Clinical trial NCT01536535
Conditions: Ulcerative Colitis
Keywords: Ulcerative Colitis mesalamine corticosteroids PROTECT
Sponsor: Connecticut Children's Medical Center
Start date: 2012-07-10; Completion date: 2018-04-30
Enrollment: 431 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a multi-center, open-label study to determine the safety and effectiveness (how well it works) of two standardized treatments called "mesalamine" (Pentasa®) and "prednisone" in children with newly diagnosed Ulcerative Colitis (UC). Standardized treatments are types of treatments agreed upon and used by many qualified doctors. The medications being used in this study are considered "standard of care". Currently the ways in which these medicines are used (doses, frequency of dosing) may vary from site to site. This study will determine response to a standardized way of giving these medicines.

This study will also identify biomarkers for ulcerative colitis. Biomarkers are things that doctors can find in blood, stool, or bowel tissue that indicate how much inflammation there is in the bowel, how the inflammation is produced, and whether the inflammation is responding to treatment. Collecting response and remission (free of symptoms) information on these standardized treatments and the "biomarkers" can possibly help doctors create a model, or plan to know which children with UC may respond quickly, or which children may develop complications.

Detailed description:
Ulcerative Colitis (UC) denotes a phenotype of chronic inflammatory bowel disease (IBD), where inflammation is localized to the colonic mucosa, and extends from the rectum proximally in varying extents. The disorder is thought to result from an inappropriate activation of the mucosal immune system by antigens derived from both the host epithelium and the enteric flora, in genetically susceptible individuals. UC is strikingly heterogeneous with respect to age of onset, anatomical extent and disease course, with some experiencing chronically active severe disease, while others have intermittent periods of clinical remission and disease exacerbation. Patients' therapeutic responses vary. The reasons underlying such variability are not well understood. Although it has been widely hypothesized that several genes may influence the development of UC, and modify its phenotypic expression and severity, to date there are few confirmed examples of such relationships.

It has been postulated that the 5-aminosalicylate drugs exert their anti-inflammatory effect locally at the intestinal mucosa. Mechanisms likely include inhibition of 5-lipoxygenase resulting in decreased production of leukotriene B4, scavenging of reactive oxygen metabolites, prevention of up-regulation of leukocyte adhesion molecules, and inhibition of Interleukin 1 (IL-1) synthesis. Though multiple studies have shown the efficacy of aminosalicylates in inducing and maintaining remission in adults with UC, there are few data in children.

Corticosteroids (CS) have been the mainstay of treatment of severe UC since efficacy was first demonstrated in the 1955 randomized controlled trial by Truelove and Witts. Recent practice guidelines developed in adults support their use because of rapid onset of action and significant efficacy though CS dependency is noted. Though no controlled data on their use have been reported in children they are frequently used in this population. A recent report from investigators leading the prospective Pediatric IBD Collaborative Research Group Registry described 97 subjects with a diagnosis of UC and a minimum of 1 year follow-up; 79% received CS. At diagnosis 81% of CS treated patients had moderate/severe disease, and 81% had pancolitis. Clinically inactive disease, determined by physician global assessment, was noted in 60% at 3 months following CS therapy, but by one year 45% were considered CS dependent despite the frequent use of immunomodulators (IM). Among those children with initially moderate to severe disease in clinical remission at 3 months, about two-thirds had stopped the CS by one year.

Arms: arm 1: Mild = Initiated on mesalazine, or on oral CS with Pediatric Ulcerative Colitis Activity Index (PUCAI) \< 45

Patients can be treated with any of the therapies noted below:

Mesalazine: doses is rounded to the nearest 500mg increment, maximum dose of 75 mg/kg/day Oral corticosteroids:1-1.5 mg/kg/day, rounded up to the nearest 5 mg value

IV corticosteroids:

Additional Therapies:

Calcineurin inhibitor (cyclosporine, tacrolimus) or anti-Tumour Necrosis Factor alpha (TNFα) therapy: These therapies may also be instituted if in the judgment of the attending physician is needed.

Immunomodulator or biologic therapy: thiopurine then dosing of azathioprine:2.5-3 mg/kg/day; 6-MP at 1-1.5 mg/kg/day Colectomy arm 2: Moderate/Severe = Initiated on IV CS, or oral CS with PUCAI ≥45

Patients can be treated with any of the therapies noted below:

Mesalazine: doses is rounded to the nearest 500mg increment, maximum dose of 75 mg/kg/day Oral corticosteroids:1-1.5 mg/kg/day, rounded up to the nearest 5 mg value

IV corticosteroids:

Additional Therapies:

Calcineurin inhibitor (cyclosporine, tacrolimus) or anti-TNFα therapy: These therapies may also be instituted if in the judgment of the attending physician is needed.

Immunomodulator or biologic therapy: thiopurine then dosing of azathioprine:2.5-3 mg/kg/day; 6-Mercaptopurine (MP) at 1-1.5 mg/kg/day Colectomy
Interventions: intervention 1: Mesalazine intervention 2: IV Corticosteroid intervention 3: Oral Corticosteroids intervention 4: Additional Therapies intervention 5: Colectomy
Intervention descriptions: intervention 1: Mesalazine (Pentasa) comes in 500mg capsules, and doses will need to be rounded to the nearest 500mg increment, with a maximum dose of 76 mg/kg/day. The average dose for the pediatric population will be approximately 70 mg/kg/day. Patients will be allowed to escalate to the final dose over 4 days to minimize side-effects such as headache. intervention 2: Treatment with IV Corticosteroid intervention 3: Treatment with oral corticosteroids intervention 4: Anti-TNFα, Calcineurin inhibitor, Immunomodulator intervention 5: Colectomy

Protocol documents:
Prot_SAP_000.pdf: 
PROTECT Study Protocol 2FEB2015 
 
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PROTOCOL 
 
 
Predicting Response to Standardized 
Pediatric Colitis Therapy  
(PROTECT) 
 
 
 
 
Funded by the National Institute of Diabetes and Digestive 
and Kidney Diseases 
National Institutes of Health 
Department of Health and Human Services 
U01 DK 095745-01 
Version:05 
 
 
 
 
 
February 2, 2015 
 
 
 
 
 
 
 
 
 
 
 
PROTECT Data Coordinating Center 
Collaborative Studies Coordinating Center 
Department of Biostatistics 
137 E. Franklin St., Ste 203, CB#8030 
The University of North Carolina at Chapel Hill 


PROTECT Study Protocol 2FEB2015 
 
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Chapel Hill, NC27514-4145 
TABLE OF CONTENTS 
 
