Conditions: Clostridium Difficile Infection
Keywords: Clostridium difficile glutamine diarrhea alanyl-glutamine
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 7
Healthy volunteers: no

Brief summary:
The aim of this study is to test the efficacy of alanyl-glutamine supplementation in the treatment of C. difficile infection. We hypothesize that alanyl-glutamine when given with standard antibiotic treatment for C. difficile infection will decrease diarrhea, mortality and recurrent disease.

Detailed description:
This is a Phase II randomized, placebo-controlled, double-blinded, dose-ranging study to determine optimal effective dose and safety of AQ between 0, 4, 24, and 44 g doses administered orally for ten days concurrent with standard treatment (oral vancomycin at UVa) among first time incident cases of uncomplicated CDI in hospitalized persons age 50 and older. Our hypothesis is that AQ will reduce recurrence (primary outcome) and mortality (secondary outcome) at 60 days post-treatment. Furthermore, we hypothesize that alanyl-glutamine supplementation will be associated with decreased intestinal and systemic inflammation and improvement of intestinal microbial and metabolic profiles. We plan to enroll 260 patients, equally divided into 4 arms. Upon enrollment, participants will be randomized to either receive AQ at 4, 24, or 44 g or placebo (water). Study agent is administered once a day, orally or enterally, if feeding tube is present. Because we are enrolling subjects over a longer period of time, we will utilize block randomization to ensure that relative temporal balance is maintained throughout the trial. Participants will be followed up daily during treatment for adverse event monitoring and weekly for 60 days post-treatment for recurrences and survival. Blood, urine and stool specimens will be collected at days 0, 10 and 70 to assay for markers of inflammation and microbial and metabolic profiling.

The data set utilized for all initial baseline feature and demographic reporting will be the Intention to Treat Analysis Dataset, which will be comprised of all randomized participants. The primary dataset will be a Modified Intention to Treat Analysis Dataset for all endpoints, comprised of all participants who took at least one dose of study intervention (placebo or treatment), regardless of completeness of follow-up outcome data. The Safety Analysis Dataset will be all participants who took at least one dose of study intervention. The Per Protocol Analysis Dataset will be those patients who took at least 9 doses of study intervention for 9 days of the treatment period (10 days). Analysis will utilize ANOVA unless statistically significant differences in the distribution of baseline characteristics or features of non-normality are detected and relevant, at which point contingency utilization of ANCOVA, logistic regression, or other approaches as appropriate will be implemented. Treatment group level rates will be presented as incidence risk ratios relative to the control (placebo) group with 95% confidence intervals.

Safety endpoints will be evaluated on an individual AE by AE event via the DSMB and utilizing summary statistics during treatment and through duration of follow up. Adverse events will be presented by System Organ Class and will include information on start and stop date, severity, projected relationship, expectedness, and outcome and duration (the latter two after the event is considered to have concluded).

Arms:
arm 1: Alanyl-glutamine, 44g, taken by mouth daily for 10 days.

Interventions: intervention 1: Alanyl-glutamine
Intervention descriptions: intervention 1: Alanyl-glutamine, 44g, taken by mouth daily for 10 days

Protocol document:
Prot_SAP_000.pdf: 
 
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1 
Alanyl-glutamine supplementation of standard treatment for 
C. difficile infection: A single arm trial 
Protocol Number: UVAHS-144404 
PI of IND/IDE: Cirle Warren, MD 
IND/IDE Number:   121545 
NCT 02053350 
Principal Investigator: Cirle Warren, MD 
Medical Monitor: Cirle Warren, MD 
Draft or Version Number:  
 June 20, 2016 
 


 
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2 
STATEMENT OF COMPLIANCE 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies 
(45 CFR Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
 
ICH E6; 62 Federal Register 25691 (1997)  
 
University of Virginia Institutional Review Board 
 
University of Virginia School of Medicine Clinical Trial Office 
 
University of Virginia Cancer Center Office of Clinical Research Data and Safety Monitoring 
Committee 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 
 


 
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3 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Principal Investigator 
 
Signed: 
 
Date: 
 
 
Cirle Warren, MD 
 
 
 


 
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4 
TABLE OF CONTENTS 
 
Page 
Statement of Compliance ............................................................................................................... 2 
Signature Page ............................................................................................................................... 3 
Table of Contents ............................................................................................................................ 4 
List of Abbreviations ....................................................................................................................... 7 
Protocol Summary .......................................................................................................................... 9 
1 
Key Roles ............................................................................................................................. 12 
2 
Background Information and Scientific Rationale ............................................................... 13 
2.1 
Background Information ........................................................................................... 13 
2.1.1 
Description of the Study Agent(s)/Intervention(s) ....................................... 13 
2.1.2 
Summary of Previous Pre-clinical Studies .................................................. 13 
2.1.3 
Summary of Relevant Clinical Studies ........................................................ 14 
2.1.4 
Summary of Epidemiological Data .............................................................. 15 
2.2 
Rationale .................................................................................................................. 16 
2.3 
Potential Risks and Benefits .................................................................................... 17 
2.3.1 
Potential Risks ............................................................................................. 17 
2.3.2 
Known Potential Benefits ............................................................................. 17 
3 
Objectives ............................................................................................................................ 18 
3.1 
Study Objectives ...................................................................................................... 18 
3.2 
Study Outcome Measures ....................................................................................... 18 
3.2.1 
Primary Outcome Measures ........................................................................ 18 
3.2.2 
Secondary Outcome Measures ................................................................... 18 
4 
Study Design ........................................................................................................................ 19 
4.1 
Study Endpoints ....................................................................................................... 19 
4.1.1 
Primary Endpoint ......................................................................................... 19 
4.1.2 
Secondary Endpoints ................................................................................... 19 
4.1.3 
Exploratory Endpoints .................................... Error! Bookmark not defined. 
4.1.4 
Substudy Endpoints ....................................... Error! Bookmark not defined. 
4.2 
Substudies (if applicable)........................................... Error! Bookmark not defined. 
5 
Study Enrollment and Withdrawal ....................................................................................... 20 
5.1 
Subject Inclusion Criteria ......................................................................................... 20 
5.2 
Subject Exclusion Criteria ........................................................................................ 20 
5.3 
Strategies for Recruitment and Retention ............................................................... 21 
5.4 
Treatment Assignment Procedures ......................................................................... 21 
5.4.1 
Randomization Procedures ......................................................................... 22 
5.4.2 
Masking Procedures .................................................................................... 22 
5.4.3 
Reasons for Withdrawal ............................................................................... 22 
5.4.4 
Handling of Withdrawals .............................................................................. 22 
5.4.5 
Termination of Study .................................................................................... 22 
5.5 
Subject Compensation ............................................................................................. 22 
6 
Study Intervention/Investigational Product .......................................................................... 23 
6.1 
Study Product Description ....................................................................................... 23 


 
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5 
6.1.1 
Acquisition .................................................................................................... 23 
6.1.2 
Formulation, Packaging, and Labeling ........................................................ 23 
6.1.3 
Product Storage and Stability ...................................................................... 23 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ..................................................................................................................... 23 
6.3 
Modification of Study Intervention/Investigational Product for a ParticipantError! Bookmark not de
6.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) ... 23 
6.5 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product ..................................................................................................................... 24 
6.6 
Concomitant Medications/Treatments ..................................................................... 24 
6.6.1 
Prohibited Medications and Procedures...................................................... 24 
6.6.2 
Precautionary Medications and Procedures ............................................... 24 
6.7 
Prophylactic Medications and Procedures ................ Error! Bookmark not defined. 
6.8 
Rescue Medications................................................... Error! Bookmark not defined. 
7 
Study Schedule .................................................................................................................... 25 
7.1 
Screening ................................................................................................................. 25 
7.2 
Enrollment/Baseline ................................................................................................. 25 
7.3 
Follow-up .................................................................................................................. 25 
7.4 
Final Study Visit ....................................................................................................... 26 
7.5 
Early Termination Visit ............................................................................................. 26 
7.6 
Unscheduled Visit .................................................................................................... 26 
7.7 
Pregnancy Visit ........................................................................................................ 26 
8 
Study Procedures/Evaluations ............................................................................................ 27 
8.1 
Clinical Evaluations .................................................................................................. 27 
8.2 
Laboratory Evaluations ............................................................................................ 27 
8.2.1 
Clinical Laboratory Evaluations ................................................................... 27 
8.2.2 
Special Assays or Procedures ..................................................................... 28 
8.2.3 
Specimen Preparation, Handling, and Shipping ......................................... 28 
9 
Assessment of Safety .......................................................................................................... 29 
9.1 
Specification of Safety Parameters ......................................................................... 29 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters ............................................................................................................... 29 
9.2.1 
Adverse Events ............................................................................................ 29 
9.2.2 
Reactogenicity (for Vaccine Studies and Some Therapeutic Trials)Error! Bookmark not de
9.2.3 
Serious Adverse Events .............................................................................. 31 
9.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings .......................................................... 32 
9.3 
Reporting Procedures .............................................................................................. 35 
9.3.1 
Serious Adverse Events .............................................................................. 35 
9.3.2 
Regulatory Reporting for Studies Conducted Under a IND ........................ 37 
9.3.3 
Regulatory Reporting for Studies Not Conducted Under an INDError! Bookmark not defin
9.3.4 
Reporting of Pregnancy ............................................................................... 37 
9.4 
Type and Duration of Follow-up of Subjects after Adverse Events ........................ 37 
9.5 
Modification of Study Agent(s)/Intervention(s) for a Participant ............................. 37 


 
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6 
9.6 
Halting Rules ............................................................................................................ 37 
9.7 
Stopping Rules for an Individual Participant/Cohort ............................................... 38 
9.8 
Premature Withdrawal of a Participant .................................................................... 38 
9.9 
Replacement of a Participant Who Discontinues Study Treatment ........................ 38 
9.10 
Safety Oversight (ISM plus SMC or DSMB) ............................................................ 39 
10 
Clinical Monitoring ................................................................................................................ 42 
10.1 
Site Monitoring Plan ................................................................................................. 40 
10.2 
Safety Monitoring Plan ............................................................................................. 40 
11 
Statistical Considerations .................................................................................................... 41 
11.1 
Study Hypotheses and Design ................................................................................ 43 
11.2 
Sample Size Considerations ................................................................................... 43 
11.3 
Randomization/Stratification .................................................................................... 43 
11.4 
Safety Monitoring ..................................................................................................... 43 
 
