient balances when the child is on a conventionally 
accepted enteral diet3, 4. The use of PN is often required in SBS to provide nutrition and sustain 
life; unfortunately PN does not promote gut adaptation and also predisposes to BSI5, 6. 
Appropriate management with PN typically prevents nutritional deficiencies, enhances weight 
gain and is associated with improved overall survival rates7. In infants, NEC is the major cause 
of surgical SBS; other causes include gastroschisis, midgut volvulus and intestinal atresia8, 9. 
Children with PN-dependent SBS exhibit a high rate of recurrent BSI 10. The quality of life 
burden and healthcare costs associated with SBS in affected children is extremely high, in part, 
due to costs and complications of PN and hospitalizations for BSI11, 12. Healthcare costs in the 
first year of life in pediatric SBS patients routinely exceed $500,000 12. Inpatient hospitalization 
accounted for most (>80%) of this expense, attributed to prolonged NICU stay, surgical 
procedures and recurrent BSI12. The cost of home health care in children with SBS increases 
during each subsequent year of PN use8, 12. In light of the clinical importance of pediatric 
SBS, the high healthcare-related costs, and the ineffectiveness of current therapy to prevent 
BSI, new treatments to improve clinical outcomes are urgently required. Data to increase 
our understanding of the putative causes of BSI in this orphan disease are also needed.  
 
Recurrent sepsis and prolonged PN are each predictors of increased morbidity and mortality in 
children with SBS13, 14. The very high rate of recurrent BSI in infants with SBS is presumably 
due to impaired gut and systemic immunity and gut barrier dysfunction. Translocation of 
indigenous intestinal microbes has been shown to occur in animal models of SBS, with the 
translocation being promoted by generalized host immunosuppression, gut-associated immune 
dysfunction and gut barrier dysfunction15, 16. The incidence of catheter sepsis is significantly 
higher in children with SBS than in those without SBS (7.8 vs. 1.3 per 1000 catheter days; 


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p<0.05) and enteric organisms being responsible for 62% of catheter sepsis in patients with SBS 
compared to only 12% in patients without SBS17.  
 
Preliminary data strongly supports the finding of increased BSI and predominance of 
enteric organisms in the bloodstream of febrile SBS infants8, 18. The SBS infants in our 
preliminary data also exhibit elevated levels of proinflammatory cytokines (tumor necrosis 
factor-α, interleukin 1-β, interleukin-6 and interleukin-8) at enrollment vs. control subjects18. 
Previous studies in adults requiring chronic home PN also showed increased blood levels of 
TNF-α and IL-619.  
 
Despite receiving adequate calories, SBS infants enrolled in the NICHD Neonatal Research 
Network had poor growth indices (length and head circumference) and also exhibited a 
high rate of BSI8. These findings are similar to those previously reported for SBS children 
requiring long term PN20. Catabolic responses due to BSI and elevated systemic cytokines are 
likely involved in the poor growth exhibited. However, the relationship between BSI and growth 
failure is conjectural. There are no previous rigorous therapeutic trials designed to decrease BSI 
and improve growth velocity in infants affected by SBS, as we here propose.  
 
GLN is a central amino acid in major metabolic processes and is also utilized as a major 
fuel/substrate by intestinal mucosal cells and by immune cells, including the gut-associated 
immune system21-24. During catabolic illness, skeletal muscle exports large amounts of GLN into 
the blood, and GLN-utilizing tissues, including gut mucosa, immune cells, and kidney markedly 
increase GLN uptake25. If stress persists, GLN requirements may exceed endogenous GLN 
production, and skeletal muscle and plasma GLN concentrations decline23. Numerous studies in 
animal models show that enteral or parenteral GLN supplementation with either the dipeptide 
alanyl-GLN or free GLN, enhances gut mucosal growth, repair, and function, decreases gut-
origin sepsis and inflammation, and improves nitrogen balance in animal models of intestinal 
atrophy, injury, and adaptation 21, 22, 26-29. Several lines of evidence suggest that GLN may 
stimulate growth and function of gut mucosal cells in humans27,,30-32. In adults, GLN has potent 
anabolic effects, enhances immune functions and decreases nosocomial infection rates 24, 30, 
32, 33. In small studies, the combination of enteral GLN, subcutaneously administered 
human growth hormone and a modified enteral diet improved absorptive capacity and 
facilitated weaning of PN in adults with SBS34, 35.  
 
Pediatric studies have shown conflicting results regarding the impact of GLN to decrease 
morbidity and improve somatic growth. Studies done in clinically stable preterm infants 
showed that GLN is well tolerated both enterally and parenterally36. Premature infants are 
susceptible to GLN depletion because placental supply ceases at birth and tolerance to enteral 
nutrition is limited especially in those with SBS. Current PN in use in the United States does not 
contain GLN due to solubility and stability issues 37. Thus, total body GLN depletion may be 
exacerbated in SBS due to intestinal malabsorption, coupled with inadequate GLN intake, and 
potentially increased endogenous GLN requirements due to recurrent BSI and other stresses such 
as surgical trauma38. In a group of 69 premature, non-SBS, very low birth weight (VLBW 
<1500g birth weight) infants between ages 8 and 120 days, enteral GLN supplementation (0.3 
g/kg/day) for four months had a significant positive effect on growth indices (weight, length; mid 
arm, mid thigh and head circumferences)39. In a randomized, double-blind, controlled single 


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center trial in 68 VLBW infants, there were no growth or hospital stay difference between the 
GLN and placebo groups38. However, infants receiving GLN had a decreased incidence of sepsis 
(p<0.05) with risk of infection nearly 4-fold higher in controls38. In that study GLN was 
administered for a median of 27 days. There is limited available data on GLN supplementation in 
SBS children. In one trial of 20 preterm infants with SBS, due to NEC, intestinal atresia, 
gastroschisis, omphalocele, intestinal volvulus or malrotation were enrolled. Enteral GLN was 
administered to only 9 infants at a dose of 0.4g/kg/day up to 120 days, or until full enteral feeds 
were achieved. GLN was well tolerated, but did not show any benefit on duration of PN use or 
tolerance of enteral feeds vs. controls40, 41. However, given the sample size and diagnosis 
variability these data should be considered as preliminary.  
 
Although GLN has systemic effects after absorption, the potential effect of this amino acid to 
decrease BSI and increase protective mucosal immune response in SBS is our goal in this study. 
In vitro studies in human intestinal cell lines show that apical-derived GLN has trophic and 
cytoprotective effects in both enterocytes and colonocytes, inhibits apoptosis and promotes gut 
mucosal integrity by markedly attenuating disruption of tight junctions and paracellular 
permeability. In vivo studies in animal models of SBS show that enteral GLN improves indices 
of gut barrier function independent of its absorption 26, 30, 42-44. Investigators have shown that 
enteral GLN upregulates mucosal and luminal sIgA in rat models27.  
 
The available data are inconsistent on the clinical efficacy of parenteral or enteral GLN 
supplementation (L-GLN or the dipeptide alanyl-GLN) in infants (and more limited data on 
enteral GLN in pediatric SBS). The reasons for this potentially include underpowered sample 
size, suboptimal dose of GLN (0.3g/kg/day - 0.5g/kg/day) or other factors. The recommended 
dose of protein administered to infants is 3.0-3.5 g/kg body weight/day. The dose of parenteral 
GLN administered to critically ill neonates in a previous NICHD-sponsored multicenter trial was 
20% of the total amino acid content of the parenteral nutrition and this GLN dose was well 
tolerated 45. We propose to administer enteral GLN at a dose of 0.6g/kg/day, which is 
approximately 20% of the 3.0-3.5g/kg total protein recommended intake. Doses of 0.5 g/kg/day 
of enterally administered L-GLN have been well tolerated in clinical trials in infants 46, 47. 
 