TABLE OF CONTENTS .................................................................................................. 21 
1.  SUMMARY ................................................................................................................. 54 
2.  STUDY DEFINITIONS .............................................................................................. 65 
2.1.  Schroeder Endoscopic Scoring System (Mayo Score) .......................................... 65 
2.2.  Pediatric Ulcerative Colitis Activity Index (PUCAI) ........................................... 65 
2.3.  Corticosteroid (CS) Status ..................................................................................... 76 
2.4.  Rescue therapy ...................................................................................................... 76 
2.5.  Abbreviations and Acronyms ................................................................................ 87 
3.  BACKGROUND AND SIGNIFICANCE ................................................................... 98 
3.1.  Overview ............................................................................................................... 98 
3.2.  Comparing Pediatric Onset to Adult Onset Ulcerative Colitis ............................. 98 
3.3.  Current Management of Pediatric Ulcerative Colitis ............................................ 98 
3.3.1.  Aminosalicylates ........................................................................................... 109 
3.3.2.  Corticosteroids .............................................................................................. 109 
3.3.3.  Adherence and Clinical Outcomes ............................................................. 1110 
3.4.  Genetic Modifiers and Genomic Predictors ...................................................... 1110 
3.4.1.  Genetic Modifiers of Host Response and Potential Effect on Disease Course1110 
3.4.2.  Genomic Predictors of Response to Therapy and Disease Course. ............ 1211 
3.5.  Environmental Factors ...................................................................................... 1211 
3.5.1.  Epidemiology and Environmental Factors ................................................. 1211 
3.5.2.  Vitamin D and Immune Regulation ............................................................ 1312 
3.5.3.  Enteric Flora ............................................................................................... 1312 
3.6.  Serological Responses and Potential Relationship with Disease Course .......... 1413 
3.7.  Ulcerative Colitis and the Inflammatory Response ........................................... 1413 
3.8.  Characterizing Patients by Corticosteroid Response ........................................ 1514 
3.8.1.  Corticosteroid Pathway and its Underlying Molecular Mechanism ........... 1514 
3.8.2.  Putative Explanations for Variation in Corticosteroid Response ............... 1615 
3.9.  Assessing Success and Monitoring Disease Activity in Ulcerative Colitis ...... 1615 
3.10.  Need for a well-designed study ....................................................................... 1817 
4.  STUDY HYPOTHESES .......................................................................................... 1918 
4.1.  Primary Hypothesis ........................................................................................... 1918 
4.2.  Secondary Hypotheses ...................................................................................... 1918 
4.3.  Exploratory Hypothesis ..................................................................................... 2019 
5.  STUDY ENDPOINTS ............................................................................................. 2120 
5.1.  Overview of Primary and Secondary Clinical Endpoints ................................. 2120 
5.2.  Clinical Endpoints ............................................................................................. 2120 
5.2.1.  Primary Endpoint: SFR at 52 Weeks on Mesalamine Only ....................... 2120 
5.2.2.  Secondary Endpoints .................................................................................. 2120 
5.2.3.  Definitions of Corticosteroid (CS) Status ................................................... 2120 
6.  EXPERIMENTAL DESIGN AND METHODS ......................................................... 22 


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6.1.  Study Subjects and Eligibility ............................................................................... 22 
6.1.1.  Eligibility ........................................................................................................ 22 
6.2.  Case Ascertainment and Consent .......................................................................... 23 
6.2.1.  Case Ascertainment ........................................................................................ 23 
6.2.2.  Consent ....................................................................................................... 2524 
6.2.3.  Ethical Considerations ................................................................................ 2524 
6.2.4.  Risks and Benefits to Participants .............................................................. 2625 
6.2.5.  When and How to Withdraw Participants from Therapy or Assessment ....... 28 
6.3.  Baseline and Follow-up Evaluation .................................................................. 2928 
6.4.  Treatment .......................................................................................................... 3029 
6.5.  Pediatric Ulcerative Colitis Activity Index (PUCAI) [2] .................................. 3029 
6.5.1.  PUCAI Determination ................................................................................ 3130 
6.5.2.  Justification for the Use of PUCAI ............................................................. 3130 
6.6.  Mayo Sigmoidoscopy Score .............................................................................. 3332 
6.7.  Other Assessments ............................................................................................ 3332 
6.7.1.  Quality of Life Assessment ........................................................................ 3332 
6.7.2.  Behavioral Assessment ............................................................................... 3433 
6.7.3.  Adherence ................................................................................................... 3433 
6.8.  Collection of Laboratory Samples ..................................................................... 3433 
6.8.1.  Blood and Stool .......................................................................................... 3433 
6.8.2.  Mucosal Biopsies ........................................................................................ 3534 
7.  TREATMENT REGIMEN ...................................................................................... 3635 
7.1.  Overview ........................................................................................................... 3635 
7.2   Mesalamine ....................................................................................................... 3635 
7.3.  Initiation of Medical Therapy ........................................................................... 3635 
7.3.1.  Ambulatory patients with mild disease at diagnosis (PUCAI 10-34)......... 3736 
7.3.2.  Ambulatory patients with moderate to severe disease (PUCAI≥35) .......... 3736 
7.3.3.  Hospitalized patients with severe UC (PUCAI≥65) ................................... 3736 
7.3.4.  CS Tapering Schedule ................................................................................ 3837 
7.4.  Definitions of CS Status .................................................................................... 3938 
7.5.  Criteria to Institute Rescue Therapy .................................................................. 3938 
7.6.  Concomitant therapy ......................................................................................... 4140 
7.7.  Intolerance to mesalamine ................................................................................. 4140 
8.  ADVERSE EVENTS ............................................................................................... 4341 
PROTECT involves the study ................................................................................... 4341 
8.1.  Definitions of Adverse Event and Serious Adverse Event ............................... 4341 
8.2.  Reporting of Serious and Other Adverse Events .............................................. 4341 
9.  CENTRAL LABORATORIES ................................................................................ 4442 
9.1.  Biospecimen Collection and Shipping .............................................................. 4442 
9.2.  Biospecimen Analysis ....................................................................................... 4442 
10.  DATA MANAGEMENT....................................................................................... 4543 
10.1.  Data Management System ............................................................................... 4543 
10.2.  Data Collection, Entry, Editing and Reporting ............................................... 4543 
10.3.  Central Laboratory Management System ........................................................ 4543 
10.4.  Confidentiality and Security ............................................................................ 4543 


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10.5.  Records Retention ........................................................................................... 4644 
10.5.1.  Data Coordinating Center ......................................................................... 4644 
10.5.2.  Central Laboratories and Biospecimen Repositories ................................ 4644 
10.5.3.  Clinical Sites ............................................................................................. 4745 
11.  QUALITY ASSURANCE ..................................................................................... 4846 
11.1.  Training and Certification ............................................................................... 4846 
11.2.  Data Reporting for Quality Assurance ............................................................ 4846 
11.3.  Site Monitoring Visits ..................................................................................... 4846 
11.4.  Replicate Measures Program ........................................................................... 4846 
12.  STATISTICAL ANALYSIS ................................................................................. 5048 
12.1.  Analysis of the Primary Endpoint ................................................................... 5048 
12.2.  Analysis of the Secondary Endpoints .............................................................. 5048 
12.3.  Prediction Models ........................................................................................... 5148 
12.4.  Adherence Data ............................................................................................... 5149 
12.5.  Endoscopic Response and Remission ............................................................. 5149 
12.6  Genotyping Method, Quality Assurance, and Analysis ................................... 5250 
12.7.  Sample Size and Statistical Power .................................................................. 5351 
13.  DATA AND SAFETY MONITORING PLAN .................................................... 5553 
13.1.  Monitoring Data and Safety ............................................................................ 5553 
13.2 Study Safety Officer .......................................................................................... 5553 
13.3.  Data and Safety Monitoring Board (DSMB) .................................................. 5553 
13.3.1.  Role and Composition of the Data and Safety Monitoring Board ............ 5553 
13.3.2.  DSMB Reports.......................................................................................... 5654 
14.  STUDY AND COMMITTEE ORGANIZATION ................................................ 5755 
14.1.  Participating Entities ....................................................................................... 5755 
14.1.1.  Clinical Treatment Centers ....................................................................... 5755 
14.1.2.  Clinical Coordinating Center .................................................................... 5755 
14.1.3.  Data Coordinating Center ......................................................................... 5755 
14.1.4.  Central Laboratories and Biospecimen Repositories ................................ 5755 
14.1.5.  National Institutes of Health ..................................................................... 5755 
14.2.  Committees ...................................................................................................... 5856 
14.2.1.  Investigators Committee ........................................................................... 5856 
14.2.2.  Executive Committee ................................................................................ 5856 
14.2.3.  Ancillary Studies Committee .................................................................... 5856 
14.2.4.  Publications Committee ............................................................................ 5957 
14.2.5.  Study Coordinators Committee ................................................................ 5957 
14.2.6.  Quality Control Committee ...................................................................... 5957 
14.2.7.  Outcome Adjudication Committee ........................................................... 5957 
14.2.8.  Data and Safety Monitoring Board (DSMB) ............................................ 5957 
15.  REFERENCES ...................................................................................................... 6058 
APPENDIX A – Mesalamine (Pentasa) Package Insert ............................................... 6967 
 