        11.4.1 Severe Adverse Events………………………………………………………….44 
 
        11.4.2 30-Day Mortality…………………………………………………………………..44 
         11.5    Futility Analysis…………………………………………………………………………   44 
         11.6    Final Analysis……………………………………………………………………………  45 
12 
Source Documents and Access to Source Data/Documents ............................................. 43 
13 
Quality Control and Quality Assurance ............................................................................. 437 
14 
Ethics/Protection of Human Subjects ................................................................................ 448 
14.1 
Ethical Standard ....................................................................................................... 45 
14.2 
Institutional Review Board ....................................................................................... 45 
14.3 
Informed Consent Process ...................................................................................... 45 
14.3.1 Informed Consent/Assent Process (in Case of a Minor) ............................. 45 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations)Error! Bookmark not defined
14.5 
Subject Confidentiality ............................................................................................. 45 
14.6 
Study Discontinuation .............................................................................................. 45 
14.7 
Future Use of Stored Specimens ............................................................................ 46 
15 
Data Handling and Record Keeping .................................................................................... 47 
15.1 
Data Management Responsibilities ......................................................................... 47 
15.2 
Data Capture Methods ............................................................................................. 47 
15.3 
Types of Data ......................................................................................................... 471 
15.4 
Timing/Reports ......................................................................................................... 48 
15.5 
Study Records Retention ......................................................................................... 48 
15.6 
Protocol Deviations .................................................................................................. 48 
16 
Publication Policy ............................................................................................................... 483 
17 
Literature References .......................................................................................................... 50 
SupplementS/Appendices ............................................................................................................ 55 
Appendix A: Schedule of Events .................................................................................................. 55 
 
 


 
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7 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
AG 
L-Alanyl-L-Glutamine 
AHRQ 
Agency for Healthcare Research and Quality 
CDC 
Centers for Disease Control and Prevention 
CDI 
Clostridium difficile infection 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTO 
Clinical Trials Office 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMC 
Data and Safety Monitoring Committee 
eCRF 
Electronic Case Report Form 
ELISA 
Enzyme-Linked Immunosorbent Assay 
EMR 
Electronic Medical Record 
FDA 
Food and Drug Administration 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IDSA 
Infectious Diseases Society of America 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
MedDRA  
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NDA 
New Drug Application 
NEJM 
New England Journal of Medicine 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
PCR 
Polymerase Chain Reaction 


 
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8 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SHEA 
The Society for Healthcare Epidemiology of America 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
TPN 
Total Parenteral Nutrition 
US 
United States 
UVA 
University of Virginia 
WHO 
World Health Organization 
 
 
 


 
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9 
PROTOCOL SUMMARY 
Limit to 1-2 pages 
 
Title: 
Alanyl-glutamine supplementation of standard treatment for 
C. difficile infection:  A single arm trial 
Phase: 
II 
Population: 
The study population will include 43 participants aged ≥ 18 
years,hospitalized patients or UVA outpatients.  Both genders 
will be included.  Participants must have mild to moderate or 
severe uncomplicated C. difficile infection, and be able to 
participate in follow-up.  The trial will be held in Central Virginia. 
Number of Sites: 
One site – University of Virginia Health System 
Study Duration: 
The study duration is four years. 
Subject Participation 
Duration: 
Each individual participant will be followed for a total of 6 
months post-treatment. 
Description of Agent or 
Intervention: 
All participants will receive 44 grams of alanyl-glutamine 
dipeptide .  The supplement will be in a powder form that will be 
mixed with any juice or fluid and taken by mouth or enteral 
feeding tube. 
Objectives: 
 
Primary Outcome:  
Clinical failure (death and/or CDI recurrence assessed at D40 
and/or lack of clinical cure as described in Section 3.2.1) –
Participants will be followed up by telephone (or in person if still 
hospitalized) at days 40, 70, 190 post-enrollment.  They will be 
asked about recurrences of diarrhea and diagnoses of CDI in 
between follow up calls.  The electronic medical record (EMR) of 
the UVa Health System will be reviewed during these telephone 
or follow up visits to identify clinic or hospital encounters for 
diarrhea or CDI that may have occurred in the interim. 
Secondary Outcomes :   
CDI recurrence at days  70 and 190 
Duration of diarrhea – Duration of diarrhea will be determined by 


 
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10 
daily visits or phone calls (if hospitalized participant is 
discharged) while participant is on the study agent (D1 to D10).   
Mortality at days 70 and 190– Mortality (CDI and all-cause) will 
be determined during the follow-up phone calls and by reviewing 
the EMR.   
Description of Study 
Design: 
See Schematic of Study Design below. 
Estimated Time to 
Complete Enrollment: 
100% of participants will be enrolled at 48 months. 
 


 
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11 
Schematic of Study Design: 
 
 
 
 


 
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12 
1 
KEY ROLES 
Principal Investigator:   
Cirle Warren, MD 
Medical Monitor: 
Cirle Warren, MD 
Institutions: 
University of Virginia Health System 
Address: Division of Infectious Diseases and International 
Health, 345 Crispell Drive, Charlottesville, VA 22908 
Phone Number: 434-924-9676 
Fax Number: 434-924-0075 
Email: ca6t@virginia.edu  
 
 
Co-investigators: 
 
 
 
 
 
 
 
Jae Shin, M.D. 
 
 
 
University of Virginia Health System  
Address: Division of Infectious Diseases and International Health, 
345 Crispell Drive, Charlottesville, VA 22908 
Phone Number: 434-924-9672 
Fax Number: 434-924-0075 
 
 
 
Email: jhs9ff@virginia.edu 
 
Clinical Pharmacist:  
Amy Adams, PharmD 
 
 
 
Address: Pharmacy Services, P. O. Box 800674,  
 
 
 
Charlottesville, VA  22908 
 
 
 
Phone Number: 434-982-1048 
 
 
 
Fax Number: 434-982-2900 
 
 
 
Email: apa4n@hscmail.mcc.virginia.edu  
 
Statistician:  
 
Gina Petroni, PhD 
 
 
Address: Public Health Sciences, P. O. Box 800717, 
Charlottesville, VA  22908 
 
 
 
Phone Number: 434-924-8363 
 
 
 
Fax Number: 434-243-5787 
 
 
 
Email: grp4c@virginia.edu  


 
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13 
2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
 
2.1.1 Description of the Study Agent(s)/Intervention(s) 
AG is a dipeptide with a glutamine amino group joined to an alanyl residue.  It has the 
following chemical structure: C8H15N3O4.  It is a non-animal product available in the form of 
white crystals or crystalline powder.  Ziegler et al. performed a series of dose response studies 
to study clinical safety, pharmacokinetics, and metabolic effects of glutamine given to humans.  
Participants in the studies included healthy individuals as well as bone marrow transplant 
patients.  Glutamine was given intravenously as well as enterally at different doses.  No adverse 
events were detected.1  Another study which evaluated post-operative patients receiving TPN 
supplemented with AG did not reveal any adverse effects compared to controls which received 
TPN without AG.2  These 2 studies were included in a review of 4 clinical trials specifically 
looking at safety of glutamine by Peter Garlick et al (2001 in The Journal of Nutrition) who 
concluded that no adverse effects of glutamine have been demonstrated in doses of up to 50-60 
grams/day.  While the long-term effects of chronic supplementation (≥ 30 days) are unknown, 
no adverse events have been demonstrated in these short-term studies.3 
The chemical properties of free glutamine limit its use in clinical trials.  Problems include 
instability during sterilization and storage as well as limited solubility.  The breakdown products 
of free glutamine include glutamate and ammonia.  AG is a stable, highly soluble dipeptide.4  In 
a study from the United Kingdom, glutamine absorption was studied in men given different 
formulations of glutamine – L-glutamine, AG, and a hydrolyzed wheat protein containing L-
glutamine.  Higher plasma levels of glutamine were measured in the men given AG.5   
 
2.1.2 Summary of Previous Pre-clinical Studies 
Glutamine is an amino acid that serves as an important energy source in the body, 
particularly for enterocytes.  It is a non-essential amino acid in healthy people but is considered 
“conditionally essential” during critical illness, injury, and other stressful states.6 Utilization is 
increased during these times.  In cellular studies involving small intestinal cell lines, glutamine 
was found to stimulate cell proliferation and inhibit apoptosis.7-9  Animal models have shown that 
glutamine and glutamine dipeptide (e. g, AG) enhance gut repair mechanisms,10,11 decrease 
enteritis/colitis,12,13 and reduce bacterial translocation.14,15  Glutamine-enriched tube feeding has 
been shown to limit weight loss in rats after small bowel resection.  This study also 
demonstrated an increase in ileal hyperplasia in the rats receiving a glutamine-enriched diet, 
suggesting that glutamine may improve intestinal adaption after surgical resection.16 


 
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14 
 
In our laboratory, the cellular effects of C. difficile toxins as well as AG supplementation 
have been studied extensively.  The toxins exert their effects by inhibiting Rho proteins involved 
in maintaining cytoskeletal structure and by activating caspase enzymes involved in apoptosis.  
Rat intestinal cells treated with C. difficile toxin A and AG demonstrate an increase in Rho 
expression as well as a reduction in apoptosis and cellular damage.17,18  In studies using rabbit 
and murine cecal loops exposed to toxin A, AG supplementation results in decreased ileal 
secretion and epithelial injury.19  AG decreases apoptosis and inhibits activation of caspase 8 in 
human intestinal epithelial cells.20  In animal studies, AG has been shown to have beneficial 
effects in CDI.  In a murine model of CDI, animals who received vancomycin initially had 
improved clinical scores and survival during treatment.  However, after vancomycin was 
discontinued, 62% of mice experienced CDI recurrence and mortality rates were similar to the 
untreated control group.21  Vancomycin treatment supplemented with AG resulted in milder 
symptoms and improved mortality.  Histopathologic analysis revealed less inflammation and 
fewer apoptotic cells.22 
 
2.1.3 Summary of Relevant Clinical Studies 
Numerous clinical trials have evaluated the effects of glutamine.  In a randomized, 
double-blind pilot trial at the University of Colorado, researchers assessed the safety and 
feasibility of oral glutamine administration.  Patients undergoing elective cardiac surgery 
(requiring cardiopulmonary bypass) were given oral AG or maltodextrin daily for 3 days pre-
operatively.  Serum troponin, creatine kinase, and myoglobin levels were measured during the 
perioperative period and clinical outcomes were recorded.  The patients receiving AG had lower 
levels of these serum markers as well as fewer clinical complications.  The authors 
demonstrated that this therapy is safe and feasible.  A larger clinical trial is needed to evaluate 
the clinical efficacy or glutamine supplementation prior to cardiac surgery.23 
In a randomized, double-blind, placebo controlled trial from northern Brazil, 10 days of 
oral AG supplementation improved intestinal absorption in a group of HIV-positive patients.  
This was demonstrated by an increase in urinary mannitol excretion.24  The same researchers 
performed another randomized, double-blind placebo controlled study to evaluate the effects of 
oral AG supplementation on absorption of anti-retroviral agents.  The study showed that HIV-
positive patients receiving glutamine experienced less diarrhea compared to the controls who 
received glycine.  Higher serum levels of anti-retroviral drugs were measured in the patients 
who received glutamine.25 
In a pilot study at Indiana University, HIV-positive men with diarrhea were treated with 
soluble fiber and probiotics.  Glutamine was added to the regimen if their diarrheal symptoms 
did not resolve.  The men who received glutamine supplementation experienced an 
improvement in their diarrhea.26  Of note, the company which supplied the glutamine for the 
study, Ajinomoto™, will also supply the AG for our study. 
In cancer patients, glutamine has been evaluated as a potential supplement in managing 
the dose-limiting side effects of chemotherapy.  It has been associated with a reduction in 
irinotecan-related diarrhea in patients with colon cancer.27  In a non-randomized study of 