Innovation 
The available data on GLN outlined above supports the need for a rigorous, double-blind clinical 
study to obtain critically needed pilot data on the efficacy of enteral GLN supplementation in 
pediatric SBS. This proposed study represents collaborative, multidisciplinary, multisite, 
comparative effectiveness clinical research. The rigorous nature of this trial of GLN 
supplementation in SBS infants is innovative compared to previous studies; our subject group is 
also more homogeneous, as we limit enrollment to children with NEC, omphalocele, 
gastroschisis and those with atresia as the primary cause of SBS. Our proposed design 
incorporates important clinical outcome measures (incidence of BSI and somatic growth 
parameters) to provide needed information on GLN efficacy in pediatric SBS. The data, in turn, 
will inform the design of subsequent definitive multicenter Phase III studies. In addition, to 
follow-up on our studies of GLN-induced upregulation of stool and mucosal sIgA in rat models 
of SBS27, we have measured stool sIgA in SBS children in a previous prospective trial and 
propose here to evaluate the impact of GLN on stool sIgA as a secondary index of enteral GLN 
efficacy. 


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The study of the gut microbiome in pediatric SBS and the response to GLN on these 
microbial populations is innovative. Commensal microbes may have a role in the immune 
conditioning of the gut, and may influence subsequent immune responses in neonates48. The 
intestinal microbiome may also be important for physiologic gut absorptive and barrier 
functions; small bowel bacterial overgrowth and/or altered luminal microbiota (e.g. due to 
anatomic disruptions after intestinal resection, dysmotility, stasis, etc.) may represent a 
potentially crucial, but as yet uncharacterized pathopysiologic factor in SBS 49. The underlying 
disease (NEC), malnutrition, and illness-associated immunodeficiency have been hypothesized 
to promote the proliferation of lumenal bacteria. Further, effects of altered nutrient intake in SBS 
on the microbiome are unknown. Therefore, in specific aim #3, we will comprehensively 
assess the gut lumenal microbiome in pediatric SBS patients versus controls to: 1) correlate 
alterations in lumenal bacteria populations with BSI risk; and 2) determine whether GLN 
supplementation influences the gut microbiome and concomitant microbial pathogenesis of 
BSI.  
 
Collaborators, Drs. Frank and Pace have developed molecular tools with which to study naturally 
occurring microbial communities without the requirement for preliminary culture 50-53. A 
number of these methods are centered on analysis of bacterial 16S ribosomal RNA gene 
(rDNA) sequences (see Figure 1, below) which vary from species to species and thus can be 
used as molecular ‘bar-codes’ for species identification54-58. rDNA can be PCR amplified with 
pan-bacterial primers from genomic DNA isolated directly from specimens, without need for 
prior culture of resident microorganisms (Figure 1, step A).  Advances in sequencing technology, 
such as the massively parallel GS-FLX pyrosequencing platform of Roche 454 Life Sciences, 
Inc. dramatically increased throughput compared to the previous generation of sequencers 
(Figure 1, step B).This platform was successfully deployed in a pilot study of the pediatric SBS 
microbiota.   
 
Figure 1: Analysis of human microbiome through culture-independent rRNA sequence 
analysis 
 
 
 
Quantitative 
analysis of 
the human 
microbiome 
data will be 
done as 
follows: DNA sequences will be phylogenetically compared to rDNA sequence databases (e.g. 
GenBank which stores >2,000,000 16S entries) to relate newly generated rDNA sequences to 
those of known organisms and thereby identify the source organisms (Figure 1, steps C and D). 
The results are a snapshot census of the microbial community within a specimen (Figure 1, step 
E) which provides a reference point from which physiology and lifestyle can be inferred 59,60. 
Many rRNA-based methods demonstrated the merits of monitoring intestinal microbes to gain 
insights into how commensals contribute to gastrointestinal pathology, including antibiotic 


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associated diarrhea and inflammatory bowel disease 61-63. Analogous to IBD, large-scale 
changes in the enteric microbiota of SBS patients may exacerbate the breakdown of gut 
mucosal immune homeostasis and barrier function, and thus contribute to BSI.  
 
Updated data on glutamine use and new procedures not in original grant   
Following the submission and approval of this application, more data regarding the potential 
benefit of enteral GLN in promoting growth and stimulating immune response against infections 
has not been published. There were no new relevant trials in humans using enteral GLN in 
infants. There is follow up data on infants enrolled in earlier trials showing that those who 
received enteral GLN had decreased for developing gastrointestinal infections and dermatitis 
likely due to the impact of GLN on the immune system83. However, new animal models suggest 
GLN supplementation may be useful in reducing the severity of weaning-related gastrointestinal 
infections, by reducing the mucosal cytokine response and altering intestinal barrier function 84, 
85. As a result of this we are proposing to evaluate as part of specific aim 1 the serum levels of 
anti-flagellin and anti-liposaccharide(LPS) immunoglobulin (Ig) G levels in the infants with 
SBS. These antibodies are markers of response to bacterial antigen and improve with advancing 
enteral nutrition 18. This proposal seeks to evaluate the impact of entral L-GLN on the mucosa’s 
immune response by measuring fecal IgA.  
 
IV. Duration of Study 
 
This anticipated duration to complete the study is 3 years (enrollment, analyses, and report 
writing). Each subject will be monitored for 6 months (± 1 month). 
 
V.  Potential Benefits 
1. Duration of PN in patients with surgical short bowel syndrome may be decreased 
2. Incidence of central line infections in these patients may be decreased 
3. Improving overall nutrition and growth 
4. Improving the gut mucosal immunity with oral GLN  
 
VI. Potential Risks  
Potential risks for GLN supplementation: 
• Stomach pain - 11% 
• Nausea  - 22% 
• Tenesmus - 11% 
• Pancreatitis - 11% 
• Constipation - 11% 
• Fever - 11% 
• Gastric Ulcer - 11% 
• Increased irritability - 11% 
• Mild skin rash or itching - 11% 
• Hyperammonimia in patients with renal and hepatic dysfunction - 11% 
• Laryngitis/Pharyngitis - 11% 
 
Other adverse events reported in the product information package  
• Gastrointestinal fistula in patients with Crohn’s disease - 11% 


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• Vaginal fungal infections – 11% 
• Psychiatric disorders – 11% 
• Pyelonephritis – 11% 
• Vascular disorders- 11% 
 
Potential risks for L-alanine (placebo): 
• Nausea and vomiting - 11% 
• Hyperammonimia in patients with renal and hepatic dysfunction - 11% 
 
Potential risks for L-Glutamine and L-alanine 
• Hyperosmolality of the feeds leading to increased stool output is a potential side 
effect of L-Glutamine and L-alanine when added to formula. However, this has 
not been reported in previous trials 37-40, 47, 86. The osmolality of L-glutamine is 
310 mosm/kg of water (data provided by Emmaus Medical Inc). 
 
Potential risks for blood draw: 
• Occurrence of discomfort and/or a bruise at the site of puncture 
• Less commonly, fainting 
• The formation of a clot or swelling in the vein and surrounding tissue 
• Bleeding from the puncture site 
• On rare occasions an infection may develop at the site where the blood is 
collected 
 
VII. Overall Risk Assessment 
The overall risk assessment is minimal based on previous pediatric and adult studies. The 
potential long term benefits of reducing the duration of PN, decreasing central line infections and 
improving growth is greater than the potential risks.  
 