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1.  SUMMARY 
 
This multicenter open-label study is designed to evaluate the safety and efficacy of 
standardized initial therapy using either mesalamine or corticosteroids then mesalamine for the 
treatment of children and adolescents newly diagnosed with ulcerative colitis. 
The study will investigate the hypothesis that response to the initial 4 weeks of therapy as well 
as specific clinical, genetic, and immune parameters determined during the initial course of 
therapy will predict severe disease as reflected by need for escalation of medical therapy or 
surgery. 
A total of up to 475 subjects will be assigned to one of two initial therapeutic plans 
(mesalamine only or prednisone/liquid equivalent prednisolone followed by mesalamine) 
depending upon initial disease severity determined by the validated multi-dimensional 
Pediatric Ulcerative Colitis Activity Index (PUCAI).  Biospecimens (blood, stool, colonic 
tissue) will be obtained at diagnosis, and subsequently following the initiation of therapy at 
weeks 4, 12, and 52 (blood and stool at weeks 4 and 12; blood, stool, and colonic tissue at 
week 52). Clinical evaluation will take place at pre-specified time points over up to two to five 
years of follow up.  Adherence to mesalamine dosing will be monitored using a state of the art 
electronic Medication Event Monitoring System (MEMS®). 
The primary endpoint is corticosteroid free remission (SFR) at 52 weeks on mesalamine 
therapy only without the need for rescue therapy with immunomodulators (IM), calcineurin 
inhibitors (CI), anti-TNFα therapy, or surgery.  Secondary endpoints include steroid free 
remission (SFR) at other time points, PUCAI<10 at 4 weeks, colectomy during follow-up, 
quality of life measures and, in a subset of patients, endoscopic remission or response at 52 
weeks. 
 
 


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2.  STUDY DEFINITIONS 
 
2.1.  Schroeder Endoscopic Scoring System (Mayo Score) 
Endoscopic severity will be assessed using the Mayo sigmoidoscopy score [1]: 
 
0 = Normal or inactive disease 
 
1 = Mild disease (erythema, decreased vascular pattern, mild friability) 
 
2 = Moderate disease (marked erythema, absent vascular pattern, friability, erosions) 
 
3 = Severe disease (spontaneous bleeding, ulceration) 
 
2.2.  Pediatric Ulcerative Colitis Activity Index (PUCAI) 
The PUCAI is scored using the following table, based on information obtained by a health 
professional who has evaluated the subject [2]: 
ITEM 
POINTS 
1. Abdominal pain: 
 
No pain 
Pain can be ignored 
Pain cannot be ignored 
0 
5 
10 
2. Rectal Bleeding 
 
None 
Small amount only in less than 50% of stools 
Small amount with most stools 
Large amount (>50% of the stool content) 
0 
10 
20 
30 
3. Stool consistency of most stools 
 
Formed 
Partially formed 
Completely unformed 
0 
5 
10 
4. Number of stools per 24 hours 
 
0-2 
3-5 
6-8 
>8 
0 
5 
10 
15 
5. Nocturnal stools (any episode causing awakening) 
 
No 
Yes 
0 
10 
6. Activity level 
 
No limitation of activity 
Occasional limitation of activity 
Severe restricted activity 
0 
5 
10 
SUM OF PUCAI (0-85) 
0-85 
 


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The range of possible scores is 0-85 and the classification within this range is: 
 
PUCAI<10 
Inactive disease 
 
PUCAI 10-34 
Mild activity 
 
PUCAI 35-64 
Moderate activity 
 
PUCAI ≥65 
Severe activity 
 
2.3.  Corticosteroid (CS) Status 
CS-free:  Subjects will be considered to be CS-free if they are taking no CS at the assessment 
and have been CS-free for a minimum of 2 weeks at that point.  Alternate day CS will not be 
considered CS-free. 
CS responsive:  Clinical improvement and termination of CS within 12 weeks of initiating 
therapy, and remaining off CS for the remainder of the first year or a minimum of six months 
following initial tapering before requiring re-treatment with CS.  
CS dependent:  Required CS >12 weeks to control symptoms, or at least one additional course 
of CS starting <6 months after initial successful CS weaning  
CS refractory:  Unresponsive to oral CS within four weeks of starting therapy and required 
rescue therapy with CI or anti-TNFα therapy; or required rescue therapy with CI or anti-TNFα 
therapy because of failure to respond to intravenous CS, or required colectomy because of 
failure to respond to CS. 
 
2.4.  Rescue therapy 
The use of immunomodulators (IM), calcineurin-inhibitors cyclosporine or tacrolimus (CI), 
anti-TNFα inhibitors, or colectomy in patients with ulcerative colitis is referred to as rescue 
therapy. 
 


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2.5.  Abbreviations and Acronyms 
 
5-ASA 
5-aminosalicylic acid (mesalamine) 
 
AUC 
area under the (receiver operating characteristic) curve 
 
BASC-2 
Behavior Assessment System for Children – Second Edition 
 
CD 
Crohn’s disease 
 
CI 
calcineurin inhibitors 
 
CS 
corticosteroid 
 
CRF 
case report form 
 
DCC 
Data Coordinating Center 
 
DSMB 
Data and Safety Monitoring Board 
 
GR 
glucocorticoid receptor 
 
IBD 
inflammatory bowel disease 
 
IM 
immunomodulators 
 
IRB 
Institutional Review Board 
 
MEMS® 
Medication Event Monitoring System 
 
MH 
mucosal healing 
 
NPV 
negative predictive value 
 
OPG 
osteoprotegerin 
 
OR 
odds ratio 
 
PPD 
purified protein derivative 
 
PPV 
positive predictive value 
 
PRO-KIIDS Pediatric Resource Organization for Kids with Intestinal Inflammatory 
Disorders 
 
PUCAI 
Pediatric Ulcerative Colitis Activity Index 
 
RDCRC 
Rare Disease Clinical Research Consortium 
 
SAE 
serious adverse event 
 
SFR 
corticosteroid free remission 
 
TPMT 
thiopurine methyltransferase 
 
UC 
ulcerative colitis 
 
 
 


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3.  BACKGROUND AND SIGNIFICANCE 
 
3.1.  Overview 
Ulcerative Colitis (UC) denotes a phenotype of chronic inflammatory bowel disease (IBD), 
where inflammation is localized to the colonic mucosa, and extends from the rectum 
proximally in varying extents.  The disorder is thought to result from an inappropriate 
activation of the mucosal immune system by antigens derived from both the host epithelium 
and the enteric flora, in genetically susceptible individuals.  UC is strikingly heterogeneous 
with respect to age of onset, anatomical extent and disease course, with some patients 
experiencing chronically active severe disease, while others have intermittent periods of 
clinical remission and disease exacerbation.  Patients’ therapeutic responses are also equally 
variable.  The reasons underlying such variability are not well understood.  Although it has 
been widely hypothesized that several genes may influence the development of UC, and 
modify its phenotypic expression and severity, to date there are few confirmed examples of 
such relationships.  Recently, several investigators have completed a genome-wide association 
study (GWAS) in which variation in TNFRSF6B was linked to risk for UC in children [3].  The 
TNFRSF6B gene product, decoy receptor 3 (DCR3), regulates monocyte and lymphocyte 
function, and prolongs lymphocyte survival by inhibiting Fas signaling [4].  As chronic 
lymphocyte activation and resistance to activation-induced cell death are felt to be fundamental 
mechanisms of mucosal inflammation in UC, it is likely that variation in TNFRSF6B regulates 
both susceptibility for UC, and behavior of established disease [5]. 
 
3.2.  Comparing Pediatric Onset to Adult Onset Ulcerative Colitis 
In comparison with adult-onset disease, it is consistently observed that UC developing during 
childhood is more likely to be severe and extensive [6, 7].  Given the higher proportion of 
extensive disease seen in children, a more frequent occurrence of severe/fulminant UC might 
also be anticipated.  Indeed, while adults with UC are reported to have a 15% lifetime risk of 
experiencing a severe exacerbation, which prior to corticosteroid (CS) therapy was associated 
with significant mortality, in a recent retrospective analysis of children with UC residing in the 
Greater Toronto Area we calculated a 28% rate of hospitalization due to severe exacerbation 
over a median follow-up of six years [8].  CS failure rate (46%) was also higher among these 
hospitalized children (n=100), than had been observed in a previous systematic review of 
published cohort studies of hospitalized adults (29%)[9].  Two of the three other previously 
reported, but very small (total n=43), studies evaluating outcome of admissions for severe 
pediatric UC mirrored the Toronto Hospital for Sick Children experience [10-12].  
Interestingly, among the 100 children hospitalized in Toronto, the colectomy rate after one-
year remained remarkably stable, confirming data suggesting that most UC-related admissions 
occur during the first few years following diagnosis. 
 