 
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patients receiving paclitaxel, the participants given glutamine experienced less weakness and 
sensory loss compared to controls.28  In another group of patients receiving oxaliplatin, less 
neurotoxicity was observed.29 
 
2.1.4 Summary of Epidemiological Data 
While CDI is classically associated with antibiotic exposure, an increasing number of 
cases have been seen in individuals who have not taken antibiotics, but have been exposed to 
the healthcare system.  The increase in incidence and mortality has been attributed to the 
emergence of the BI/NAP/027 strain.  Government agencies including the Centers for Disease 
Control and Prevention (CDC) and the Agency for Healthcare Research and Quality (AHRQ) 
are closely monitoring this infection.  The Healthcare Cost and Utilization Project (HCUP), 
sponsored by the AHRQ, has published data on the rates of CDI.  HCUP consists of a group of 
databases which contain information related to diagnoses, procedures, discharge status, and 
patient demographics.  Per data from HCUP, there were 336,000 hospitalizations which 
involved CDI in 2009.  This is an increase from 139,000 hospitalizations in 2000.  CDI as the 
principal diagnosis increased from 32,800 hospital stays to 110,600 during the same time 
period.30  Patients hospitalized with CDI are typically more ill than other hospitalized patients.  In 
addition to diarrhea, the infection is associated with dehydration, electrolyte imbalances, 
sepsis/septic shock, renal failure, intestinal perforation, and toxic megacolon. 
Data collected the by CDC’s Emerging Infections Program indicates that 94% of CDI 
was associated with some type of healthcare exposure (Figure 1).  25% of patients have 
symptoms while hospitalized whereas the remaining 75% develop symptoms in a nursing home 
or after receiving care at a clinic.31  When this latter group of patients presents at an acute care 
hospital, they are considered to have CDI that is ‘present on admission.’  Depending upon the 
diagnostic test used at the facility (i.e. toxin assay or PCR), the diagnosis may not be confirmed 
for 24-48 hours.  During that period, the patients may not be placed on appropriate isolation 
precautions and the risk for transmission to other patients by healthcare workers is increased.  
CDI that is ‘present on admission’ serves as an important source of intra-hospital transmission 
and is a risk factor for higher hospital-acquired CDI rates.31  The incubation period for CDI is 
approximately 2-3 days so patients are more likely to have acquired CDI in the setting in which 
symptom began. 


 
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16 
 
Source: CDC31 
 
2.2 
Rationale 
Hypothesis: Supplementation of standard CDI treatment with AG will decrease recurrence, 
improve symptoms, and reduce mortality associated with the disease. 
Participants will be given AG 44 grams by mouth (or by enteral feeding tube) daily for 10 
days.  AG  will be prepared in powder form, mixed with any juice or fluid, and taken orally (or 
enterally).  These supplements will be given in addition to standard antibiotic treatment for CDI.  
The dose was chosen based on a prior study.25  Daily dosing was chosen for ease of 
administration in participants who are no longer hospitalized – i.e. the participant will be 
responsible for taking the drug rather than administration by nursing staff.  The duration of 
intervention is 10 days, regardless of the duration of antibiotic treatment for CDI. 
Our study population includes all adult inpatients, including those diagnosed with cancer 
– currently diagnosed or a prior history as well as outpatients seen in UVA clinics.  These 
individuals have many exposures to the healthcare system, a known risk factor for acquiring C. 
difficile.  This particular population with malignancy is also at an increased risk for the infection 
due to being immunocompromised from chemotherapy, radiation, or surgical procedures. 


 
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17 
2.3 
Potential Risks and Benefits 
2.3.1 Potential Risks 
In a study published in the New England Journal of Medicine, critically ill patients with 
multiorgan failure were given glutamine supplementation (intravenously and enterally) or 
placebo.  At 28 days, a trend toward increased mortality was noticed in the glutamine group 
compared to controls (32.4% vs 27.2%; adjusted odds ratio 1.28; 95% confidence interval, 1.00-
1.64, P=0.05).  In-hospital mortality and mortality at 6 months were higher among patients given 
glutamine.32  Serious adverse events recorded during this study include: nervous system 
disorders (encephalopathy, intracranial hemorrhage, seizures, stroke), respiratory disorders 
(respiratory distress), cardiac disorders (acute cardiac failure, cardiac arrest, cardio-respiratory 
arrest, pericarditis, arrhythmia), gastrointestinal diseases (acute pancreatitis, gastrointestinal 
hemorrhage, ileus, ischemic bowel), hepatobiliary disorders (cholestasis), vascular disorders 
(peripheral vascular disease), eye disorders (blindness), and procedural complications 
(embolism at central line, tube malposition).  There were no differences among the groups with 
respect to serious adverse events (P=0.83).32 
Another study of 7 patients identified liver toxicity as a potential risk associated with 
glutamine supplementation.  However, these patients were receiving TPN.  The authors 
hypothesized that the elevation in transaminases was possibly a complication of intravenous 
nutrition.  No control group was used in the study.33  Patients with liver disease may have 
decreased ammonia excretion and may be predisposed to the accumulation of ammonia, a 
breakdown product of glutamine.  Similarly, patients with kidney disease may have decreased 
renal excretion of ammonia and glutamic acid.34 
Of note, in this proposal, patients who are critically-ill are excluded.  Alanyl-glutamine will 
be given orally or enterally only and limited to 10 days of administration.  Per review of clinical 
trials specifically addressing safety, glutamine at a dose of up to 60 g/day given as short as 4 
hours to up to 30 days, administered to either healthy volunteers, bone marrow transplant 
patients, or preterm neonates, was considered safe and not associated with adverse events3. 
2.3.2 Known Potential Benefits 
Based upon prior research involving cellular and animal models, potential benefits 
include decreased C.difficile toxin-mediated effects subsequently leading to less infection-
induced inflammatory damage and increased epithelial repair.  Participants may experience 
decreased diarrhea, reduced recurrence of CDI, and improved survival.  These latter benefits 
may lead to an improved quality of life and fewer hospitalizations.  
 


 
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18 
3 
OBJECTIVES 
3.1 
Study Objectives 
We propose to perform a single arm trial to estimate the effect of AG supplementation in 
participants receiving antibiotic treatment for CDI.  The dosing regimen for AG is as follows: 44 
grams of AG given by mouth (or by enteral feeding tube) daily for ten days.  The primary 
objective is to estimate the effect of AG on day 40 clinical failure.  Secondary objectives include 
estimation of CDI recurrence at days 70 and 190, duration of diarrhea, and CDI-associated and 
all-cause mortality.  The exploratory objectives (only for inpatient participants) include 
determining the extent of (1) intestinal inflammation (fecal lactoferrin), (2) intestinal tissue 
integrity, (3) systemic inflammation, and (4) intestinal infection. 
3.2 
Study Outcome Measures 
3.2.1 Primary Outcome Measures 
Day 40 clinical failure – clinical failure includes death or CDI recurrence assessed 40 days post 
initiation of study agent  or lack of clinical cure as described below. 
Death – death from any cause. 
CDI recurrence –CDI recurrence is determined by documentation of diarrhea and stool positive 
for C. difficile toxin.   
Clinical cure – resolution of diarrhea (i.e., 3 or fewer unformed stools for 2 consecutive days), 
with maintenance of resolution for the duration of therapy or marked reduction in the number of 
unformed stools at the end of treatment and no further requirement (in the investigator’s 
opinion) for therapy for CDI as of the second day after the end of the course of therapy (36).   
3.2.2 Secondary Outcome Measures 
CDI recurrence at days 70 and 190 
Duration of diarrhea – Duration of diarrhea will be determined using a symptom diary of 
gastrointestinal symptoms.  Mortality – Mortality (CDI and all-cause) will be determined during 
the follow-up phone calls and by reviewing the EMR.   
Mortality – Mortality (CDI and all-cause) will be determined during the follow-up phone calls and 
by reviewing the EMR.   


 
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4 
STUDY DESIGN 
We propose to perform a single arm trial to estimate the effect of AG supplementation on 
the resolution of diarrhea during treatment, on recurrence of CDI after treatment, and on 
mortality at Days 40+7, 70+7 and 190+7 post-enrollment. The study will include hospitalized 
patients or outpatients seen in UVA clinics. 
 
4.1 Study Endpoints 
 
4.1.1 Primary Endpoint 
CDI recurrence – The primary outcome will be measured by monitoring length of time to CDI 
recurrence post-enrollment.  CDI recurrence is determined by documentation of diarrhea and 
stool positive for C. difficile toxin.  Participants will be followed up by telephone (or in person if 
still hospitalized) at Days 40, 70 and 190 post-enrollment.  During these “visits” or telephone 
calls, the participant and investigator will refer to the symptom diary for recurrence of diarrhea 
and associated symptoms, clinic/doctor visit or not, stool testing or any interventions that may 
have occurred since the last encounter. The EMR will be reviewed during these telephone or 
follow up visits to identify clinic or hospital encounters for diarrhea or CDI that may have 
occurred in the interim. For consultation and stool testing done outside of the UVA Health 
System, we shall obtain documentation of the clinic/hospital visits and test results. 
 
4.1.2 Secondary Endpoints 
Duration of diarrhea – Duration of diarrhea associated with the initial CDI diagnosis will be 
determined using a symptom diary of gastrointestinal symptoms.  This diary will be given to the 
participants at enrollment.  If the participant is discharged during the treatment period (days 1 to 
10), the participant will be called daily to ask for occurrence of diarrhea and other symptoms 
such as abdominal pain, nausea and vomiting, and others. Mortality – Mortality (CDI and all-
cause) will be determined during the follow-up phone calls and by reviewing the EMR.   
 