VIII. Risk Category 
 Minimum 
 
IX. Methods 
 
A. Subjects and controls Selection 
 
Inclusion criteria for age matched controls (n=12: 6 per site) 
• 
Males and females will be recruited as controls for this study if they are ≤ 12 months of 
age (corrected for gestational age) 
• 
Normal small bowel length without any intestinal resection or primary intestinal disease 
• 
Not currently on TPN and if ever on TPN this should have been discontinued for at least 
4 weeks 
 
Exclusion criteria for age matched controls 
• Major congenital or chromosomal anomalies 
• Inability to tolerate enteral nutrition/ regular cow’s milk, breast milk or soy formula  


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• History of liver/intestinal transplantation 
• Active milk protein allergy 
• Inflammatory bowel disease 
• Multiorgan failure 
 
Inclusion Criteria for glutamine and placebo group of SBS patients (n=36; 18 per site)  
• Males and females ≤ 12 months of age (corrected for gestational age) 
• Patients who have undergone small bowel resection due to complications of or related to 
NEC, omphalocele, gastroschisis or intestinal atresia with known small bowel length  
• Patients who have been PN dependent for more than 42 consecutive days (6 weeks) and 
currently on TPN at time of enrollment. This would include patients who were off TPN 
for < 7 consecutive days. 
• Patients who have the ability to take partial enteral nutrition of at least 5ml/hr  breast milk 
or elemental formula  (Elecare® is the preferred formula at both clinical sites but use of 
other elemental formula does not exclude the patient) (via gastric or intestinal tube, or 
orally) to allow the appropriate dose of GLN (0.6 g/kg/day) or placebo (L-alanine) 
• Signed informed consent for the use of GLN or placebo obtained 
 
Exclusion Criteria for glutamine and placebo group of SBS patients 
• Major congenital or chromosomal anomalies 
• Inability to tolerate enteral nutrition that will preclude treatment with enteral GLN or L-
alanine placebo for >2 consecutive weeks  
• Liver/intestinal transplantation 
• Multiorgan failure  
 
B. Withdrawal Criteria 
1. Patient cannot complete evaluation visit assessments  
2. Patient develops any of the exclusion criteria detailed above 
3. Patient develops significant hepatic dysfunction (defined as INR > 2.5 or direct 
serum bilirubin level > 10 mg/dL)  
4. Parent(s)/legal guardian are unwilling to administer the GLN or placebo at the 
required dosage 
5. Patient is lost to follow-up 
6. Patient has 3 missed study visits 
7. It is in the patients best interest 
 
 
C. Dropouts 
 
Data collected from patients who voluntarily withdraw from the study or who are withdrawn 
from the study will be included in the final analysis using intent to treat. Patient and control 
enrollment will continue until 32 patients complete all requested study requirements outlined. 
The reason for withdrawal will be documented.  
 
D. Stopping Rules  
 


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An early stopping rule pending further investigation for this study will be invoked if any of the 
following adverse events occur: 
• The rate of BSI in SBS patients in either the GLN supplementation or the placebo is an 
absolute 20% higher compared to the historical rate in SBS patients. The rate of catheter 
associated BSI is assessed monthly in all SBS patients at both CCHMC and the 
University of Michigan as part of the current clinical improvement process, If the rate of 
the BSI is higher in either group by 20% then study enrollment will be halted until further 
evaluation by the Data Safety Monitoring Board (DSMB). 
• If two subjects develop the same grade-3 adverse event (including liver function tests, 
coagulation profile) related to the study medication that is irreversible upon de-escalation 
(does not reverse after 10 days) or upon discontinuation of the study drug 
• Any subject develops a related grade-4 adverse event. 
 
Since this is a double blind placebo control design, the data will only be unmasked by the 
research pharmacy at the request of the DSMB, if any of the above events occur. The statistician 
on the DSMB will evaluate the data. The unmasked data will not be revealed to the investigators 
unless the DSMB requests that the study be terminated. 
 
E.  Monitoring for toxicity due to GLN  
 
Potential toxicity will be assessed by analyzing clinical and laboratory parameters using the 
following PN labs (Complete blood count, complete metabolic profile (Renal, Calcium, 
Phosphorus, Magnesium, Liver panel, Total Protein, Albumin) monthly.  Additional blood 
samples will be done  in accordance with current institutional standards for monitoring chronic 
PN use in infants.  Plasma GLN levels will be obtained at baseline and at 6 months.  
 
F. Evaluating compliance 
 
All of the subjects will be instructed to bring to the monthly visits, the containers containing the 
supplement supplied at the previous visit. The study team will count the remaining bags left 
over. We will also evaluate the diary provided to the families. Compliance will be evaluated 
based on the diary and the number of returned containers. Families are expected to be 90% 
compliant. If the child/family is less than 90% compliant, the family will receive additional 
counseling to identify reasons and how to attain this level of compliance. Children will remain in 
the study as long as they receive at least 70% of the expected amount for 3 consecutive months.   
The level of compliance on enteral GLN effect will be included in the analysis. Children who 
receive less than 70% or more than 120% of the prescribed glutamine or placebo will considered 
to be non-compliant. The reason for non-compliance will be documented and included in the 
final analysis. Average intake/day of glutamine by the patients will be included in the analysis 
including the response based on dose received.  
   
 
G.  Patient Recruitment and Consenting Process 
 
Patient Recruitment 
 


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A total of 36 patients with short bowel syndrome (SBS) ≤ 12 months of age will be recruited for 
this study.  Of the 36 patients, approximately 24 patients will have colon in continuity and 12 
will have colon not in continuity. Assuming a 10% dropout rate, it is expected that 32 SBS 
patients will complete the study.   
 
For Aim 3 of the study, an additional 12 infants without SBS will be enrolled as the non-SBS 
control patients.   Therefore, a total of 48 patients will be enrolled across the two sites, 
University of Michigan and Cincinnati Children’s Hospital Medical Center. 
 
The study will be explained in detail by the study coordinator or other study team member and 
the consent form will be reviewed per institutional IRB requirements.  These subjects will be 
recruited through the Division of Gastroenterology, Hepatology, and Nutrition, the Department 
of Surgery, or the Division of Neonatology.  The prospective patients will be identified through 
the clinical staff within all three divisions working collaboratively.  The clinical staff will 
initially inform potential subjects and families about the research study. The research staff will 
notify and obtain permission from the patient’s primary gastroenterologist, surgeon, or 
neonatologist before contacting the family. 
 
Subjects who drop out prior to drug initiation may be replaced with new subjects until target 
enrollment is achieved, as these will not have been given study medication. 
 
Consenting Process 
 
For those patients meeting inclusion criteria, the study purpose, procedures, costs, risks, benefits, 
and alternatives to participation will be thoroughly explained and presented to the patient and 
their family by the designated study coordinator and/or investigator, and subjects will be 
screened for willingness to participate.  Subjects and parents will be explicitly informed that 
choosing not to participate will in no way affect the quality of the medical care that they will 
receive.  Once patients and families have had enough time to consider participation and have 
expressed a willingness to participate, consent will be obtained from the parents or legal 
guardian.   
 
One copy of the consent will be given to the family and one will be retained in the study records.  
One copy will also be placed in the child’s medical record.   
 