3.3.  Current Management of Pediatric Ulcerative Colitis 
Basic induction therapy usually includes aminosalicylates in subjects with mild to moderate 
disease, and aminosalicylates and CS in those with moderate to severe disease; inadequate 
response often prompts the use of immunomodulators (IM), calcineurin inhibitors (CI; 
cyclosporine, tacrolimus), or infliximab. 
 


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3.3.1.  Aminosalicylates 
It has been postulated that the 5-aminosalicylate drugs exert their anti-inflammatory effect 
locally at the intestinal mucosa [13, 14]. Mechanisms likely include inhibition of 5-
lipoxygenase with resulting decreased production of leukotriene B4, scavenging of reactive 
oxygen metabolites, prevention of up-regulation of leukocyte adhesion molecules, and 
inhibition of IL-1 synthesis. Because 5-ASA is rapidly absorbed from the upper gastrointestinal 
tract on oral ingestion, different delivery systems have been used to prevent absorption until 
the active drug can be delivered to the distal small bowel and colon. Pentasa® (mesalamine) 
the 5-ASA preparation to be used in this study coats microgranules of mesalamine with 
ethylcellulose, releasing it in a time-dependent fashion. 
 
Though multiple studies have shown the efficacy of aminosalicylates in inducing and 
maintaining remission in adults with ulcerative colitis [15-17], there are few data in children.  
Indeed, with the exception of one small study comparing olsalazine to sulfasalazine in mild to 
moderate UC [18], there have been no controlled studies.  Other reports have all been single 
center anecdotal experiences with small numbers of patients [19, 20].  The ideal dose of 5-ASA 
in the treatment of pediatric ulcerative colitis is also uncertain with doses from 30-80 
mg/kg/day being noted [20]. In adults it has been suggested that higher doses are more 
effective than lower doses [21, 22].  
 
Adverse reactions to all of the 5-ASA preparations have been described, requiring 
discontinuation in 5-15% of cases. More common side-effects include headache and abdominal 
pain, which often improve with time and infrequently require cessation of therapy. 
Approximately 5-10% of treated cases of ulcerative colitis develop paradoxical worsening of 
diarrhea and/or bleeding on 5-ASA requiring that the medication be stopped. Pancreatitis is 
seen in up to 3% of treated cases and does not appear to be dose-dependent; it usually occurs 
within the first month or two of starting therapy. Nephritis is rare but periodic urinalysis and 
measurement of renal function are recommended. 
 
3.3.2.  Corticosteroids 
Corticosteroids have constituted the mainstay of treatment of severe UC since efficacy was 
first demonstrated in the 1955 randomized controlled trial by Truelove and Witts [22].  Recent 
practice guidelines developed in adults support their use because of rapid onset of action and 
significant efficacy [23] though CS dependency is noted [24].  Though no controlled data on 
their use have been reported in children they are frequently used in this population [25].  A 
recent report from investigators leading the prospective Pediatric IBD Collaborative Research 
Group Registry [26] described 97 subjects with a diagnosis of UC and a minimum of 1 year 
follow-up; 77 (79%) received CS (62 within 30 days of diagnosis (early) and 15 between 31 
days and 6 months (late)).  At diagnosis 81% of CS treated patients (age 11.3 ± 3.5 years) had 
moderate/severe disease, and 81% had pancolitis.  Clinically inactive disease as determined by 
physician global assessment was noted in 60% at 3 months following CS therapy, but by one 
year 45% were considered CS dependent despite the frequent use of IM.  Among those 
children with initially moderate to severe disease in clinical remission at 3 months, about two-
thirds had stopped the CS by one year.  In this patient cohort there were no standardized CS 
dosing regimens or tapering schedules. 
 


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3.3.3.  Adherence and Clinical Outcomes 
Treatment regimens for pediatric UC involve many tablets at different times of the day and can 
have unpleasant side effects.  Our research has identified non-adherence prevalence rates 
ranging from 50-88% across medications in pediatric IBD [36-38].  These data are alarming 
given that the risk of relapse in UC is 5.5 times greater in non-adherent patients than in 
adherent patients, and the annual costs of health care for non-adherent UC patients are 12.5% 
higher than for adherent patients [39].  Work from our group has shown that internalizing 
problems such as depression are often associated with non-adherence in pediatric IBD patients; 
other barriers to adherence include forgetting, being away from home, and interference with an 
activity [40].  Data from an ongoing study of self-management behavior in pediatric IBD in a 
sample of 62 adolescents, led by our co-investigator Dr. Hommel, has confirmed that reduced 
medication adherence is associated with increased disease severity after statistically controlling 
for the effects of age, sex, disease sub-type, and internalizing behavioral symptoms (e.g., 
depression) [41].  However, no study has prospectively defined rates or consequences of non-
adherence to aminosalicylates in pediatric UC. 
 
3.4.  Genetic Modifiers and Genomic Predictors 
3.4.1.  Genetic Modifiers of Host Response and Potential Effect on Disease Course 
Although the major goal of genetic research has been to elucidate etiopathogenesis, it is 
hypothesized that an understanding of genetic variation may also help explain much of the 
tremendous heterogeneity that has been observed in phenotypic expression, natural history, and 
response to therapy.  While the molecular determinants of UC progression and treatment 
response remain to be fully elucidated, it seems reasonable to assume that at least some of the 
factors and processes that originally modulate disease initiation will also be at play in disease 
progression.  For instance, the most consistently replicated HLA associations with UC are 
DRB1*0103 and DRB1*15 [42-46].  The association with both of these variants is particularly 
strong in patients with extensive or severe disease.  A biallelic structural polymorphism in the 
inhibitor of ĸB-like (IKBL) gene, in the central MHC region has also been associated with 
severe UC [45].  While some of the polymorphisms of the cellular export pump MDR1 have 
been generally associated with UC susceptibility, other polymorphisms (C3435T) have been 
specifically associated with CS treatment response [47, 48].  This polymorphism is associated 
with increased intestinal MDR1 expression, and presumably altered cellular CS metabolism.  
Recently, results of a nonsynonymous SNP scan for ulcerative colitis identified a previously 
unknown susceptibility locus at ECM1 [49].  It was also shown that several risk loci are 
common to UC and Crohn’s disease (IL23R, IL12B, HLA, NKX2-3 and MST1).  A recent 
German study identified 3p21.31, NKX2-3, and CCNY as susceptibility factors for both 
Crohn’s disease and UC, and variants in PTPN2, HERC2, and STAT3 associated with UC only 
[50].  The recent meta-analysis which included both adult- and pediatric-onset cases increased 
the number of UC risk loci to 47, and implicated biologic pathways involved in barrier 
function, autophagy, and adaptive immune responses [31]. 
Most relevant to the current study, a recent report examined Genome Wide Association Study 
(GWAS) data from 324 adult UC patients who required colectomy for refractory disease 
compared to 537 adult UC patients who did not require colectomy [51].  Colectomy was 
associated with pancolitis and a positive family history of UC, two factors which are more 
common in pediatric-onset disease.  Colectomy was not associated with age at onset or gender.  


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A risk score based on the combination of 46 single nucleotide polymorphisms (SNPs) 
associated with refractory UC accounted for 48% of the variance for colectomy risk in this 
cohort.  Risk scores divided into quartiles showed the risk of colectomy to be 0%, 17%, 74%, 
and 100% in the four groups.  The area under the curve (AUC) for the risk score was equal to 
0.91, with a sensitivity and specificity over 90%.  Whether the genetic variants identified in 
this cohort or the recent GWAS meta-analysis may also influence disease behavior including 
the response to therapy in pediatric UC is not known. 
 