 
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20 
5 
STUDY ENROLLMENT AND WITHDRAWAL 
5.1 
Participant Inclusion Criteria 
Participants must meet all of the inclusion criteria in order to be eligible to participate in the 
study.   
Inclusion Criteria 
• 
Adult of either gender, 18 years or older 
• 
C. difficile positive stool 
• 
Diarrhea at time of stool testing 
• 
Within 72  hours of receiving either oral metronidazole for mild-moderate disease or oral 
vancomycin for severe uncomplicated disease 
• 
Admitted in the hospital or outpatient at the time of enrollment  
• 
Ability to provide informed consent or if with cognitive impairment, has a surrogate 
• 
Have an understanding of study procedures 
• 
Ability to comply with study procedures for the duration of study agent administration 
Contraceptive methods – Given the current rates of CDI at UVA Health System, we expect that 
most participants will be beyond their reproductive years (mean age 61 years).  Female 
participants will be excluded from the trial if they become pregnant.  Based upon current 
studies, we do not expect any adverse events to the fetus.  Female participants can use 
whichever method of contraception that they prefer. 
 
5.2 
Participant Exclusion Criteria 
Participants that meet any of the exclusion criteria at baseline will be excluded from study 
participation.  
Exclusion Criteria 
• 
Hypotension or shock 
• 
Megacolon or moderate to severe ileus 
• 
Acute abdomen 
• 
Severe leukocytosis (WBC > 25,000 cells /µL) 
• 
Admission to intensive care unit on enrollment 


 
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21 
• 
Inability to tolerate oral or enteral medication 
• 
Other known etiology of diarrhea (e.g. other enteric pathogen, other intestinal disease) 
• 
Inflammatory bowel disease (e.g. Crohn’s disease, ulcerative colitis) 
• 
Enrollment in another clinical trial that may interfere with this protocol 
• 
Receiving other alternative treatment for CDI during course of study agent administration 
(e.g. antibiotics other than oral metronidazole or oral vancomycin; probiotics; 
immunoglobulin therapy; fecal transplant) 
• 
Cirrhosis or ALT > 3x normal. 
• 
End stage renal disease or in participants with or without known kidney disease, 
estimated Creatinine clearance or glomerular filtration rate of < 30 ml/min, even after 
adequate rehydration. 
• 
Pregnancy 
• 
Unavailable for follow up visits 
• 
Absolute neutrophil count of < 500/µL 
• 
History of organ or hematologic transplantation within the past year 
• 
 
 
 
 
5.3 
Strategies for Recruitment and Retention 
 
Patients will be recruited if they meet the criteria listed above upon review of the medical 
record.  While receiving the treatment, the study team will follow the participant daily if 
hospitalized.  Outpatients or discharged inpatients  will be followed-up by phone calls (or in 
person if still hospitalized) daily if discharged before day 10 and at Days 40, 70 and 190 post-
enrollment. 
5.4 
Treatment Assignment Procedures 
Participants who meet all eligibility criteria and consent to participation will be stratified 
according to disease severity (mild-moderate or severe disease) and recurrent CDI (yes or no).  
Mild-moderate disease is defined as a leukocyte count of ≤ 15,000 cells/µL and a serum 
creatinine <1.5 times the premorbid level.  Severe disease is defined as a leukocyte count of > 
15,000 cells/µL or a serum creatinine level ≥ 1.5 times the premorbid level.35   


 
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22 
5.4.1 Randomization Procedures 
There is no randomization for this trial.  All participants enrolled in the study will  receive the 
study agents described in Section 11.3. 
5.4.2 Masking Procedures 
None.  
5.4.3 Reasons for Withdrawal 
A study participant will be discontinued from protocol treatment if any of the following occurs: 
 
Any clinical adverse event (AE), laboratory abnormality, intercurrent illness, or other medical 
condition or situation occurs such that continued protocol treatment would not be in the best 
interest of the subject. 
 
Development of any exclusion criteria  
Participants are free to withdraw from participating in the study at any time.  Participants may 
also withdraw voluntarily from receiving the study intervention for any reason. 
5.4.4 Handling of Withdrawals 
For participants who voluntarily withdraw from the study, we will continue to follow them (if they 
agree) in the same manner as trial participants – daily follow-up for the 10 days after starting the 
treatment and telephone follow-up at Days 40, 70 and 190 (using the same questions on the 
symptom diary) post-enrollment.  
5.4.5 Termination of Study  
Reasons for termination of the study treatment include development of laboratory toxicities not 
attributable to other causes, study closure as recommended by the UVA IRB or Cancer Center 
DSMC, and increased number of SAEs as described in Section 9.2.3. 
5.5 
Participant Compensation 
Participants will be given $50 to compensate for time spent during follow-up visits.  $25 will be 
mailed upon completion of Day 10 follow-up and another $25 will be mailed following Day 190 
follow-up. 
 


 
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23 
6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
6.1 
Study Product Description 
6.1.1 Acquisition 
We will purchase the Study Agent- AG, from Ajinomoto™. 
6.1.2 Formulation, Packaging, and Labeling 
AG  will be in white crystalline formulation and will be appropriately labeled as L-alanyl-
L-glutamine in 20 kgs packages when received from Ajinomoto.  This stock agent will be 
directly shipped to the UVA Pharmacy Services.  Upon receipt of the study agent, the 
Pharmacy will repackage the agent into 44 g (AG) or  doses placed in coded  containers.  
6.1.3 Product Storage and Stability 
The product is shipped at ambient temperature and humidity.  It must be stored in a dry 
place at ambient temperatures away from strong odors.  The retest date is 48 months 
from the date of manufacture. 
6.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
The Pharmacy will prepare the 44g packets of AG  in  containers and coded appropriately.  
Once in containers , agents will be discarded if not used within 6 months.  The agents will be 
dissolved in 250ml of any juice or fluid, and will be administered by mouth or enteral tube daily 
for 10 days. There will be no make up for missed doses. 
6.3 
Accountability Procedures for the Study 
Intervention/Investigational Product(s) 
The study agent will be directly shipped, repackaged, stored in our pharmacy (drug repository) 
and distributed on as needed basis.  The pharmacy will dispense the study agent in containers, 
daily while participants are in the hospital and will prepare the  containers with study agent to be 
sent home for participants discharged before day 10 post-enrollment or for outpatient 
participants  Any unused products will be transferred to our research laboratory at the 
conclusion of the study. 


 
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24 
6.4 
Assessment of Participant Compliance with Study 
Intervention/Investigational Product  
While participants are hospitalized, the ward nurse-in-charge of the participant for that day will 
directly observe administration of the study agents. A clinical pharmacist will also follow-up daily 
with the participant in the hospital while taking the study agent per standard of care. A member 
of the study team will visit the participant daily to confirm intake of the study agent from the 
nurse and documentation in EPIC and record intake of study agent daily in the CRF. For 
outpatients and discharged inpatients,if the participant is discharged before completion of 
treatment a member of the study team will call the participant daily, to ask if study agent was 
taken, throughout the rest of the 10 day treatment period. 
6.5 
Concomitant Medications/Treatments 
6.5.1 Prohibited Medications and Procedures  
 
Prohibited medications/procedures include investigational drugs in other trials, anti-C. 
difficile antibiotics other than oral metronidazole and oral vancomycin, probiotics, 
immunoglobulin therapy, and fecal transplant. Patients who are on prohibited 
medications and procedures are excluded from the study. 
 
6.5.2 Precautionary Medications and Procedures 
 
We do not expect any drug-study agent interaction.  Any medications or procedures not 
mentioned under the exclusion criteria are allowed. 


 
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25 
7 
STUDY SCHEDULE 
7.1 
Screening  
Study researchers will review clinical microbiology laboratory reports in hospital’s 
Microbiology laboratory daily to identify patients whose stool tested positive for C. difficile.  Both 
genders will be eligible for the study. The EMR will be reviewed to determine if the patient meets 
the inclusion and exclusion criteria listed in Sections 5.1 and 5.2.  If the patient meets eligibility 
criteria, the team member will meet with the patient to discuss the trial including the purpose of 
the study, the study intervention and other study procedures including follow-up visits, specimen 
collection, time commitment, and compensation.  Signed informed consent will be obtained from 
all participants.  Participants will be consented as long as the study agent can be administered 
within 72 hours of initiation of oral metronidazole or oral vancomycin. 
7.2 
Enrollment/Baseline 
The EMR will be used to assess whether or not the patient meets eligibility criteria and 
may be enrolled.  The criteria are listed in Sections 5.1 and 5.2.  At baseline, the participant will 
be asked questions regarding his current gastrointestinal symptoms.    The supplement will be 
taken by mouth or by enteral tube, if applicable.  It is unlikely that the participant will experience 
any adverse reactions to the intervention so vital signs will be monitored as ordered by the 
treating physician during the admission process.  
7.3 
Follow-up  
Please refer to the Schedule of Events in Appendix A for a list of the follow-up visits.  
The Schedule of Events also includes which labs will be drawn at each visit.  For outpatients, or 
 inpatients, if  discharged during the treatment period (days 1 to 10), the participant will be called 
daily to ask for occurrence of diarrhea and other symptoms such as abdominal pain, nausea 
and vomiting, and other gastrointestinal symptoms. The participant and the investigator will refer 
to the symptom diary for Days 40, 70 and 190-follow up “visits” or telephone calls, for 
recurrence of diarrhea and associated symptoms, clinic/doctor visit or not, stool testing or any 
interventions that occurred.   The EMR will be reviewed during the visits to identify clinic or 
hospital encounters for diarrhea or CDI that may have occurred in the interim.  We will also 
assess AEs at the follow-up visits during the treatment period.  Allowable windows for follow-up 
visits will include +2 days for Day 10 follow-up/specimen collection and +1 week for Days 40, 70 
and 190 follow-up visits. 


 
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26 
7.4 
Final Study Visit 
The final study visit (via telephone call) will occur at day 190 post-enrollment.  The 
participant will be asked about recurrence of diarrhea or CDI diagnoses as described above 
(Section 7.3).  The EMR will be reviewed to identify clinic or hospital encounters for diarrhea or 
CDI that may have occurred in the interim. 
7.5 
Early Termination Visit 
There are no specific evaluations which should be done at a termination visit. 
7.6 
Unscheduled Visit  
Participants will be evaluated at the Infectious Diseases Clinic at UVA. 
7.7 
Pregnancy Visit 
As mentioned previously, given the mean age of participants with CDI, pregnancy is 
unlikely to be a common event.  Participants who become pregnant will be excluded from the 
trial and will continue routine prenatal care with their obstetrician. 
 