Vulnerable Population 
 
This study will enroll children.  To protect this population from coercion or undue influence, 
consent will be obtained only after a full discussion of the research protocol and of the risks and 
benefits that may occur from participation.  Patients and parents will be explicitly informed that 
choosing not to participate will in no way affect the quality of the medical care that they will 
receive. 
 
H.  Study Procedures 
 
Randomization and study groups  


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At the baseline visit, SBS subjects will be block randomized in a 1:1 scheme by site and strata 
(i.e the presence or absence of colon in continuity) to receive either L-GLN powder or 
isonitrogenous, isocaloric placebo [L-alanine (ALA)]. Randomization will be done by the 
Cincinnati Children’s Hospital Medical Center research pharmacist who will not be blinded. 
Distribution will be done by the site study personnel. The subjects, site investigators and study 
personnel at all sites will be blinded to the randomization.  Since patients will be on breast milk 
or Elemental formula (Elecare® being the preferred elemental formula at both institutions; 
however use of other elemental formula does not exclude the patient from the study), the 
randomization process will take into account whether breast milk or formula is been consumed 
by the infant. The research pharmacist will make decisions regarding randomization 
based on the use of formula versus breast milk to ensure adequate distribution within groups. 
 
Glutamine group: Infants randomized to the GLN group will receive L-GLN  administered 
enterally at a dose of 0.6g/kg body weight/day (0.3g/kg/dose) in 2 divided daily doses for 6 
months (figure 2). GLN will be dissolved in water, breast milk or formula and administered to 
the subject orally or through their enterostomy tube approximately every 12 hours (twice a day). 
This can be administered as a bolus or a continuous infusion lasting a maximum of 2 hours. We 
will use NutrestoreTM (Emmaus Medical, Inc.), a commercially available, FDA approved pure L-
GLN product in the study. In the event that this is not available we will use commercially 
available 100% USP grade L-GLN. The minimal volume L-GLN will be dissolved in at least 10 
ml of water, breastmilk or formula. 
    
 
Placebo group: Infants in the placebo group will receive ALA at a dose of 0.6g/kg body 
weight/day in 2 divided doses (0.3g/kg/day twice a day), exactly as outlined above for the GLN 
group. 
ALA is a non-essential amino acid and will be used as the placebo in this study because it is a 
nutritional supplement without any known impact on gastrointestinal permeability or local effect 
on the mucosa or infections. It will balance out the administration of an amino acid (L-GLN) to 
the study population.   
 


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GLN/Placebo groups-Visits to clinical research site /clinic: baseline, days 30, 60, 90, 120, 
150 and 180: Infants will be evaluated at the clinical research site at baseline, 30 (± 7 days), 60 
(± 7 days), 90 (± 7 days), 120 (± 7 days), 150 (± 7 days) and 180 (± 7 days) after the baseline 
visit (figure 2).  There will be a 7 day grace period before and after the desired clinic visit dates.   
 
Approximately, two weeks prior to the all of the visits (except baseline), the site coordinator will 
mail a detailed 3-days food record to the family. This form will be returned for analysis of oral 
diet and fluid intake, including formula and tube feeding intake (see below). During each visit, 
the following procedures will be performed with data entered without subject identifiers into the 
case report form:  
 
1. Complete medical history (including information on BSI): Gestational age, age at 
diagnosis of NEC, omphalocele, gastroschisisor atresia. age at surgery, type of surgery, 
dates/age for BSI events, types of organisms cultured (genus and species) and 
susceptibility to antibacterials;  
2. Anthropometric measurements of weight, length, mid arm circumference and head 
circumference in duplicate, when possible (see Aim 2);  
3. Enteral nutrition assessment: The subject’s usual nutrient intake (PN, solid and liquid oral 
diet provided by food sources, supplements and/or tube feedings) will be assessed by the 
site staff and investigators. All of the primary caregivers will receive a 3-days’ food 
record to complete about 1-2 weeks prior to their clinic visit. The nutritionist at each site 
and flagellin and LPS specific IgG 
and flagellin and LPS specific IgG 
and flagellin and LPS specific IgG 


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will evaluate this form and the data will be entered into the ESHA Nutrient Analysis 
Program to calculate the enteral total caloric and protein intake. The mean daily intake of 
PN and enteral nutrition calories and grams of protein and fat during the week prior to 
each evaluation visit will be determined and change (∆) in these variables from baseline 
will be calculated. Enteral diet optimization will occur throughout the study as 
clinically indicated using our comprehensive feeding advancement principles and 
algorithm for SBS children, as previously outlined18. The percent of total energy and 
protein needs that are met enterally will be calculated. PN will also be assessed for total 
calories and proportion of calories provided by dextrose, protein and lipids.  
4. The family will be instructed on how to administer GLN and placebo. During the 
baseline visit, the initial teaching will be done and they will be provided with the 
premeasured powder. The dose (0.6g/kg/d (0.3g/kg/dose) of GLN or ALA) will be 
measured out in the research pharmacy based on the weight of the child at the visit into 
individual packets. Children hospitalized during the study will receive GLN or ALA from 
the research pharmacy delivered to the patient’s bedside to be administered by nursing 
staff.  At subsequent visits (except visit on day 180), this teaching will be repeated as the 
dose to be administered will change based on the weight of the child. The family will also 
be given a weekly drug diary to complete in between visits. They will return the drug 
diary to the study staff during subsequent visits.  
5. During the initial visit, stool will be collected into sterile containers and stored at -80ºF 
until analysis for sIgA and bacterial composition evaluation. It is anticipated that the 
majority of stool will be collected in clinic during the study visit. If stool is collected in 
clinic, the stool will be stored immediately in the -80ºF freezer without any need for 
DNA stabilizer.   
If stool is not collected during this visit, families will be instructed on how to collect stool 
for evaluation into a sterile container. They will put this in a storage bag and kept cold 
until picked up by the home health team or delivered to the research team.  
Patients already enrolled will be provided with a sterile container with instructions to 
collect stool and bring in to the clinic visit for storage and analysis. The family will be 
asked to collect stool 24-48 hours before their clinic visit and put the stool in a sterile 
container which will be supplied to them by the study team. They will put this in a 
storage bag and keep cold.  They will bring the sample to the clinic visit.  RNAlater may 
be used with samples when samples are unable to be obtained within 24 hours of the 
clinic visit.   
 
Stool sIgA analysis: sIgA will be determined by ELISA as described 27,64.  
 
During baseline visit and visit on day 180 the following additional procedures will be done:  
Extra 2 ml of blood will be collected for plasma glutamine, proinflammatory cytokines analysis, 
flagellin and LPS specific IgG levels.  
 
During a BSI the following procedure will be done: stool collection for bacterial analysis and 
sIgA measurement. Extra 2 ml of blood will be collected for flagellin and LPS specific IgG 
levels. The blood will be obtained as soon as possible when the patient is being investigated for a 
suspected CA-BSI. In the event that the episode does not meet the criteria for a CA-BSI the 
samples will not be evaluated. 


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All bacterial isolates obtained during the study from patients will be saved at their respective 
institutions (CCHMC and University of Michigan) until the end of the study in the event further 
work with them is required. 
 
Plasma glutamine analysis: Plasma GLN will be performed using quantitative ion exchange 
chromatography65. 
 
Proinflammatory cytokine evaluation: Analysis will be performed utilizing Luminex xMAP 
technology (Austin TX) as previously described66, 67. 
 