3.4.2.  Genomic Predictors of Response to Therapy and Disease Course. 
Several studies from our group and others have examined the global pattern of gene expression 
in the colon of UC patients at diagnosis and during therapy.  We found that colonic STAT3 
activation and biologic networks driving leukocyte recruitment, HLA expression, angiogenesis, 
and tissue remodeling are enriched in the colon of children with UC at diagnosis [52].  
Subsequently, genetic variation in STAT3 was found to control risk for the development of 
IBD.  Investigators from our group have utilized microarray from RNA isolated from 
peripheral blood mononuclear cells (PBMC) of children with UC receiving intravenous 
corticosteroids to identify a panel of 10 genes which predicted clinical response with over 80% 
sensitivity and specificity [53].  These included ABC4, MMP8, and CEACAM1.  More recently, 
microarray has been used to identify colonic genes which may predict the response to 
inflixmab in adults with UC [54].  A panel of five genes (osteoprotegerin (OPG), 
stanniocalcin-1, prostaglandin-endoperoxide synthase 2 (COX2), interleukin 13 receptor alpha 
2 and interleukin 11) separated responders from non-responders with 95% sensitivity and 85% 
specificity.  Remarkably, our group has now reported that elevated fecal OPG is associated 
with lack of response to intravenous corticosteroids in pediatric UC [55].  Whether 
determination of the global pattern of colon gene expression by RNA-Seq or microarray may 
be used to identify a panel of colonic genes at diagnosis of pediatric UC which will predict 
clinic outcomes is not known.   
 
3.5.  Environmental Factors 
3.5.1.  Epidemiology and Environmental Factors 
The incidence of both CD and UC has continued to increase during the past decade, 
particularly in pediatric populations reported from both North America and Europe [56]. In a 
retrospective study of pediatric IBD in Northern California from 1996-2006, the incidence of 
UC increased significantly by 2.7 fold and CD increased two fold [57].  Similarly, an 
epidemiologic review of pediatric IBD globally revealed increased incidence of both UC and 
CD [58].  Specifically, IBD appears to be increasing in parts of the world including Asia and 
South America where it was previously uncommon [59]. 
This heightens the importance of ongoing studies to define environmental triggers for disease 
development.  Studies in Scotland have implicated higher socioeconomic status (without 
change in genetic susceptibility) as an important factor in the development of IBD [60].  This 
suggests early life environmental exposures may potentially be associated with IBD.  Microbial 
exposures in childhood related to the “hygiene hypothesis” have been explored including H 
pylori, helminthes, and other childhood infections.  Other factors modifying the response to 
infection including childhood vaccinations, antibiotic exposure, mode of delivery and 
breastfeeding have also been considered.  However, currently only two environmental 


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influences, smoking and appendectomy, are well established risk factors for IBD [61, 62]. 
While a detailed consideration of environmental factors which may contribute to the 
pathogenesis of pediatric UC is beyond the scope of PROTECT, we will consider the role of 
vitamin D deficiency in Aim 2. 
 
3.5.2.  Vitamin D and Immune Regulation 
Recent registry studies have demonstrated a high rate of vitamin D deficiency in children with 
CD and UC [27], and vitamin D regulates innate and adaptive components of the mucosal 
immune response which could contribute to IBD clinical outcomes.  For example, the 
expression of cathelicidin and defensins is under the control of vitamin D.  These natural 
antibiotics are part of the innate immune system that controls the microbial populations that 
colonize intestinal mucosal surfaces [27, 28].  Patients with UC have elevated -defensin-2 and 
cathelicidin in the colon, a protective response that may be blunted in vitamin D deficiency 
[29].  Vitamin D induces autophagy, a process that recycles defective intracellular organelles 
and plays a role in bacterial killing.  Loss-of-function polymorphisms in autophagy ATG16L1 
and IRGM1 genes are susceptibility factors for CD and defects in autophagy secondary to 
vitamin D deficiency may worsen the course of UC [30].  Importantly, a recent meta-analysis 
has identified DAP (death-associated protein), a negative regulator of autophagy, as a 
susceptibility gene for UC [31].  Vitamin D also regulates T cell activation and the phenotype 
and function of antigen-presenting cells (APC), particularly dendritic cells (DCs) [32].  In a 
rodent model of colitis, DCs preferentially polarize naïve T cells toward a regulatory 
phenotype under the influence of vitamin D [32].  Consistent with this, resistance to vitamin D 
results in fulminant disease in murine colitis [33].  Supplementation with 1,25(OH)2D reduced 
both serum CRP and clinical disease activity by week 6 in adult CD patients [34].  In a recent 
randomized controlled trial, oral vitamin D supplementation reduced the one year rate of 
relapse in adult CD patients from 29% to 13% [35].  It is not known if vitamin D deficiency is 
associated with dysregulated immune responses and worse clinical outcomes in children with 
UC. 
 
3.5.3.  Enteric Flora 
Studies using newer molecular methods have begun to characterize fundamental differences in 
the enteric flora between children with IBD and healthy controls [63].  The microbiota of the 
ileum and colon contains a variety of metabolically active bacteria that interact with the host 
mucosal immune system.  Inappropriate activation by the intestinal microbiota is thought to 
play an important role in the etiology of IBD [64]. A breakdown in the balance between 
protective and harmful bacteria (dysbiosis) is a current prevailing hypothesis for the 
development of disease [65].  Bacterial load and the nature of the commensal flora can 
influence both the site and degree of GI inflammation [66-68].  Some changes in the intestinal 
microbial community are shared in UC and CD including increased concentrations of E. coli 
[69, 70], presence of non-commensals [65, 71, 72] and reduced diversity [71].  Bacterial 
populations have also been found to vary based on CD phenotype.  Patients with ileal disease 
may have disappearance of Faecalibacterium and Roseburia with increased numbers of 
Enterobacteriacea and Ruminococcus gnavus [73].    The role the microbiota plays in the 
pathogenesis of IBD will continue to be a very active area of investigation.  Results of a large 
prospective cohort study of high-risk families, and of the composition of the enteric flora in 
twins divergent for IBD, are eagerly awaited.  While a detailed characterization of the enteric 


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flora would be beyond the scope of PROTECT, we will store mucosal DNA and fecal samples 
which may be used in future ancillary microbiome studies.  These will likely be conducted 
using new molecular and bioinformatics approaches which are being developed by the recently 
initiated CCFA Microbiome Consortium. 
 
3.6.  Serological Responses and Potential Relationship with Disease Course 
The relationship between serological response to microbial and auto-antigens and disease 
course in UC remains largely unexplored.  In principle, serological status may be an indicator 
of ongoing antigen exposure due to disrupted mucosal integrity and, as such, may be predictive 
of more persistent disease that will require an escalation in therapy.  In CD, ASCA, antibodies 
to Escherichia coli outer membrane porin C (anti-OmpC) and antibodies to CBir1 flagellin 
(anti-CBir1) have been shown to predict disease progression [74].  One study in UC suggested 
that pANCA (perinuclear anti-neutrophil cytoplasmic antibody) positivity was associated with 
increased resistance to treatment in left-sided colitis [75]; another has shown that the response 
to infliximab is better in adult UC patients who are pANCA sero-negative [76].  The presence 
of OmpC and pANCA seem to be predictive of pouchitis following ileoanal pouch procedure 
[77].  Immunization of mice to flagellin has been shown to exacerbate colitis due to dextran 
sodium sulfate exposure, suggesting that these antibodies may play a role in disease 
pathogenesis [78].  Recently, we have reported for the first time that high titers of circulating 
Granulocyte Macrophage Colony Factor (GM-CSF) auto-antibodies (Ab) are associated with 
refractory disease requiring surgery in pediatric and adult CD [79].  We found that GM-CSF 
was required for gut barrier function in both animals and pediatric CD patients, and that loss of 
GM-CSF bioactivity exacerbated experimental gut injury [80].  In our recent preliminary 
studies we have determined that GM-CSF Ab are produced by lamina propria cells (LPMC) 
isolated from the inflamed colon in UC, and that adult UC patients with higher circulating 
levels of GM-CSF Ab are more likely to require colectomy.  Whether neutralizing GM-CSF 
Ab are also associated with disease outcomes in pediatric UC is not known.  We will examine 
the relationship between these serologic markers and longitudinal disease course in UC. 
 