 
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27 
8 
STUDY PROCEDURES/EVALUATIONS 
8.1 
Clinical Evaluations 
Medical History – The EMR will be reviewed as part of the enrollment process to determine if 
the patient meets the inclusion and exclusion criteria (listed in Sections 5.1 and 5.2).  At each 
follow up visit, the participant will be interviewed (via telephone or in person) about the presence 
of any gastrointestinal symptoms or CDI diagnoses.  The EMR will also be reviewed during the 
follow-up visits to identify any clinic or hospital encounters for diarrhea or CDI that may have 
occurred in the interim. 
Medications history – During enrollment, the participant will be asked about currently taken 
medications including prescription and over-the-counter medications.  At each follow-up visit, 
the participant will be asked about new medications (prescription and over-the-counter). 
Physical examination – The participant’s vital signs will be reviewed – blood pressure, heart 
rate, respiratory rate, and temperature. 
Review of symptom diary – The participant will be provided with a symptom diary during 
enrollment.  The symptom diary will be reviewed at each follow-up visit. 
8.2 
Laboratory Evaluations 
8.2.1 Clinical Laboratory Evaluations 
University of Virginia Clinical Laboratory 
Hematology: White blood cell count, Red blood cell count, Hemoglobin, Hematocrit, 
platelet count 
Biochemistry: Sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose, 
albumin, total bilirubin,  alkaline phosphatase, AST, ALT  erythrocyte sedimentation rate, 
C-reactive protein, and if needed, serum pregnancy test (HCG qualitative) 
University of Virginia Clinical Microbiology Laboratory 
Stool: C. difficile toxin B PCR 
Blood volume: 5-10ml at baseline will be collected if there are no leftover sera for add-on 
laboratory tests for screening purposes. Stool volume: Leftover stool specimens from baseline 
C.difficile positive sample will be collected from the Clinical laboratory. 


 
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28 
 
**In order to exclude pregnant patients, female participants <50 years of age will be questioned 
regarding prior surgeries including tubal ligation and hysterectomy.  The EMR will also be 
reviewed.  For those who may be pregnant, a serum pregnancy test will be completed within  7 
days prior to study agent administration.  Results must be available prior to administration of the 
study product. 
8.2.2 Special Assays or Procedures 
8.2.3 Specimen Preparation, Handling, and Shipping 
8.2.3.1 
Instructions for Specimen Preparation, Handling, and Storage 
Stool – On Day 0, any remaining stool will be collected from the Clinical Microbiology 
Laboratory within 24 hours of collection date. There will be no additional stool collection 
if specimens are not available from the clinical laboratory. It will be brought to the 
Warren Lab, de-identified, coded, and stored in the -80°F freezer until needed for future 
studies.  Future studies include lactoferrin assay, C. difficile toxin B PCR, C. difficile toxin 
A and B ELISA, C. difficile culture and ribotyping.. 
8.2.3.2 
Specimen Shipment 
 


 
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29 
9 
ASSESSMENT OF SAFETY 
9.1 
Specification of Safety Parameters 
CDI recurrence – CDI recurrence will be assessed at Days 40, 70 and 190 post-enrollment.  
CDI recurrence is determined by documentation of diarrhea and stool positive for C. difficile 
toxin.  Participants will be followed up by telephone (or in person if still hospitalized) at Days 40, 
70 and 190 post-enrollment.  They will be asked about recurrences of diarrhea and diagnoses of 
CDI in between follow up calls.  The EMR will be reviewed during these telephone or follow up 
visits to identify clinic or hospital encounters for diarrhea or CDI that may have occurred in the 
interim. 
Duration of diarrhea – Duration of diarrhea will be assessed daily while participant is on the 
study agent.  This will be performed by actual visits while participant is in the hospital or by 
phone calls if the participant has been discharged prior to Day 10 or is an outpatient participant. 
  
Mortality – Mortality (CDI and all-cause) will be determined during the follow-up phone calls and 
by reviewing the EMR.   
9.2 
Methods and Timing for Assessing, Recording, and 
Analyzing Safety Parameters 
9.2.1 Adverse Events 
Adverse Event:  ICH E6 defines an AE as any untoward medical occurrence in a clinical 
investigation participant administered a pharmaceutical product regardless of its causal 
relationship to the study treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom, or disease temporally associated with 
the use of medicinal (investigational) product. The occurrence of an AE may come to the 
attention of study personnel during study visits and interviews of a study recipient presenting for 
medical care, or upon review by a study monitor.  
All AEs including local and systemic reactions not meeting the criteria for “serious adverse 
events” should be captured on the appropriate CRF. Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis, which would 
include MD, PA, Nurse Practitioner, DO, or DDS), and time of resolution/stabilization of the 
event. All AEs occurring while on study must be documented appropriately regardless of 
relationship. All AEs should be followed to adequate resolution.  


 
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30 
Any medical condition that is present at the time that the participant is screened should be 
considered as baseline and not reported as an AE. However, if it deteriorates at any time during 
the study, it should be recorded as an AE. 
The adverse event collection interval for this study is from the start of treatment until a minimum 
of 7 days following last dose of study treatment. SAEs considered related to study drug may be 
reported until the 190 day follow-up.  
All AEs must be graded for severity, seriousness and relationship to study product, as defined 
below.  
 
Severity of Event:  The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. 
All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy 
of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov). 
To assess severity of adverse events not included in the CTCAE version 4.0, use Table below.  
Table. Adverse Event Severity Grading Scale for Adverse Events Not Specifically Listed in the NCI 
CTCAE 
Grade 
Severity 
1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated. 
2 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental 
ADLa 
3 
Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of 
hospitalization indicated; disabling; limiting self care ADLb 
4 
Life-threatening consequences; urgent intervention indicated. 
5 
Death related to AE 
a. 
Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing money, etc. 
b. 
Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not bedridden. 
 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed. Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
Relationship to Study Products:  The clinician’s assessment of an AE's relationship to test 
article (vaccine or study drug) is part of the documentation process, but it is not a factor in 
determining what is or is not reported in the study. If there is any doubt as to whether a clinical 
observation is an AE, the event should be reported. All AEs must have their relationship to study 
product assessed using the terms: associated or not associated. In a clinical trial, the study 
product must always be suspect. To help assess, the following guidelines are used. 


 
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31 
 
Associated – The event is temporally related to the administration of the study product and 
no other etiology explains the event. For the purposes of reporting to the DSMC, this is 
consistent with the definitions of possible, probable and definite. 
 
 
Not Associated – The event is temporally independent of study product and/or the event 
appears to be explained by another etiology. For the purposes of reporting to the DSMC, 
this is consistent with the definitions of unrelated and unlikely. 
 
9.2.2 Serious Adverse Events 
Serious Adverse Event (SAE): An SAE is defined as an AE that meets one of the following 
conditions: 
 
Death during the period of protocol defined surveillance 
 
Life-threatening event (defined as a participant at immediate risk of death at the time of the 
event) 
 
An event requiring inpatient hospitalization for more than 24 hours or prolongation of 
existing hospitalization during the period of protocol defined surveillance*  
 
Results in congenital anomaly or birth defect 
 
Results in a persistent or significant disability/incapacity 
 
Any other important medical event that may not result in death, be life threatening, or require 
hospitalization, may be considered a serious adverse experience when, based upon 
appropriate medical judgment, the event may jeopardize the participant and may require 
medical or surgical intervention to prevent one of the outcomes listed above. Examples of 
such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
All SAEs will be: 
 
recorded on the appropriate SAE CRF 
 
followed through resolution by a study clinician 
 
reviewed and evaluated by a study clinician 
 
*Hospitalization or prolonged hospitalization should be documented and reported as a serious 
adverse event, except if the hospitalization meets at least one of the following criteria: 
 
The hospitalization is less than 24 hours without an admission 
 
Hospitalization for respite care 


 
 ____________________________________________________________________________________________ 
32 
 
Hospitalization for a preexisting condition, provided that all of the following 
criteria are met: 
o The hospitalization was planned prior to the study or was scheduled 
during the study when elective surgery became necessary because of the 
expected normal progression of the disease.  
o The patient has not suffered an adverse event. 
If the hospitalization meets any of these criteria, then it is not considered a serious adverse 
event. 
 
 
9.2.3 Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
Collection of laboratory data will be limited to those laboratory parameters that are relevant to 
safety, study outcome measures, and/or clinical outcome.  
The toxicity tables are adapted from the Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0 published by the US DHHS.  A grade 3 or higher abnormality related to 
study agent in the table below will be defined as a SAE. For any other adverse event, we shall 
use the table in Section 9.2.1.  
 
Grade 
Adverse Event 
1 
2 
3 
4 
5 
Leukocytosis 
- 
- 
>100,000/mm3 
 
 
Abdominal distension 
Asymptomatic; clinical 
or diagnostic 
observations only; 
intervention not 
indicated 
Symptomatic; limiting 
instrumental ADL 
Severe discomfort; 
limiting self care ADL 
 
- 
- 
Abdominal pain 
Mild 
Moderate; limiting 
instrumental ADL 
Severe pain; limiting 
self care ADL 
- 
- 
Ascites 
Asymptomatic; clinical 
or diagnostic 
observations only; 
intervention not 
indicated 
Symptomatic; 
medical intervention 
indicated 
Severe symptoms; 
invasive intervention 
indicated 
Life-threatening 
consequences; 
urgent operative 
intervention indicated 
Death 
Bloating  
No change in bowel 
function or oral intake 
Symptomatic, 
decreased oral 
intake; change in 
bowel function 
- 
- 
- 
Colitis 
Asymptomatic; clinical 
or diagnostic 
observations only; 
intervention not 
indicated 
Abdominal pain; 
mucus or blood in 
stool 
Severe abdominal 
pain; change in 
bowel habits; 
medical intervention 
indicated; peritoneal 
signs 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Colonic hemorrhage 
Mild; intervention not 
indicated 
Moderate symptoms; 
medical intervention 
or minor 
cauterization 
indicated 
Transfusion, 
radiologic, 
endoscopic, or 
elective operative 
intervention indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Colonic perforation 
- 
Symptomatic; 
Severe symptoms; 
Life-threatening 
Death 