Measurement of Flagellin and Lipopolysaccharide- specific Immunoglobulin G level: This 
will be done using enzyme-linked immunosorbent assay (ELISA) as previously described 18, 87-88. 
 
Safety evaluation: The study coordinator will contact the family by phone to assess for the 
development of any serious adverse events on days 15(±7 days), 45 (±7 days), 75 (±7 days), 105 
(±7 days), 135 (±7 days) and 165 (±7 days).  There will be a 7 day grace period before and after 
the desired date of safety evaluation phone calls. The phone interview will assess safety and 
evaluate for any symptoms suggestive of adverse side effects related to L-GLN.   If hospitalized, 
the safety evaluation will occur with the primary caregiver (bedside nurse or parent).   
 
Additional safety precautions: If while enrolled in the study the patient develops multiorgan 
failure or requires vasopressors, the study drug will not be administered. 
  
Clinical GI endoscopy 
Patients with short bowel syndrome/Intestinal failure may have clinical indications for 
gastrointestinal endoscopies. These indications include: 
- 
Gastrointestinal bleeding 
- 
Persistent feeding intolerance leading to failure to advance enteral feeds  
When the patient is scheduled for a clinically indicated gastrointestinal endoscopy, duodenal 
fluid (approximately 1 ml) will be aspirated into a sterile container. An additional tissue biopsy 
will also be obtained. 
Patients will not have GI endoscopies only as part of the study procedures. 
 
The samples of duodenal fluid and additional tissue collected will be stored in - 80º C freezer. 
They will subsequently be sent for bacterial composition evaluation at the University of 
Colorado using pyrosequencing techniques described below. 
 
 
 
 
 
 
 
 
 
 


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The following table summarizes Study Procedures: 
 
Study Procedures Table for GLN/Placebo group 
 
 
Screening 
and 
Consent 
Baseline 
(Day 0) 
Day 
15 
30 
day 
visit 
Day 
45 
60 
day 
visit 
Day 
75 
90 
day 
visit 
Day 
105 
120 
day 
visit 
Day 
135 
150 
day 
visit 
Day 
165 
180 
day 
visit 
(final 
visit) 
When 
clinically 
indicated 
Signed informed 
consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Physical 
Examination 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Blood collection for 
glutamine,   
proinflammatory 
cytokines (TNF-α, 
IL-1β, IL-6, IL-8)  and 
Antiflagellin and 
Anti-LPS IgG 
(2 ml))  
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
X (During 
suspected 
or 
confirmed 
BSI) 
Weight, length mid 
arm and head 
circumference 
velocities 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Nutritional intake 
evaluation 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
stool collection for 
bacterial and sIgA 
analysis 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X (During 
suspected 
or 
confirmed
BSI) 
Instruction on how 
to measure & 
administer 
GLN/placebo 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
3-day food record 
completed by family 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Phone interview 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
Aspiration of 
duodenal fluid (~1 
ml) and 1 additional 
biopsy from the 
duodenum  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 


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Study procedures of control group:   
Healthy age-matched infants (n=12; 6 at each site) will have serial stools collected on 4 
occasions, each separated by 60 days [baseline, days 60(±15), 120(±15) and 180(±15)]. This is 
abbreviated compared to the study subjects. 
 
A week prior to the scheduled date of stool collection, families will receive a brief medical 
questionnaire and stool container. They will be instructed to complete the questionnaire and send 
in with the stool specimen.  
 
They will be provided with a sterile container with instructions to collect stool and bring in to the 
clinic for storage and analysis. They will be asked to collect stool on the day of the next 
anticipated clinic visit, put the stool in a sterile container (which may contain RNAlater) which 
will be supplied to them by the study team. They will put this in a storage bag, keep cool  and 
bring the sample to the clinic visit.     
 
Parents/Primary caregivers drop off stool specimen to the research team at the study site or will 
arrange with the team for the specimen to be collected from their homes. 
Study Procedures Table for Control group 
 
 
Screening 
and 
Consent 
Baseline  
(Day 0) 
60-day 
visit 
120-day 
visit 
180-day 
visit 
(final 
visit) 
Signed informed consent 
X 
X 
 
 
 
Medical History/questionnaire 
 
X 
X 
X 
X 
stool collection for bacterial and 
sIgA analysis 
 
X 
X 
X 
X 
 
I.  Data Collection 
 
Demographic and clinical information will be collected whenever clinical data are available via 
uniform baseline and follow up clinical data extraction forms to ensure accuracy and 
consistency.  The essential demographic information includes age at diagnosis, gender, race and 
ethnicity, birth history and past medical history.  The clinical information includes bowel length, 
dates and type of surgeries, nutrition data (formula type, amount, weaning foods, parenteral 
nutrition type and amount), infection type  (genus, species and susceptibility to antibacterials) 
date infection is diagnosed) hospital admissions and hospital length of stay. 
 
J.  Compensation 
 


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Participants will receive $10 for every stool collected for analysis during study participation.  
Subjects will receive study medication, clinical supplies, and laboratory evaluations related to the 
study free of charge.   
 
 
 
X.  Assessment of Efficacy 
 
Hypothesis 1: Enteral GLN supplementation in infants with SBS after massive bowel resection 
for NEC will decrease BSI, and thereby decrease systemic proinflammatory cytokine 
concentrations.  
 
Specific Aim 1: To perform a double-blind, randomized, placebo controlled, 6-months pilot 
study of enteral GLN (0.6g/kg) vs. isocaloric, isonitrogenous alanine (placebo) treatment in PN-
dependent SBS children due to NEC to evaluate the efficacy of GLN to decrease:  a) BSI 
(primary endpoint) and b) serial serum concentrations of TNF-α, IL1- β, IL-6 and IL-8 
(secondary endpoint). The primary outcome is the incidence of BSI during the 6 months on 
GLN supplementation. BSI is defined as laboratory-confirmed, culture-proven bloodstream 
infections that meet the Centers for Disease Control and Prevention (CDC) definition68, 91.  This 
allows for the distinction between catheter related-BSI and other causes of BSI.  
 
Secondary outcomes: 1) Plasma proinflammatory cytokine levels (TNF-α, IL-1β, IL-6, IL-8); 2) 
readmissions and hospital length of stay; 3) proportion of nutrition delivered enterally versus 
parenterally; 4) stool sIgA levels; 5) Plasma glutamine. 
 
Expected results: We expect that that the SBS children on enteral GLN supplementation will 
have significantly fewer BSI and greater decline in blood cytokine levels during the period of 
therapy.   
 
Hypothesis 2: Enteral GLN supplementation will improve somatic growth parameters in Aim 1 
patients.  
 
Specific Aim 2: To assess the efficacy of 6 months of therapy with enteral GLN on somatic 
growth parameters [length velocity (primary endpoint), weight velocity, mid arm circumference 
and head circumference] in Aim 1 subjects and the impact of BSI on these growth parameters. 
 
The primary outcome for specific aim #2 is length velocity. Secondary outcomes are growth 
parameters of weight mid arm circumference and head circumference velocities. These are 
clinically relevant growth parameters identified by our preliminary data in which SBS children 
had growth delay with short lengths and smaller head circumferences than infants without NEC 
or SBS8.  
 
Data collection: During CNC/clinic visits (baseline, days 30, 60, 90, 120, 150 and 180) as 
described above, each child will have weight, length mid arm circumference and head 
circumference measured in duplicate. These measurements will be done by a trained member of 
the study staff at each center. The individual at each site will be trained to perform these 


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measurements according to standard methods previously described69, 70. Institutional scales, 
length boards and measuring tapes will be used at each center.  
 