3.7.  Ulcerative Colitis and the Inflammatory Response 
In the normal host, inflammation is generally a two-phase process.  Cytokines such IL-6, IL-1β 
and TNF-α are released in response to an initial inflammatory trigger and activate a series of 
pro-inflammatory transcription factors including nuclear factor- (NF-) and activator 
protein 1 (AP-1).  Consistent with this, we have recently identified IL-6:STAT3 dependent 
biologic networks up-regulated in the colon of children with UC at diagnosis which drive 
leukocyte recruitment and survival [52].  These in turn lead to the production of  various pro-
inflammatory chemokines and cytokines including IL-8, IL-12, IL-15, IL-17 and IL-18 [81].  
In balance, anti-inflammatory agents including cytokines, such as INFα and IL-10, as well as 
cortisol, secreted in response to ACTH from the pituitary, are also released [82].  In patients 
with UC, a variety of cytokine abnormalities have been reported, even during periods of 
clinical remission.  For instance, the over-expression of IL-1β and IL-8 in colonic mucosa has 
been repeatedly demonstrated [83-91].  Similar observations for the inflammatory cytokines 
IL-6  and TNFα have also been reported, but less consistently[84, 87, 88, 90-92].  Interestingly, 
elevation of the anti-inflammatory cytokine IL-10 within the inflamed colonic mucosa of 
patients with UC has also been demonstrated, although, again, not universally [83, 89, 91, 93].  
To date, however, alterations in IL-18 and IL-12 have not been reported in UC [94].  Lamina 


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propria T cells from UC patients produce significantly greater amounts of IL-5 and IL-13 than 
control cells and little IFN-gamma, whereas comparable cells from CD patients produce large 
amounts of IFN-gamma and small amounts of IL-13 [95].  Stimulation of UC lamina propria T 
cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing 
transfected CD1d induces significant IL-13 production.  These studies suggest that UC is 
associated with an atypical Th2 response mediated by non-classical NKT cells producing IL-13 
and having cytotoxic potential for epithelial cells [95].  Another critical feature is the resistance 
of the mucosal T lymphocytes to activation-induced cell death, which has been linked to 
increased production of decoy receptors including DCR3 [5].  Collectively, these studies are 
quite consistent with our discovery of TNFRSF6B, the gene which encodes DCR3, as a 
susceptibility gene for pediatric UC [3].  However, whether a specific mucosal cytokine profile 
is associated with clinical outcomes in pediatric UC is not known. 
 
3.8.  Characterizing Patients by Corticosteroid Response 
CS have been the mainstay of therapy for moderate or severe UC; disease course is often 
characterized in terms of being CS-responsive, CS-dependent, or CS-refractory. 
 
3.8.1.  Corticosteroid Pathway and its Underlying Molecular Mechanism 
CS readily cross the cell membrane and bind to the cytosolic glucocorticoid receptor alpha 
(GRα).  In the absence of CS, the inactivated GRα is bound to a multi-protein structure within 
the cytoplasm which includes, among others, the proteins HSP90 and calreticulin [82, 96, 97] .  
In the presence of CS, GRα dissociates from this multi-protein structure, and the joint CS/GRα 
complex migrates to the nucleus and binds to the glucocorticoid responsive elements (GRE).  
Within the nucleus, the CS/GRα complex mediates various positive and negative reactions.  
Through an interaction with transcription factors such as AP-1 and NF-, the complex 
inhibits the transcription of pro-inflammatory factors [81, 98], thus decreasing systemic 
inflammation. 
Some of the variability in CS-responsiveness may stem from differences in disease severity; 
but underlying heterogeneity in molecular pathways involved in the CS response may also be 
important.  It has been demonstrated in clinical studies that CS therapy failure is more common 
in patients with severely active disease [8].  Furthermore, there have been in vitro studies of T-
cells isolated from UC patients who had failed to respond to CS therapy, whereby the cells 
obtained during the acute flare demonstrated CS resistance, while those obtained 3 months 
later, following colectomy, did not [99].  In contrast, there have been numerous clinical studies 
in both IBD and rheumatoid arthritis which have demonstrated no correlation between disease 
activity and in vitro CS response [82, 100]; whereas there have been various in vitro studies on 
samples from patients with a variety of inflammatory diseases where abnormal T-cell 
proliferation patterns have persisted during periods of inactive disease [101-103].  Even 
lymphocytes collected from healthy volunteers have a heterogeneous in vitro response to CS 
stimulation.  Taken together, these observations suggest that it is a combination of factors, 
relating to both intrinsic individual mechanisms and to underlying disease activity, which 
account for CS resistance in UC.  Any investigation examining predictors of CS response must 
consider both. 
 


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3.8.2.  Putative Explanations for Variation in Corticosteroid Response 
Variation in CS response has been related to both underlying genetic variation, as well as 
specific cytokine profiles. 
The Glucocorticoid Receptor (GR):  The GR (encoded by NR3C1 on chromosome 5 at 5q31) 
has various isoforms (, P, A); however, only GRα is pharmacologically functional [82, 
96].  Although produced in much smaller quantities, GRβ has a much longer half-life than 
GRα.  Interestingly, it has been demonstrated to inhibit GRα [104, 105].  While its physiologic 
role remains uncertain, increased GRβ expression has been associated with CS resistance in 
both asthma and UC patients [106-109].  To date, various genetic polymorphisms within the 
GR gene have been shown to differentiate CS responsive from resistant patients [96].  While 
some variants have been associated with CS hypersensitivity, others have been associated with 
a reduced CS response.  In some instances, this has been related to increased GRβ stability, but 
in most instances the mechanism of associated CS resistance has not been ascertained [96, 110-
117]. 
Macrophage migration inhibitory factor (MIF):  MIF is excreted from the pituitary gland and 
various immune cells including macrophages and dendritic cells.  It induces the secretion of 
various pro-inflammatory cytokines including TNFα, IL-6 and IL-1B.  It also regulates IL-2 
secretion as well as T-cell proliferation [118, 119].  As such, the induction of MIF can 
effectively counteract the immunosuppressive effects of CS.  Indeed, the importance of this 
molecule in the development of colitis per se has been clearly demonstrated in a murine model 
[120].  An association with a single polymorphism within the gene for this molecule (MIF-
173G>C) and CS resistance has been variably reported in children with juvenile arthritis [121-
124]. 
Cytokine stimulation and up-regulation:  Various studies have demonstrated the tight interplay 
between the levels of specific cytokines and CS resistance.  For instance, the joint effect of IL-
2 and IL-4 is to induce the phosphorylation of GR by p38MAPK, thereby reducing its affinity 
for CS, and increasing the levels of GRβ [124, 125].  Indeed, even when healthy donor 
lymphocytes are incubated with IL-2 and IL-4, the expression of GRβ increases and steroid 
resistance is induced [108].  Similarly, TNFα, IL-8 and IL-1β have also all been shown in vitro 
to induce GRβ expression (and thus steroid resistance) [126, 127] with clinical trials 
demonstrating higher colonic levels of these cytokines in UC patients refractory to CS 
compared to those responsive to such therapy [87].  Of note, IL-10 acts in an opposite manner 
and increases CS sensitivity [126, 127].  A sub-group of asthmatic patients resistant to CS 
therapy have been recognized as having a defect in IL-10 secretion [128].  In fact, carriage of 
the -1082 AA IL-10 genotype (low producer) may be a relevant risk factor for developing 
steroid-dependent IBD [129]. 
Given the above observations, temporary CS resistance mediated by pro-inflammatory 
cytokines, or defective up regulation of IL-10, may well occur, leading in turn to an 
observation of steroid resistant disease. 
 