 
 ____________________________________________________________________________________________ 
33 
 
Grade 
Adverse Event 
1 
2 
3 
4 
5 
medical intervention 
indicated  
elective operative 
intervention indicated 
consequences; 
urgent intervention 
indicated 
Colonic obstruction 
Asymptomatic; clinical 
or diagnostic 
observations only; 
intervention not 
indicated 
Symptomatic; altered 
GI function 
Hospitalization 
indicated; elective 
operative 
intervention 
indicated; disabling 
Life-threatening 
consequences; 
urgent operative 
intervention indicated 
Death 
Constipation 
Occasional or 
intermittent symptoms; 
occasional use of stool 
softeners, laxatives, 
dietary modification, or 
enema 
Persistent symptoms 
with regular use of 
laxatives or enemas; 
limiting instrumental 
ADL 
Obstipation with 
manual evacuation 
indicated; limiting 
self care ADL 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Diarrhea 
Increase of <4 stools 
per day over baseline; 
mild increase in 
ostomy output 
compared to baseline 
Increase of 4 - 6 
stools per day over 
baseline; moderate 
increase in ostomy 
output compared to 
baseline 
Increase of >=7 
stools per day over 
baseline; 
incontinence; 
hospitalization 
indicated; severe 
increase in ostomy 
output compared to 
baseline; limiting self 
care ADL 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Dyspepsia 
(uncomfortable, often 
painful feeling in the 
stomach, resulting from 
impaired digestion. 
Symptoms include 
burning stomach, 
bloating, heartburn, 
nausea and vomiting) 
Mild symptoms; 
intervention not 
indicated 
Moderate symptoms; 
medical 
intervention indicated 
Severe symptoms; 
surgical 
intervention indicated 
- 
- 
Flatulence 
Mild symptoms; 
intervention not 
indicated 
Moderate; persistent; 
psychosocial 
sequelae 
- 
- 
- 
Gastritis 
 Asymptomatic; clinical 
or diagnostic 
observations only; 
intervention not 
indicated 
Symptomatic; altered 
GI function; medical 
intervention indicated 
Severely altered 
eating or gastric 
function; TPN or 
hospitalization 
indicated 
Life-threatening 
consequences; 
urgent operative 
intervention indicated 
Death 
Gastroparesis (A disorder 
characterized by an 
incomplete paralysis of 
the muscles of the 
stomach wall resulting in 
delayed emptying of the 
gastric contents into the 
small intestine.) 
Mild nausea, early 
satiety and bloating, 
able to maintain caloric 
intake on regular diet 
Moderate symptoms; 
able to maintain 
nutrition with dietary 
and lifestyle 
modifications; may 
need pharmacologic 
intervention 
Weight loss; 
refractory to medical 
intervention; unable 
to maintain nutrition 
orally 
- 
- 
Nausea 
Loss of appetite 
without alteration in 
eating habits 
Oral intake 
decreased without 
significant weight 
loss, dehydration or 
malnutrition 
Inadequate oral 
caloric or fluid intake; 
tube feeding, TPN, 
or hospitalization 
indicated 
- 
- 
Proctitis 
Rectal discomfort, 
intervention not 
indicated 
Symptoms (e.g., 
rectal discomfort, 
passing blood or 
mucus); medical 
Severe symptoms; 
fecal urgency or 
stool incontinence; 
limiting self care ADL 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 


 
 ____________________________________________________________________________________________ 
34 
 
Grade 
Adverse Event 
1 
2 
3 
4 
5 
intervention 
indicated; limiting 
instrumental ADL 
Typhlitis (inflammation of 
the cecum) 
- 
- 
Symptomatic (e.g., 
abdominal pain, 
fever, change in 
bowel habits with 
ileus); peritoneal 
signs 
Life-threatening 
consequences; 
urgent operative 
intervention indicated 
Death 
Vomiting 
1 - 2 episodes 
(separated by 5 
minutes) in 24 hrs 
3 - 5 episodes 
(separated by 5 
minutes) in 24 hrs 
>=6 episodes 
(separated by 5 
minutes) in 24 hrs; 
tube feeding, TPN or 
hospitalization 
indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Alanine aminotransferase >ULN - 3.0 x ULN 
>3.0 - 5.0 x ULN 
>5.0 - 20.0 x ULN 
>20.0 x ULN 
- 
Alkaline phosphatase 
>ULN - 2.5 x ULN 
>2.5 - 5.0 x ULN 
>5.0 - 20.0 x ULN 
>20.0 x ULN 
- 
Aspartate 
aminotransferase 
increased 
>ULN - 3.0 x ULN 
>3.0 - 5.0 x ULN 
>5.0 - 20.0 x ULN 
>20.0 x ULN 
- 
Blood bilirubin increased 
>ULN - 1.5 x ULN 
>1.5 - 3.0 x ULN 
>3.0 - 10.0 x ULN 
>10.0 x ULN 
- 
Creatinine increased 
>1 - 1.5 x baseline; 
>ULN - 1.5 x ULN 
>1.5 - 3.0 x baseline; 
>1.5 - 3.0 x ULN 
>3.0 baseline; >3.0 - 
6.0 x ULN 
>6.0 x ULN 
- 
Weight loss 
5 to <10% from 
baseline; intervention 
not indicated 
10 - <20% from 
baseline; nutritional 
support indicated 
>=20% from 
baseline; tube 
feeding or TPN 
indicated 
- 
- 
Dehydration 
Increased oral fluids 
indicated; dry mucous 
membranes; 
diminished skin turgor 
IV fluids indicated 
<24 hrs 
IV fluids or 
hospitalization 
Indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Hyperglycemia 
Fasting glucose value 
>ULN - 160 mg/dL; 
Fasting glucose value 
>ULN - 8.9 mmol/L 
Fasting glucose 
value >160 - 250 
mg/dL; Fasting 
glucose value >8.9 - 
13.9 mmol/L 
>250 - 500 mg/dL; 
>13.9 - 27.8 mmol/L; 
hospitalization 
Indicated 
>500 mg/dL; >27.8 
mmol/L; 
life-threatening 
consequences 
Death 
Hyperkalemia 
>ULN - 5.5 mmol/L 
>5.5 - 6.0 mmol/L 
>6.0 - 7.0 mmol/L; 
hospitalization 
indicated 
>7.0 mmol/L; life-
threatening 
Consequences 
Death 
Hypernatremia 
>ULN - 150 mmol/L 
>150 - 155 mmol/L 
>155 - 160 mmol/L; 
hospitalization 
indicated 
>160 mmol/L; life-
threatening 
Consequences 
Death 
Hypoalbuminemia 
<LLN - 3 g/dL; <LLN - 
30 g/L 
<3 - 2 g/dL; <30 - 20 
g/L 
<2 g/dL; <20 g/L 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Hypoglycemia 
<LLN - 55 mg/dL; 
<LLN - 3.0 mmol/L 
<55 - 40 mg/dL; <3.0 
- 2.2 mmol/L 
<40 - 30 mg/dL; <2.2 
- 1.7 mmol/L 
<30 mg/dL; <1.7 
mmol/L; life-
threatening 
consequences; 
seizures 
Death 
Hypokalemia 
<LLN - 3.0 mmol/L 
<LLN - 3.0 mmol/L; 
symptomatic; 
intervention 
indicated 
<3.0 - 2.5 mmol/L; 
hospitalization 
indicated 
<2.5 mmol/L; life-
threatening 
Consequences 
Death 
Hyponatremia 
<LLN - 130 mmol/L 
- 
<130 - 120 mmol/L 
<120 mmol/L; life-
threatening 
consequences 
Death 


 
 ____________________________________________________________________________________________ 
35 
9.3 
Reporting Procedures 
Adverse events will be collected from Day 1 of study agent  through a minimum of 7 days after 
last day of study agent. Serious Adverse Events that are related to the study agent will be 
reported through Day 190 follow-up.  
9.3.1 Serious Adverse Events 
The study clinician will complete a Serious Adverse Event Form within the following timelines:   
Type of Event 
To whom will it 
be reported: 
Time Frame for 
Reporting 
How reported? 
Any internal event 
resulting in death that is 
deemed DEFINITELY 
related to (caused by) 
study participation 
(Note:  An internal event is 
one that occurs in a 
participant enrolled in a 
UVa protocol.) 
IRB-HSR 
 
 
 
 
FDA 
Within 24 hours 
 
 
 
 
Within 7 days 
IRB Online and phone call 
 
www.irb.virginia.edu/ 
 
 
Phone call, email and letter 
Internal, Serious, 
Unexpected adverse event 
  
 
IRB-HSR 
 
 
 
 
 
FDA 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event. 
 
Within 15 days 
 
Timeline includes 
submission of signed 
hardcopy of AE form. 
IRB Online 
www.irb.virginia.edu/ 
 
 
 
 
Email and letter 
Unanticipated Problems 
that are not adverse 
events or protocol 
violations  
This would include a Data 
Breach.   
IRB-HSR 
 
 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event.  
Unanticipated Problem report 
form.  
 
http://www.virginia.edu/vprgs/i
rb/HSR_docs/Forms/Reporting_
Requirements-
Unanticipated_Problems.doc ) 


 
 ____________________________________________________________________________________________ 
36 
Protocol Violations  
The IRB-HSR only requires 
that MAJOR violation be 
reported, unless otherwise 
required by the sponsor 
 
OR 
 
Enrollment Exceptions 
IRB-HSR 
 
 
Within 7 calendar 
days from the time 
the study team 
received knowledge 
of the event.  
 
Protocol Violation and 
Enrollment Exception Reporting 
Form 
 
 
http://www.virginia.edu/vprgs/i
rb/hsr_forms.html 
Go to 3rd bullet from the bottom 
Data Breach  
The UVa 
Corporate 
Compliance and 
Privacy Office, a 
 
 
ITC:  if breach 
involves  
electronic data-  
 
 
 
UVa Police if 
breach includes 
such things as 
stolen 
computers.  
As soon as possible 
and no later than 24 
hours from the time 
the incident is 
identified. 
 
As soon as possible 
and no later than 24 
hours from the time 
the incident is 
identified. 
 
IMMEDIATELY.  
 
 
UVa Corporate Compliance and 
Privacy Office- Phone 924-9741 
 
 
 
 
ITC:  Information Security 
Incident Reporting procedure,  
http://www.itc.virginia.edu/sec
urity/reporting.html 
 
 
Phone- (434) 924-7166 
UVa PI HELD IND 
Life-threatening and/or fata
unexpected events related 
or possibly related to the 
use of the investigational 
agent. 
FDA 
Within 7 calendar 
days of the study 
team learning of the 
event 
Form FDA 3500A (MedWatch) 
or narrative 
Serious, unexpected and 
related or possibly related 
adverse events 
FDA 
Within 15 calendar 
days after the study 
team receives 
knowledge of the 
event 
Form FDA 3500A (MedWatch) 
or narrative 
All adverse events 
FDA 
Annually 
IND annual report 
 
Other supporting documentation of the event may be requested by the IRB-HSR, Cancer Center 
DSMC and FDA and should be provided as soon as possible. 