Expected results: We expect that SBS children on enteral GLN supplementation will have more 
rapid growth as measured by the parameters above. The group with no BSI or decreased BSI (2 
episodes) during the study period is expected to have a greater increase in growth parameters 
compared to the group with > 2 BSI.  
 
 
Hypothesis 3: The gut lumenal microbiome is altered in pediatric SBS versus non-SBS controls, 
correlates with BSI and is altered by enteral GLN supplementation. 
 
Specific Aim 3: To comprehensively assess the gut lumenal microbiome (using state-of-the art 
molecular methods) in Aim 1 SBS patients versus non-SBS, age-matched control subjects 
(primary endpoint), to correlate major bacterial dynamics present in the lumen with BSI due to 
specific microbes and to explore whether the gut lumenal microbiome is altered by enteral GLN 
treatment. 
Primary outcome: Increased K. pneumonia and E. faecalis in the gut microbiome of SBS 
infants with BSI.  
Secondary outcome: Alteration in other commensals in the gut lumenal microbiome of SBS 
infants.  
 
Subjects: Infants recruited in specific aim 1 (n=48 – subjects- 36; controls- 12)  
Healthy age matched infants (n=12) will be recruited from the Gastroenterology and surgery 
clinics.  
 
Sample Procurement:  Stool samples (~500 mcg), will be obtained from infants’ diapers or 
ostomy bags. Stool specimens will be collected prospectively during visits at baseline, days 30, 
60, 90, 120, 150 and 180. Stool will also be collected whenever the patient has a BSI.  
The controls (12 healthy infants) will have serial stools collected on 4 occasions, each separated 
by 60 days (baseline, days 60, 120 and 180).   
It is anticipated that stool samples will be collected during the clinic visit. All stool samples will 
be scooped into a stool container and stored immediately in a -80°C freezer. For samples 
collected at home, subjects will be instructed to collect in a container containing RNAlater tissue 
reagent. This will be stored in a cold at home and when delivered to the study team will be stored 
in -80°C freezer until processed. RNAlater provides immediate stabilization of DNA, RNA, and 
protein in samples at room temperature. This preserves the in vivo profile of DNA, RNA, and 
proteins, allowing reliable downstream analysis. Stabilized samples can be transported at 15–
25°C for up to 7 days, or stored at 2–8°C for up to 12 months. For longer storage, stabilized 
samples can be archived at –80°C. 
Small bowel fluid and tissue procurement: If the patient is scheduled for a clinically indicated 
gastrointestinal endoscopic procedure; duodenal fluid (~1 ml) will be aspirated and an additional 
duodenal biopsies will be collected. Specimens will then be stored at -80°C within 10 minutes of 
obtaining until processing. They will subsequently be sent for bacterial composition evaluation at 
the University of Colorado using pyrosequencing techniques described below. 


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Genomic DNA Preparation: Total genomic DNA will be prepared from specimens using a 
robust protocol that entails chemical and mechanical disruption of cells, which is performed in a 
HEPA-filtered, UV-sterilized PCR hood71-74. Sham extractions are performed in parallel to detect 
contaminated reagents.  Broad-Range PCR: DNA lysates are subjected to PCR with pan-
bacterial 16S rDNA primers (27FYM+3 and 338R), yielding libraries of PCR amplicons 
representative of all bacteria in a specimen74, 75. Triplicate PCR reactions will be performed and 
amplicons pooled for each sample. To alleviate representational bias in PCR amplicon libraries, 
we will determine empirically the minimum number of PCR cycles required to generate the 
quantities of DNA required for direct sequencing by broad-range rDNA Q-PCR.  High 
throughput DNA Pyrosequencing: Subject PCR amplicon libraries will be sequenced using the 
high-throughput Genome Sequencer™ FLX pyrosequencing platform of Roche/454 Life 
Sciences, Inc., available through the University of Colorado’s Consortium for Comparative 
Genomics. Barcoded PCR primers will be used to multiplex ~200 libraries per pyrosequencer 
run76-79. Approximately 5000 rRNA gene sequences will be determined for each sample library, 
which based on our preliminary studies will comprise a comprehensive census of the resident 
microbes. Additional sequences will be screened if coverage is less than 95%79-81.  
 
XI. Assessment of Safety    
 
A DSMB will monitor the study for the occurrence of adverse events (both serious and 
otherwise).  A significant increase in the rate of adverse events would be cause for concern for 
the safety of participants in the study.  Information on adverse events will be presented in several 
ways:  (1) listings of serious adverse events with accompanying narrative summary by the PI: (2) 
summaries of adverse events by body system and type of event.  This information will be 
presented to the DSMB. 
 
Interim monitoring reports for the DSMB will include, but will not be limited to, such 
tabulations as:   
 
      1.   Frequency of BSI and hospitalization 
2. coagulation studies 
3. liver profiles 
4. renal profiles 
5. Frequency of adverse events  
 
The DSMB will meet at the start of the study, then after 5 patients have received treatment. 
Subsequent meetings will be at the discretion of the chair  given the expected speed of 
recruitment and study medication administration. 
 
The DSMB chair (or designee) will be asked to review SAE reports within 48 hours after initial 
receipt of the information by the investigator(s), to review the PIs assignment of SAEs as related 
or unrelated to treatment, to confirm the grading of any toxicities. This will also be reported to 
the site IRB and the study sponsor. 
 


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A safety analysis will be performed after approximately 5 patients have received treatment or, if 
enrollment begins slowly, 12 months after the first patient is enrolled.  Safety data will be shared 
with the Data and Safety Monitoring Board (DSMB).  An investigator in this study will not be a 
member of the DSMB.  Members of the DSMB will receive safety data approximately every 6 
months for review or more frequently if requested by the chair.  Safety assessments will consist 
of vital signs, AEs, and laboratory evaluations.  A cumulative listing of patient withdrawals, dose 
adjustments, and serious adverse events (SAEs) will also be reviewed.  DSMB members will be 
notified of all SAEs reported expeditiously to regulatory authorities.   
 
Dr. James E. Heubi, Division of Gastroenterology, will act as the chair of the DSMB. There will be 
2 other members appointed to the DSMB: a statistician (Dr. Christopher Lindsell) and a 
neonatologist (Dr. Kurt Schibler), who are not involved in the conduct of the study.  Dr. Sean 
Moore will serve as the safety officer for this study. The safety officer is not a member of the 
DSMB and he is an expert on glutamine metabolism and use in children with chronic and persistent 
diarrhea. 
 
 
Study Monitoring 
 
An investigational new drug (IND) application was submitted by the PI for the use of L-GLN in 
the management of infants with SBS. An independent monitor from the CCHMC Clinical Trials 
Office will conduct routine independent monitoring visits that shall occur one or more times 
during the period after study initiation but before study closeout. Guidelines for scheduling 
monitoring visits shall be determined according to the stage of development, complexity of the 
study, the rate of subject accrual and other factors. Monitoring visits are conducted for routine 
monitoring only and are intended to ensure that the protocol and applicable regulatory 
requirements are being followed, that subjects' rights and safety are protected, and to confirm 
data integrity and quality. 
 
XII. Adverse Events  
 
An adverse event (AE) is any untoward medical occurrence in a patient participating in this trial.  
An adverse event can be an unfavorable and/or unintended sign (including an abnormal 
laboratory finding), symptom, syndrome or disease associated with or occurring during the use 
of an investigational product whether or not considered related to the investigational product.   
 