3.9.  Assessing Success and Monitoring Disease Activity in Ulcerative Colitis 
Disease course is generally described in terms of a patient’s response to therapy: the ability to 
induce remission, the frequency of relapse and the dependence on CS.  Escalation in therapy is 
usually due to an inadequate response to previous therapeutic interventions.  Thus, the ‘natural 


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history’ of UC is intimately entwined with therapeutic exposure.  Ideally, any study aimed at 
predicting outcome must recognize this and endeavor to incorporate both features into its 
design. 
In order to study the longitudinal disease course of a chronic relapsing and remitting disease, a 
reliable method of assessing disease activity is required.  The Pediatric Ulcerative Colitis 
Activity Index (PUCAI) has been developed by and validated for use as a non-invasive 
measure of disease activity in pediatric UC [2].  The PUCAI scores 6 areas (abdominal pain, 
degree of rectal bleeding, stool consistency, number of stools per 24 hours, nocturnal stooling, 
and activity level) with a minimum and maximum score of zero and 85, respectively.  It has 
been shown to have an excellent correlation with physician global assessment, colonoscopic 
appearance, and Mayo endoscopic severity score [2]. 
The relevance of specifically ascertaining mucosal healing (MH) in addition to clinical 
response, and its relationship to ‘disease activity’ is being increasingly studied [130-133].  As 
confirmed in a recent meta-analysis [134], clinical trials in UC that use endoscopy as an 
endpoint have a significantly lower placebo rate, highlighting the degree of discord that exists 
between disease activity indices and the condition of a patient’s mucosa.  Current data, mostly 
gained from adult trials, indicate that MH, particularly with CS, is more often achieved in UC 
than in CD.  What is less certain, however, is whether early MH improves long-term disease 
outcomes in UC.  Certainly, observations in CD (where MH has been associated with longer 
periods of clinical remission, fewer hospitalizations and fewer surgical interventions [131, 135]  
support the longer term utility of achieving MH.  Minimal similar data, however, are available 
for UC, although a recent cohort study of adult patients from Norway did observe a notably 
lower rate of colectomy within 5 years for patients with early MH compared to those without 
[136].  This was recently confirmed by a prospective cohort study from Italy in which mucosal 
healing at week 12 following the initial course of CS for adult UC was associated with 
significantly less requirement for immunosuppressive therapy or colectomy during follow-
up[137].  No similar pediatric data exist. 
Unfortunately, routine endoscopic re-assessment to ascertain MH is more difficult in children 
than in adults given the need for sedation/anesthesia in the former group.  Thus, if achieving 
early MH does, indeed, prove an important determinant of longer-term UC outcomes, 
identifying an effective non-invasive marker of MH will be paramount.  To date, there remains 
no single, universally accepted, non-invasive biological marker of disease activity in Pediatric 
IBD.  While patients with active IBD can have altered levels of acute phase proteins, elevated 
ESR and thrombocytosis [138, 139], these markers are not always reliable in children and 
adolescents [138].  Members of the S100 family (a group of binding proteins specifically 
expressed by granulocytes) may potentially provide better biological evidence of gut 
inflammation [140].  One of the proteins from this family is calprotectin.  Calprotectin is a 
calcium binding protein found within the neutrophil cytosol that is released with cell death or 
activation, and has therefore been studied as a marker of intestinal inflammation.  It is stable at 
room temperature for one week, and up to one year if frozen.  It can be measured from a 
random, spot stool using a commercially available ELISA which has excellent inter- and intra-
observer reproducibility.  Fecal levels of calprotectin have been shown to be elevated in active 
disease, to fall with treatment, and to correlate very well with endoscopic findings [140, 141].  
Furthermore, a cutoff of 150 mcg/gm in patients with quiescent IBD is a highly sensitive 
(90%) and specific (83%) predictor of disease relapse within 12 months [142].  Minimal data 


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are available from pediatric clinical trials utilizing biological and formal disease activity 
indices concurrently. 
 
3.10.  Need for a well-designed study 
Multi-center registry data concerning incident cases of pediatric UC have served to 
characterize the spectrum of illness at presentation.  There are no studies in which drug 
treatment has been standardized according to a set protocol, nor has there been an attempt to 
better characterize the inflammatory process and to consider how serologic or genetic factors 
influence the presentation and severity of disease.  In no outpatient study to date have 
biological and molecular data been prospectively collected in parallel with thorough clinical 
data.  The DNA collected in PROTECT will be available for assessment of UC susceptibility 
genes, currently and yet to be identified, and, equally importantly, for assessment of 
polymorphisms in genes associated with CS and aminosalicylate response.  The value of 
serologic and fecal inflammatory markers in predicting disease course as well as relapse off CS 
in those subjects who previously responded to CS will be assessed. 
Overall, this multi-center cohort of pediatric UC will facilitate assessment of outcomes with 
best current treatments, will allow study of factors important to pathogenesis, and will form the 
basis of ongoing modifications of therapeutic regimens based on clinical, genetic and serologic 
variables.  As such, it will represent the first large-scale attempt to advance knowledge and 
improve outcomes in this rare and often devastating pediatric disease.  Importantly, members 
of the PROTECT investigators group have been actively involved in developing a North 
American pediatric IBD Quality Improvement (QI) network over the past several years, 
ImproveCareNow (ICN) [143].  ICN members care for over 10,000 children at more than 30 
pediatric hospitals in the United States and Canada.  This has provided a framework for 
translation of clinical trials of 6-mercaptopurine and infliximab in pediatric CD to practice, 
with substantial improvements in remission rates across the collaborative.  It is anticipated that 
ICN will provide the infra-structure to rapidly spread findings from PROTECT to the larger 
pediatric IBD community.   
 


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4.  STUDY HYPOTHESES 
 
We hypothesize that in children presenting with ulcerative colitis initial response to the initial 4 
weeks of therapy as well as specific clinical, genetic, and immune parameters determined 
during the initial course of therapy will predict subsequently severe disease as reflected by 
need for escalation of medical therapy or surgery. 
 
4.1.  Primary Hypothesis 
Hypothesis 1.  Relative to other patients, those who have a PUCAI<10 at 4 weeks from start of 
therapy will be more likely to be in SFR (PUCAI<10) at 52 weeks while receiving the 
aminosalicylate mesalamine only as maintenance therapy without the need for rescue therapy 
with IM, CI, anti-TNFα, or surgery. 
 
4.2.  Secondary Hypotheses 
Hypothesis 2. Relative to other patients, those who have  a PUCAI<10 at 4 weeks from start of 
therapy will be more likely to be in corticosteroid free remission (PUCAI<10) at 12, 26 and 
104 weeks while receiving mesalamine only as maintenance therapy without the need for 
rescue therapy with IM, CI, anti-TNFα, or surgery. 
 
Hypothesis 3.  Relative to other patients, those who have a PUCAI<10 at 4 weeks from start of 
therapy  will  have lower colectomy rates through 104 weeks. 
 
Hypothesis 4.  Relative to other patients, those who have a PUCAI<10 at 4 weeks from start of 
therapy will be more likely to have Mayo endoscopy sub-score of 0 or 1 at 52 weeks. 
 
Hypothesis 5.  A prediction model based on factors supported by preliminary data and 
measured at weeks 0, 4, and/or 12 will have good sensitivity and specificity (each ≥ 0.75) for 
the 52-week outcome of SFR. 
 
Hypothesis 6.  Relative to patients who have poor mesalamine adherence (< 80% of prescribed 
medication taken), those with good adherence (≥ 80% of prescribed medication taken) will be 
more likely to be in SFR (PUCAI<10) at 52 weeks while receiving mesalamine only as 
maintenance therapy without the need for rescue therapy with IM, CI, anti-TNFα, or surgery 
 
Hypothesis 7. Relative to patients who are not in SFR at 52 , those who are in SFR at 52 
weeks are less likely to require colectomy by terminal follow up visit (2 to 5 years from 
diagnosis). 
 
Hypothesis 8.  Relative to patients who do not achieve a Mayo endoscopy sub-score of 0 or 1 
at week 52, those who achieve a Mayo endoscopy sub-score of 0 or 1 at week 52 are less likely 
to require colectomy by terminal follow up visit (2 years (104 weeks) to 5 years from 
diagnosis). 
 