 
 ____________________________________________________________________________________________ 
37 
9.3.2 Regulatory Reporting for Studies Conducted Under a IND 
All SAEs will be reported to the UVA IRB, UVA Cancer Center Oncore/DSMC, and FDA, as 
described in Section 9.3.1. 
9.3.3 Reporting of Pregnancy 
Pregnant patients will be excluded from the trial.  Participants who become pregnant during the 
duration of study agent administration will be excluded but will continue to be followed daily until 
day 10 and at days 40, 70, and 190 by the study team. 
9.4 
Type and Duration of Follow-up of Participants after 
Adverse Events 
AEs will be followed until resolved or considered stable. Follow-up will be performed daily for 
hospitalized participants and by telephone after discharge and for outpatients. For participants 
seen by health care providers as outpatient, the EMR will be reviewed for symptom, sign or 
laboratory abnormalities. 
 
9.5 
Modification of Study Agent(s)/Intervention(s) for a Participant 
9.5.1 Dose / Schedule Modifications for a Participant 
There will be no dose modification for a participant. Schedule of intake of study agent may be 
delayed if there are procedures or participant-related activities that are in conflict with the 
schedule of intake.  In these cases, the study agent can be taken as soon as possible but 
should be within the same day as scheduled.  There will be no make-up for missed doses. 
9.6 
 Halting Rules 
 
Safety findings that would temporarily suspend study agent until a safety review is convened 
(either routine or ad hoc) include occurrence of an unexpected adverse event or development of 
a SAE which cannot be attributed to other causes. The result of a safety review will guide 
decision as to whether the study agent (for an individual or study) should continue per protocol, 
proceed with caution, be further investigated, be discontinued, or be modified and then proceed. 
 Suspension of enrollment (for a particular group or for the entire study) is another potential 
outcome of a safety review. 
Safety review will be triggered as described in Section 11.4. 
Initial safety review will be performed by the PI and the Cancer Center DSMC.  All SAEs will be 
reported (Section 9.3.2) and reviewed in consultation with the UVA IRB. 


 
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38 
Subsequent review of serious, unexpected and related adverse events by the PI, Cancer Center 
DSMC ethics review committee or IRB, the FDA, and other regulatory authorities may also 
result in suspension of further trial interventions/administration of study agent at a site. The 
FDA, other regulatory authorities, and the study sponsor(s) retain the authority to suspend 
additional enrollment and Study Agent(s)/Intervention(s) administration for the entire study as 
applicable. Theoretically, participants with liver disease may have decreased ammonia 
excretion and may be predisposed to the accumulation of ammonia, a breakdown product of 
glutamine. Similarly, patients with kidney disease may have decreased renal excretion of 
ammonia and glutamic acid34.  Therefore, the following are risks that may be expected to occur 
in participants who have underlying liver or kidney dysfunction and taking alanyl-glutamine: 
elevation of liver enzymes, increased ammonia levels and increased BUN. Although we expect 
that these are low risk events and will have a maximum grade of 3, participants who have liver 
and kidney disease are excluded from the study. Enrolled subjects will be tested for ammonia 
levels if clinically indicated. 
 
9.7 
Stopping Rules for an Individual Participant 
Criteria for Discontinuation of Study Agent(s)/Intervention(s) for Withdrawal of a Participant. 
The study agent for an individual will be discontinued if there is development of laboratory 
toxicities, study closure due to DSMC review, or discretion of IND holder. 
A study participant will be discontinued from further Study Agent if any clinical adverse event, 
laboratory abnormality, intercurrent illness, other medical condition or situation occurs such that 
continued participation in the study would not be in the best interest of the participant. 
Development of any exclusion criteria at the time of administration of the study agent will be 
cause for discontinuation. 
Note that the participant will continue to be followed with participant’s permission if Study Agent 
is discontinued. There will be no modifications to the schedule and duration of continued follow-
up even if study agent is discontinued. 
 
Discontinuation of the study will only occur if the PI, DSMC or IRB deem it necessary. 
 
9.8 
Premature Withdrawal of a Participant 
Study participants may voluntarily withdraw any time after enrolment. However, with their 
permission, they will still be followed up at Days 40, 70 and 190 post-enrollment to gather 
information on recurrent diarrhea and documented CDI. 
 
9.9 
Replacement of a Participant Who Discontinues Study 
Treatment 
Discontinued participants will not be replaced. 


 
 ____________________________________________________________________________________________ 
39 
9.10  Safety Oversight (ISM plus SMC or DSMC) 
The Cancer Center Data and Safety Monitoring Committee will be the safety oversight body for 
this investigator-initiated trial. Adverse events, audit results and protocol violations will be 
reported to the DSMC per the Cancer Center NIH approved institutional plan. The DSMC will 
meet every month for aggregate review of AE data.  Tracking reports of the meetings are 
available to the PI for review.  Issues of immediate concern by the DSMC are brought to the 
attention of the PI (and if appropriate to the PRC and IRB) and a formal response from the PI is 
requested. 
 


 
 ____________________________________________________________________________________________ 
40 
10 
CLINICAL MONITORING 
10.1 Site Monitoring Plan 
This clinical trial will be performed in one site only.  The PI and the study coordinator shall 
monitor this site by reviewing 15% of all subjects randomly selected for 100% source 
documentation verification  semiannually to  determine if any breach in the protocol occurred 
regarding human participant protection, study procedures, laboratory, study intervention 
administration, and data collection processes. The principal investigator, assisted by the study 
coordinator, will be responsible for conducting the monitoring and will be responsible for 
ensuring that monitoring findings are addressed. The UVA Post Approval Monitoring (PAM) 
office will audit the study on a randomly selected basis. 
 
10.2 Safety Monitoring Plan 
 
Data and safety monitoring will be performed under the direction of the PI or her designee in this 
pilot study.   All AEs, protocol violations and study data will be entered into Oncore/DSMC and 
will be reviewed by the Cancer Center Data and Safety Monitoring Committee monthly. 
 


 
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41 
11 
STATISTICAL CONSIDERATIONS 
11.1 Study Design 
This is a single arm phase II trial to determine whether the data support the underlying 
hypothesis that supplementation of standard CDI treatment with AG will decrease the 40-day 
rate of clinical failure by 50%; and to obtain preliminary estimates of secondary endpoints.  
Study objectives are described in section 3.2 and endpoint in section 4.1.  
11.2 Sample Size Considerations 
A recent clinical epidemiologic study at the UVA Health System recruited 362 participants who 
were admitted and diagnosed with CDI from October 2008 to August 2011.  For 80% of these 
participants, this was their first episode of CDI. Forty-seven percent had concurrent or a history 
of solid organ or hematologic malignancy. Louie et al 2011 report results from a randomized trial 
of fidaxomicin versus vancomycin for CDI where 40-day clinical failure was observed in 36% 
(95% CI(31, 42%) of participants treated with vancomycin (36). For this study, target sample 
size is based upon detecting a 50% reduction in 40-day clinical failure (to 18%) compared to the 
null of 36% with a one-sided 10% significance level using an exact test. Accrual of 40 eligible 
participants provides approximately 90% power to test for a null 40-day clinical failure rate 
against the alternative of 18%.  Adjusting for a 5% ineligibility rate maximum sample size is 
estimated at 43 participants.  Accrual is estimated at 3-4 participants a month, thus accrual to 
the study should be completed in 4 years.  
11.3 Randomization/Stratification 
Randomization is not part of this single arm study.  
11.4 Safety Monitoring 
Safety monitoring guidelines have been generated for the study. The stopping boundary will be 
monitored and if a stopping boundary is crossed then accrual to the study will be suspended 
until the study PI, co-investigators and the DSMC can review the data, and determine if whether 
the study should continue, be amended or be closed to further accrual.  
11.4.1 SAEs 
Safety will be assessed by monitoring the number of participants who experience an 
SAE with AG treatment. This will be performed by the PI and Cancer Center DSMC. 
Results from Louie et al 2011 reported an SAE rate of approximately 24.1% (95% CI(20, 
29%)) for participants treated with vancomycin. The upper boundary of a sequential 


 
 ____________________________________________________________________________________________ 
42 
probability ratio test (SPRT) based upon a binomial test of proportions for SAE will be 
used for monitoring to protect against excessive SAE rates.  The stopping boundary is 
for a SPRT contrasting an assumed SAE rate of 25% versus an unacceptable SAE rate 
of 35%, with nominal type I and II errors of 10% and 10%, respectively and results in a 
regression coefficient of 0.298.  Thus, the safety boundary will be considered crossed if 
at any time > 28.9% of accrued participants experience a SAE  
11.4.1 30 day mortality 
30-day mortality (Day 40 post-enrollment) will be assessed by monitoring the number of 
participants who die within 40 days of starting protocol treatment. Results from 
Hensgens et al 2013 reported an all-cause mortality risk of 13.1% (95% CI(11, 15%)) 
after 30 days (37). The stopping boundary is for a SPRT contrasting an assumed 30-day 
mortality rate of 13% versus an unacceptable rate of 20%, with nominal type I and II 
errors of 10% and 10%, respectively and results in a regression coefficient of 0.163.  
Thus, the safety boundary will be considered crossed if at any time > 16.3% of accrued 
participants  die within 40 days of starting protocol treatment.  
11.5 Analyses 
Primary analyses will be based upon intention-to-treat where all eligible participants are 
included in the analysis regardless of amount of treatment received or compliance. Secondary 
analyses will assess the impact of non-compliance to treatment if 10% or more of participants 
do not comply with the treatment schedule. 
Adverse events and SAEs will be tabulated by frequency, severity and attribution. The one-
sample exact test for proportions will be used to test the hypothesis. 90% confidence intervals 
for a one-sample proportion will be estimated. Point estimates and 90% confidence intervals will 
be calculated for clinical failure rates at 70 and 190 days post-enrollment. CDI recurrence will be 
estimated by a cumulative incidence curve with death as a competing risk.  Duration of diarrhea 
will be calculated from the day the participant reports the onset of diarrhea to the day it stops.  
Maximum duration will be reported for multiple episodes. 
The product limit method of Kaplan and Meier will be used to estimate overall survival and the 
Cox proportional hazards  maximum likelihood estimate will be used to estimate the association 
of baseline factors and overall survival. 


 
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43 
12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of participants. All participants will be coded in official record and their identities 
only known to the PI and selected personnel assisting the PI in enrollment, screening, and 
follow-up of the enrolled participants. Participants will be referred to by code only, keeping their 
identities confidential from outside parties.  All information in which names or personal identifiers 
of Participants are linked to the donor code is kept on a password-protected computer device or 
in a locked file cabinet in the PI’s office.  Data arising from the project and publicly presented will 
be independent of any personal identifier.  All clinical specimens will be coded.  Personnel 
processing clinical specimens will not have access to the code. 
This site will permit authorized representatives of regulatory agencies to examine (and when 
required by applicable law, to copy) clinical records for the purposes of quality assurance 
reviews, audits, and evaluation of the study safety and progress. 
Data gathered will come from the electronic medical records (EPIC) (for clinical progress), 
symptom diary (specific signs and symptoms), and laboratory results (clinical microbiology for 
screening stool tests) and research lab for sera citrulline and LPS assays, stool lactoferrin 
assay, clostridial PCR and toxin assay, and select microbial assay. Data will be compiled for 
each participant in a Case Report Form (CRF) and encoded in the database.   