A serious adverse event (SAE) is one that: 
 
1. results in death 
2. is life threatening 
3. requires an inpatient hospitalization or prolonged existing  hospitalization 
4. is a persistent or significant incapacity or substantial disruption of ability to conduct 
normal life functions 
5. is a congenital anomaly/birth defect 
6. is an event that does not meet the above criteria but may jeopardize the patient or may 
require medical or surgical intervention to prevent one of the outcomes listed above. 


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IRB Notification of SAEs 
 
Per Institutional Review Board (IRB) Requirements, the Investigator will report serious adverse 
events to the IRB Office within the time period required at each institution (at CCHMC this is 
outlined in research policy R-18).  All other adverse events that are not serious will be reported 
to the IRB at the time of continuing review, per IRB requirements.   
 
 
AE’s require prompt (within 7 days after knowledge of the problem) reporting to the 
IRB when meeting the following criteria: 
• Any unanticipated problem involving risk to subjects 
• Deviations from or changes to, the protocol to eliminate immediate hazards to the 
trial participants 
• Changes increasing the risk to subjects and/or significantly affecting the conduct 
of the trial. 
• All adverse drug reactions that are both serious and unexpected 
• New information that may adversely affect the safety of the subjects or conduct of 
the trial. 
 
These events will be reviewed by the safety officer and are required to be reported to 
the FDA within 7 calendar days of the sponsor’s initial receipt of the information if 
characterized as fatal or life-threatening.  Non-fatal or non-life threatening events 
must be reported in 15 calendar days or at the time for annual review (reporting will 
be based on the FDA criteria for reporting AEs). In addition, these events are to be 
reported to the IRB based on IRB reporting guidelines.  . 
 
Recording of Adverse Events  
 
Adverse event recording will begin after administration of study medication.   
 
Diagnostic and therapeutic non-invasive and invasive (i.e. surgical) procedures will 
not be reported as adverse events. However, the medical condition for which the 
procedure was performed must be reported if it meets the definition of an adverse 
event, unless it is pre-existing (prior to initiating GLN). 
 
Medical conditions/diseases present before starting drug are only considered adverse 
events if they worsen after starting treatment.  
 
Symptoms of the original or targeted disease are not to be considered adverse events 
in this treatment.  The following symptoms are indicative of underlying disease and 
will not be reported as adverse events:  Malabsorption, growth failure, liver disease 
and BSI. 
 


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Abnormal laboratory values or test results constitute adverse events if they induce 
clinical signs or symptoms or require therapy.  They are to be recorded on the 
Adverse Events CRF under the signs, symptoms or diagnoses associated with them.   
CA-BSI will only be reported as an adverse effect if the rate is higher than 20% of 
what is standard for the center. 
 
As far as possible, each adverse event will be assessed by the Investigator with 
respect to its duration (start and end dates), the severity grade (mild, moderate, 
severe, life-threatening, fatal), its relationship to the drug (unrelated, unlikely to be 
related, possibly, probably and definitely related), the action(s) taken, the outcome 
(i.e. resolved, resolved with sequelae, continuing), gravity (SERIOUS or NOT 
SERIOUS), and expected/unexpected on the basis of drug insert. 
 
Grading (Severity) of Adverse Events 
 
Each adverse event will be graded to describe its intensity according to the Severity 
scale listed for each AE found on the 
 NCI’s Common Terminology Criteria for Adverse Events Version 4.0.    
 
Relationship to Drug 
 
For all adverse events that occur while the patient is on drug, the relationship to the 
drug must be assessed by the Investigator using the following scale: 
 
1 = not related   
 
2 = unlikely 
 
 
3 = possibly related  
 
4 = probably related   
5 = definitely related   
 
For Grade > 3 adverse reactions which are possibly (3), probably (4) or definitely (5) 
related to the drug, study medication should be discontinued as described above. 
 
Please note: For safety data analysis, not related and unlikely are considered “not 
attributable;” possible, probable and definite are considered “attributable.”  
Assessment of relationship to drug must be carried out by the Investigator according 
to the 5-point scale above. 
 
Gravity (Seriousness) of Adverse Events 
 
Each adverse event is to be classified by the Investigator as SERIOUS or NOT 
SERIOUS.   
 
Follow-Up after Adverse Events 
 


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All adverse events will be followed until they are resolved or the patient’s 
participation in the treatment ends (i.e., until a final report is completed for that 
patient).  
 
In addition, all SAEs and those non serious events assessed by the Investigator as 
attributable (i.e. possibly, probably, definitely related to the drug) will continue to be 
followed even after the patient’s participation in the treatment is over. Such events 
will be followed for 30 days post treatment. Resolution of such events is to be 
documented in the CRF. 
 
Expedited Reporting 
 
SAE’s require expedited reporting when meeting the following criteria: 
• Serious 
• Unexpected 
• At least possibly related to the study agent or other protocol specific activity 
 
Serious Adverse Events meeting the above criteria are required to be reported to the FDA as 
follows: 
• If characterized as fatal or life-threatening, within 7 calendar days of the sponsor’s initial 
receipt of the information  
• If non-fatal or non-life threatening, within 15 calendar days.  
 
In addition, the sponsor is required to submit serious adverse events to the DSMB for review, 
and serious adverse events requiring expedited reporting will be reported to the IRB 
concurrently.  
 
Significant unplanned deviations from the protocol will be reviewed and will be reported as 
stipulated above.   
 
XIII. Statistical Analysis Plan  
 
To assess the comparability of the two treatment groups, demographic and baseline data will be 
summarized for each treatment group.  For continuous data, mean, standard deviation, median, 
min, max, first quartile and third quartile will be presented.  Comparisons will be made using the 
two-independent sample t-test or the Wilcoxon Rank sum test, depending on the distribution of 
the variable.  For categorical data, frequencies and percentages in each group will be presented.  
Comparisons will be made using Fisher’s exact test. These tests will be conducted at the two-
sided, 5% level of significance. In addition, demographic and baseline data for the control group 
will be summarized and presented as described above. The statistical analysis plan for each aim 
is presented below.  
Aim 1 Hypothesis:  Enteral GLN supplementation in infants with SBS after massive bowel 
resection for NEC will decrease BSI, and thereby decrease systemic proinflammatory cytokine 
concentrations. 
 


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The primary outcome variable is the incidence of BSI during the 6 months on GLN 
supplementation.  BSI is defined as laboratory-confirmed, culture-proven bloodstream infections 
that meet the Centers for Disease Control and Prevention (CDC) definition69. To compare the 
incidence of BSI during the 6 month time period, the percent of patients with at least one BSI 
will be calculated in each group.  Since this is a pilot study, the desire is to detect a trend in 
improvement of BSI with GLN supplementation. Therefore the 2 groups will be compared using 
the Fisher’s Exact test at the one-sided 10% level of significance which will indicate a significant 
trend.  As an additional analysis, time to first BSI will be displayed using Kaplan-Meier (K-M) 
curves. The log-rank test will be used to test for differences in curves between treatment groups. 
Patients will be censored if they exit the study prior to having their first BSI. As a further 
exploratory analysis, time to multiple-event analyses techniques will be used to compare groups 
where the multiple events represent multiple episodes of BSI within patients. 
 