 
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44 
13 
QUALITY CONTROL AND QUALITY ASSURANCE 
The PI and study coordinator shall review 15% of the data semiannually and determine if any 
breach in the protocol occurred regarding human participant protection, study procedures, 
laboratory, study intervention administration, and data collection processes. During these 
reviews all data fields in the OnCore database will be monitored.  The principal investigator, 
assisted by the study coordinator, will be responsible for conducting the review of the data 
source and database. All research personnel will be required to complete CITI training. 
See Section 10.1 for detail on Site Monitoring. 


 
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45 
14 
ETHICS/PROTECTION OF HUMAN PARTICIPANT S 
14.1 Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the US National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the 
ICH E6; 62 Federal Regulations 25691 (1997).   
14.2 Institutional Review Board 
The study protocol will be reviewed and approved by the Cancer Center PRC and the UVA IRB. 
Any amendments to the protocol consent materials must also be approved before they are 
placed into use. 
14.3 Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation. Extensive discussion of 
risks and possible benefits of this therapy will be provided to the participants and their families. 
Consent forms describing in detail the study interventions/products, study procedures, and risks 
are given to the participant and written documentation of informed consent is required prior to 
starting intervention/administering study product. Consent forms will be IRB-approved and the 
participant will be asked to read and review the document. Upon reviewing the document, the 
investigator will explain the research study to the participant and answer any questions that may 
arise. The participants will sign the informed consent document prior to any procedures being 
done specifically for the study. The participants should have the opportunity to discuss the study 
with their surrogates or think about it prior to agreeing to participate. The participants may 
withdraw consent at any time throughout the course of the trial. A copy of the signed informed 
consent document will be given to the participants for their records. The rights and welfare of 
the participants will be protected by emphasizing to them that the quality of their medical care 
will not be adversely affected if they decline to participate in this study.  If the participant has 
cognitive impairment, consent will be deferred to the patient’s surrogate. 
14.4 Subject Confidentiality 
Participant confidentiality is strictly held in trust by the participating investigators and staff. This 
confidentiality is extended to cover testing of biological samples and genetic tests in addition to 
the clinical information relating to participating participants. 


 
 ____________________________________________________________________________________________ 
46 
The study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records. 
14.5 Study Discontinuation 
In the event that the study is discontinued, all participants will continue their standard therapy as 
prescribed by their health care provider or team.  There will be no crossover to study drug for 
placebo recipients at the completion of the study. 
14.6 Future Use of Stored Specimens 
Residual specimens will be maintained after the study is complete. The consent form will 
include storage of specimens for future studies, for which the participant may opt out from.   The 
specimens will not contain any participant identifiers and will be maintained at UVA only. If 
shipped for further testing elsewhere, the receiving institution will ship back or dispose leftover 
specimens after appropriate processing. These specimens will be coded and delinked from the 
medical records at the completion of the study, data analyses and publication of results.  The 
IRB will review future studies, and protections of confidentiality for any future studies with the 
stored specimens, eg, specimens will be coded, bar-coded, delinked.  Unless approved by the 
IRB, genetic testing will not be performed.  
 


 
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47 
15 
DATA HANDLING AND RECORD KEEPING 
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness 
of the data reported. All source documents should be completed in a neat, legible manner to 
ensure accurate interpretation of data. Black ink is required to ensure clarity of reproduced 
copies. When making changes or corrections, cross out the original entry with a single line, and 
initial and date the change. DO NOT ERASE, OVERWRITE, OR USE CORRECTION FLUID 
OR TAPE ON THE ORIGINAL. 
Copies of the electronic CRF (eCRF) will be provided for use as source documents and 
maintained for recording data for each participant enrolled in the study. Data reported in the 
eCRF derived from source documents should be consistent with the source documents or the 
discrepancies should be explained 
15.1 Data Management Responsibilities 
All source documents and laboratory reports must be reviewed by the clinical team and data 
entry staff, who will ensure that they are accurate and complete. Adverse events must be 
graded, assessed for severity and causality, and reviewed by the PI or designee. 
Data collection is the responsibility of the clinical trial staff under the supervision of the PI. 
During the study, the investigator must maintain complete and accurate documentation for the 
study. 
The PI, study coordinator, and the biostatistician for this study and will be responsible for data 
management, quality review, analysis, and reporting of the study data. 
15.2 Data Capture Methods 
Clinical data (including AEs, concomitant medications, interventions, and other clinical data) and 
clinical laboratory data coming from EPIC, participant symptoms and follow-up information from 
the symptom diary and the results of specimen testing from the research laboratory will all be 
entered into Oncore as required by the Cancer Center. The data system includes password 
protection and internal quality checks to identify data that appear inconsistent, incomplete, or 
inaccurate. Clinical data will be entered directly from the source documents.  


 
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48 
15.3 Types of Data 
Data for this study will include safety, laboratory (hematologic, chemistry and microbiologic), 
and outcome measures (eg, duration of diarrhea, adverse events, recurrence, and death). 
15.4 Timing/Reports 
Daily clinical data will be reviewed while inpatient participants are in the hospital and receiving 
study agent through Day 10.  A semi-annual review of CRFs and source documents will be 
performed as described in Sections 10, 11 and 13. Stopping rules and reports are described in 
Section 9. The PI will be reviewing the study for data and safety semi-annually.  The Cancer 
Center DSMC reviews data and safety monthly and reports annually. An interim analysis for 
adverse events will be performed half-way through enrollment.   
15.5 Study Records Retention 
Study documents should be retained for a minimum of 6 years after the completion of the study. 
These documents should be retained for a longer period, however, if required by local 
regulations.   
15.6 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements. The noncompliance may be either on the part of 
the participant, the investigator, or the study site staff. As a result of deviations, corrective 
actions are to be developed and implemented promptly.  
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, section 5.1.1 
5.20 
Noncompliance, sections 5.20.1, and 5.20.2. 
It is the responsibility of the site to use continuous vigilance to identify and report deviations 
within 5 working days of identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity.  
All deviations from the protocol must be addressed in study participant source documents. A 
completed copy of the Protocol Deviation Form must be maintained in the regulatory file, as well 
as in the participant’s source document. Protocol deviations must be sent to the IRB per their 
guidelines. The site PI/study staff is responsible for knowing and adhering to the IRB 
requirements. 


 
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49 
16 
PUBLICATION POLICY 
Following completion of the study, the investigator is expected to publish the results of this 
research in a scientific journal.  The International Committee of Medical Journal Editors (ICMJE) 
member journals have adopted a trials-registration policy as a condition for publication. This 
policy requires that all clinical trials be registered in a public trials registry such as 
ClinicalTrials.gov*, which is sponsored by the National Library of Medicine. Other biomedical 
journals are considering adopting similar policies.  It is the responsibility of the overall PI to 
register this trial in an acceptable registry.  Any clinical trial starting enrollment after 01 July 
2005 must be registered on or before participant enrollment 
 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
participants to intervention or comparison groups to study the cause-and-effect relationship 
between a medical intervention and a health outcome.  Studies designed for other purposes, 
such as to study pharmacokinetics or major toxicity (eg, Phase I trials), would be exempt from 
this policy. 
 
 
 
 
*Journal Citation: 
De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, Horton R, et al. Clinical trial registration: a statement from the 
International Committee of Medical Journal Editors. N Engl J Med. 2004;351:1250-1. 
 


 
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50 
17 
LITERATURE REFERENCES 
1. Ziegler TR, Benfell K, Smith RJ, et al. Safety and metabolic effects of L-glutamine 
administration in humans. JPEN J Parenter Enteral Nutr. 1990;14(4 Suppl):137S-146S. 
2. Jian ZM, Cao JD, Zhu XG, et al. The impact of alanyl-glutamine on clinical safety, nitrogen 
balance, intestinal permeability, and clinical outcome in postoperative patients: A randomized, 
double-blind, controlled study of 120 patients. JPEN J Parenter Enteral Nutr. 1999;23(5 
Suppl):S62-6. 
3. Garlick PJ. Assessment of the safety of glutamine and other amino acids. J Nutr. 2001;131(9 
Suppl):2556S-61S. 
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SUPPLEMENTS/APPENDICES 
APPENDIX A: SCHEDULE OF EVENTS 
 
Follow-Up Schedule 
 
Procedures 
Screening 
Baseline/Day 0 
Days 1-4 
Day 5 
Days 6-9 
Day 10+ 2  
Day 40 + 7  
Day 70 + 7 
Day 190 + 7 
Study 
Completion 
Premature 
Discontinuation 
Signed Consent 
Form 
X 
X 
 
 
 
 
 
 
 
 
 
Assessment of 
Eligibility Criteria 
X 
X 
 
 
 
 
 
 
 
 
 
Review of Medical 
History 
 
X 
 
 
 
 
 
 
 
 
 
Review of 
Concomitant 
Medications  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study Intervention 
 
X 
X 
X 
X 
X 
 
 
 
 
 
Clinical Monitoring 
and Follow-Up 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
Adverse Events 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical 
Laboratory 
Chemistry 
 
X 
 
 
 
 
 
 
 
 
 
Hematology 
 
X 
 
 
 
 
 
 
 
 
 
Stool 
X 
 
 
 
 
 
 
 
 
 
 
Research 
Laboratory 
 
 
 
 
 
 
 
 
 
 
 
 
Stoola 
X 
 
 
 
 
 
 
 
 
 
 
 
 
aThis specimen will come from the clinical laboratory and no additional collection will be performed if 
not available. 
 
 
 
UVA Clinical Laboratory 
Hematology –White blood cell, Red blood cell, Hemoglobin, Hematocrit, platelet count  


 
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56 
Biochemistry – Sodium, potassium, chloride, bicarbonate, BUN, creatinine, glucose, 
albumin, total bilirubin, alkaline phosphate, AST, ALT, erythrocyte sedimentation rate, C-
reactive protein, serum pregnancy test (HCG qualitative) 
 
UVA Clinical Microbiology Laboratory 
Stool: C. difficile toxin B PCR 
 
Warren Research Laboratory at UVA 
 
 
Stool: Lactoferrin assay, C. difficile toxin B PCR, C. difficile toxin A and B ELISA, anaerobic 
culture and ribotyping of isolated C. difficile 
  
 
 
 

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