Secondary outcomes include the following and will be tested at the two-sided 5% level of 
significance:   
1) Plasma proinflammatory cytokine levels: TNF-α, IL1-β, IL-6, and IL-8. Change from 
baseline values at 180 days will be computed for cytokine levels. Change from baseline 
to 180 days values will also be calculated for the flagellin and LPS IgG levels. 
Appropriate transformations of the data (e.g. log) will be done prior to calculating change 
from baseline in order to meet the assumptions of the analyses (i.e. normality, equal 
variance).  Analysis of covariance techniques will be used to compare the change from 
baseline scores of the treatment groups. Covariates included in the analysis will be 
baseline value, age, gender, ostomy status, feeding type (formula, breast milk).  
2) Length of hospital stay and number of readmissions: For length of hospital stay, 
treatment groups will be compared using the Wilcoxon Rank Sum test. For number of 
readmissions, either a Wilcoxon Rank Sum test or a Chi-Square test for homogeneity will 
be used to compare groups, depending on the variability of the number of readmissions.   
3) Stool sIgA levels: Treatment groups will be compared using the random effects repeated 
measures analysis of covariance. The response variables for each of the parameters will 
be change from baseline. The time points included in the analyses will be 30, 60, 90, 120, 
150, 180 days post-baseline. If required to meet the assumptions of the analyses 
(normality, equal variances), appropriate transformations will be used. Treatment groups 
will be compared at each individual time point using appropriate contrasts and the error 
term from the repeated measures model. Covariates will include baseline value, age, 
gender, ostomy status, feeding type (formula, breast milk).  
4) Proportion of nutrition delivered enterally versus parenterally:  The statistically analysis 
approach for this endpoint will be the same as for the Stool sIgA levels discussed above.  
Transformations appropriate for proportions may be used, if necessary, to meet 
assumptions of the analyses. 
5) Plasma glutamine: The statistical analysis approach for this endpoint will be the same as 
for the proinflammatory cytokine levels discussed above. 
6) Impact of level of compliance on outcome will be evaluated for a dose effect analysis. 


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Aim 2 Hypothesis: Enteral GLN supplementation will improve somatic growth parameters in 
Aim 1 patients. 
7) The two treatment groups will be compared using the random effects repeated measures 
analysis of covariance for the following parameters:  length velocity, weight velocity, 
mid arm circumference and head circumference. Length velocity and weight velocity will 
be analyzed as the post baseline scores. Mid arm circumference and head circumference 
will be analyzed as change from baseline. The time points included in the analyses will 
be 30, 60, 90, 120, 150, 180 days post-baseline. If required to meet the assumptions of the 
analyses (normality, equal variances), appropriate transformations will be used (e.g. log).  
Treatment groups will be compared at each individual time point using appropriate 
contrasts and the error term from the repeated measures model. Covariates will include 
baseline value, age, gender, ostomy status, feeding type (formula, breast milk). For length 
velocity and weight velocity, the baseline value will be the baseline length and weight 
values. 
To evaluate the impact of BSI on the growth parameters listed above, point biserial 
correlation coefficients will be constructed overall and within each treatment group and 
tested to determine significance.  
  
Aim 3 Hypothesis: The gut luminal microbiome is altered in pediatric SBS versus non-SBS 
controls, correlates with BSI and is altered by enteral GLN supplementation. 
 
8) Intestinal microbial groups (e.g., species, genera) will be correlated with the treatment or 
control arms of the GLN supplementation trial and with bacteria identified during any 
diagnosed BSI.  The frequency of rRNA sequences will be interpreted as the relative 
abundances of organisms in samples of stool and standardized by gram of stool weight. 
Bacteria present in specimens will be preliminarily identified by use of the Naïve 
Bayesian Classifier Tool of the Ribosomal Database Project82.83. The strength of 
association of individual microbial groups with treatment arms and/or main outcome 
measures will be measured by comparing the abundances of these groups using the non-
parametric Kruskal-Wallis test. Experimental reproducibility and inter- and intra-
individual similarities in microbial populations will be assessed by calculating and 
comparing the sequence-abundance-based Morisita-Horn index81, . In all statistical 
analyses 2-sided p-values less than 0.05 will be considered statistically significant. 
Results will be corroborated through the phylogeny-based UniFrac metric, which 
assesses population-wide differences in microbial lineages between different groups of 
subjects (SBS vs. control) 82. Stool-associated bacteria collected at BSI incidence will be 
compared to the species identified on blood culture to determine whether intestinal 
bacteria are potential sources for BSI. Alternatively, absence of particular species from 
the stool at the time of BSI diagnosis will indicate that lack of specific bacterial species 
from the stool predisposes to BSI. Tissue samples collected during endoscopy or surgery 
and intestinal fluid aspirated during endoscopy will be analyzed in parallel with stool 
samples in order to assess the use of stool samples as proxies for intestinal samples. 
Patterns will also be reviewed to assess for relationships to enteral feeding rate. 
 


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These methods have been used and demonstrated in 2 published studies by one of the 
collaborators/co-investigators 89, 90. 
 
 
Sample size  
This is a pilot study and will provide estimates of effect sizes and variability to be used to power 
a future definitive study.  The power of this pilot study was investigated based on the primary 
endpoint of BSI infection rate and the study was powered to detect a trend toward improvement 
in BSI rates.  For this study, we defined “trend” as a significant improvement tested at the one-
sided, 10% level of significance. A total of 18 patients per treatment group will be enrolled in the 
study.  Assuming a 10% dropout rate, it is expected that 16 patients per treatment group will 
complete the study.  A sample size of 16 patients per treatment group will provide at least 85% 
power to detect a trend if the true BSI rates are 75% versus 30% BSI rate at the one-sided 10% 
level of significance.  This calculation is based on the Fisher’s exact test for two proportions. 
A sample size of 16 patients per treatment group will also provide at least 80% power to detect a 
geometric mean ratio of 2.0 for the two treatment groups for the proinflammatory cytokines 
assuming a coefficient of variation of 0.7 using a two-sided, 5% level of significance test.     
We anticipate applying for a follow up R01 /U01 to attract other centers that will allow a larger 
more definitive study regarding the use of GLN in pediatric SBS. 
 
IX.   Data Management 
 
Each subject will be given a unique identifier. Only members of the study team will be able to 
link this identifier to the subject. No patient identifiers will be published. The specific data items 
to be captured will be stored in a secure area that will be a locked filing cabinet or in a password 
protected electronic file.  
 
REDCap (Research Electronic Data Capture) is a secure, web-based application designed 
exclusively to support data capture for research studies. This application will be used for data 
entry during the study. REDCap provides: 1) an intuitive interface for data entry (with data 
validation); 2) audit trails for tracking data manipulation and export procedures; 3) automated 
export procedures for seamless data downloads to common statistical packages (SPSS, SAS, 
Stata, R); and 4) procedures for importing data from external sources  
 
Deidentified samples and data will be retained until all the final data analysis and publication. 
Data/specimens/identifier will be destroyed at the earliest opportunity consistent with the 
research plan.  
 
Unused plasma, stool and mucosal tissue will be deidentified and stored for future analysis if the 
participant provides consent for this purpose. These will be used in the future for further analysis 
of bacterial DNA analysis as newer methods are developed in the field of pyrosequencing, 
nutrition metabolism and microbial DNA analysis. 
 
X.  Financing 


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Funding is by the NIH/NIDDK under the R-21 mechanism (Grant #1R21DK088027 - 01A1)  
pending IRB approval. The Division of Pediatric Gastroenterology and the CCTST will also 
provide ancillary support. GLN will be provided by Emmaus Medical Inc.  
 
 
 
 
 
 
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