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Hypothesis 9. Relative to other patients, those  who achieve SFR on mesalamine only without 
the need for rescue therapy at 52 weeks are less likely to require the addition of 
immunomodulators or anti-TNFα therapy by week 104. 
 
Hypothesis 10.  A panel of genes can be identified in rectal mucosa at diagnosis whose 
expression will accurately predict week 52 SFR. 
 
4.3.  Exploratory Hypothesis 
Hypothesis 11.  The prediction model of hypothesis 5 for week 52 SFR can be further 
improved by incorporating additional genetic, genomic and immune factors. 
 


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5.  STUDY ENDPOINTS 
 
5.1.  Overview of Primary and Secondary Clinical Endpoints 
The primary clinical endpoint is being in corticosteroid free remission (SFR) at 52 weeks on 
mesalamine only without prior recourse to medical or surgical rescue therapy.  The proportion 
of patients in SFR at 52 weeks will serve as the primary analysis variable.  Secondary 
endpoints include SFR at other time-points, endoscopic outcomes at week 52, quality of life 
measures at 52 weeks,  and colectomy during follow-up.  For endpoints ascertained at a 
particular time-point, the proportion of patients having the endpoint will serve as the principal 
analysis variable.  For colectomy, time to colectomy will serve as the principal analysis 
variable. 
 
5.2.  Clinical Endpoints 
5.2.1.  Primary Endpoint: SFR at 52 Weeks on Mesalamine Only 
The primary efficacy endpoint is being in SFR (PUCAI<10) at 52 weeks from start of therapy 
while receiving mesalamine only as maintenance therapy without the need for rescue therapy 
with IM, CI, anti-TNFα, or surgery. 
 
5.2.2.  Secondary Endpoints 
The secondary endpoints are defined relative to start of therapy: 
 PUCAI <10 at 4 weeks  
 SFR at 12 weeks without the need for rescue therapy 
 SFR at 26 weeks without the need for rescue therapy 
 SFR at 104 weeks without the need for rescue therapy 
 Endoscopic response (Mayo score reduced by ≥1) and being 0,1 at week 52 
 Endoscopic remission (Mayo score 0) at week 52 
 IMPACT – III at 52 and 104 weeks 
 Colectomy free status during the follow-up period 
 
5.2.3.  Definitions of Corticosteroid (CS) Status 
CS-free:  Subjects will be considered to be CS-free if they are taking no CS at the assessment 
and have been CS-free for a minimum of 2 weeks at that point.  Alternate day CS will not be 
considered CS-free. 
CS responsive:  Clinical improvement and termination of CS within 12 weeks of initiating 
therapy, and remaining off CS for the remainder of the first year for a minimum of six months 
following initial tapering before requiring re-treatment with CS.  
CS dependent:  Required CS >12 weeks to control symptoms, or at least one additional course 
of CS starting <6 months after initial successful CS weaning  
CS refractory:  Unresponsive to oral CS within four weeks of starting therapy and required 
rescue therapy with CI or anti-TNFα therapy; or required rescue therapy with CI or anti-TNFa 


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therapy because of failure to respond to intravenous CS, or required colectomy because of 
failure to respond to CS.  
6.  EXPERIMENTAL DESIGN AND METHODS 
 
6.1.  Study Subjects and Eligibility 
A total of up to 475 children and adolescents will be recruited after being newly diagnosed 
with ulcerative colitis. We anticipate that patient recruitment will take place over the initial 4 
years of the study. Patient follow-up will be for a minimum of one year, and will continue until 
the termination of the study allowing for up to 1 to 5 years of observation. 
 
6.1.1.  Eligibility 
The eligibility criteria are defined to reflect the typical patient population with ulcerative colitis 
for whom initial therapy with either mesalamine or corticosteroids followed by mesalamine is 
considered standard of care.  Patients with proctitis, who only represent approximately 5-7% of 
pediatric patients with ulcerative colitis are not eligible for study, as they are generally 
managed with topical and not oral therapy. 
 
6.1.1.1.  Inclusion Criteria 
 Age ≥ 4years and ≤17 years at initiation of therapy (achieved 4th birthday, not yet 18th) 
 Weight ≥15 kg 
 New diagnosis of ulcerative colitis established by standard clinical, endoscopic, and 
histologic features at the PROTECT study site (see Case Ascertainment below) 
 Colitis extending beyond the rectosigmoid (Paris classification E2, E3, or E4)[144]. If a 
patient is seriously ill and the clinician does not advance the colonoscope beyond the 
sigmoid colon but the clinical condition of the patient highly suggests more extensive 
disease then that patient is eligible for study.  
 Disease activity by PUCAI of ≥10 at diagnosis 
 No therapy previously initiated to treat the newly diagnosed ulcerative colitis 
 Stool culture negative for routine enteric pathogens (Salmonella, Shigella, 
Campylobacter, E. coli 0157:H7) and Clostridium difficile toxin. Recent successful 
treatment for Clostridium difficile does not exclude a patient if toxin now absent. 
However, the patient must be a minimum of 5 weeks from the time treatment was 
started at the time toxin is absent.  
 Stool study negative for enteric parasites (ova and parasites) 
 Parent/guardian consent and patient assent 
 Ability to remain in follow-up for a minimum of one year from diagnosis 
 Female patients of child bearing age must have a negative urine pregnancy test and 
practice acceptable contraception (e.g., abstinence, intramuscular or hormonal 
contraception, two barrier methods (e.g., condom, diaphragm, or spermicide), 
intrauterine device, verbal report of the partner with history of vasectomy, or be 
surgically sterile). All female patients of childbearing potential (post-menarche) will 
undergo urine pregnancy testing at screening and must not be lactating.  


PROTECT Study Protocol 2FEB2015 
 
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6.1.1.2.  Exclusion Criteria 
 Clinical, endoscopic, radiologic, or histologic evidence suggesting CD consistent with 
Paris and NASPGHAN criteria[144, 145], (see Case identification below)  
 A previous diagnosis of inflammatory bowel disease for which treatment was given 
 Evidence of any active enteric infection at the time of study entryUse of any oral CS for 
non-gastrointestinal indication within the past 4 weeks (e.g., asthma). Use of inhaled 
CS does not exclude a patient.  
 History of use of IM or anti-TNFα agent for other medical conditions (e.g., juvenile 
rheumatoid arthritis) within the past 6 months 
 Use of Accutane within the past 4 weeks 
 Use of any investigational drug within the past four weeks 
 Use of any 5-aminosalicylate within the past 4 weeks 
 Pregnancy 
 Subjects with poorly controlled medical conditions (e.g. diabetes, congestive heart 
failure)  
 Proctitis or proctosigmoiditis only (Paris classification E1) on colonoscopic evaluation 
 Chronic renal disease (BUN and serum creatinine >1.5 times the upper normal limit)  
 Hepatic disease (AST or ALP greater than 3 times the upper normal limit in the absence 
of concomitant liver disease associated with IBD following full evaluation)  
 History of allergy or hypersensitivity to salicylates, aminosalicylates, or any component 
of the Pentasa capsule. 
 History of coexisting chronic illness or evidence of significant organic or psychiatric 
disease on medical history or physical examination, which, in the Investigator’s 
opinion, would prevent participation in the study 
 History or presence of any condition causing malabsorption or an effect on 
gastrointestinal (GI) motility, or history of extensive small bowel resection (greater 
than half the length of the small intestine). 
 The finding of Helicobacter pylori at the time of evaluation does not exclude the patient 
from the study. Whether to treat this patient for Helicobacter pylori and when will be 
left to the discretion of the site.  
 
 
6.2.  Case Identification and Consent 
Subjects will be recruited from the clinical population served by up to 30 high volume pediatric 
IBD centers in the United States and Canada.  
 
6.2.1.  Case Identification 
Patients with a confirmed or suspected diagnosis of UC are eligible for enrollment. All patients 
must be newly diagnosed at a PROTECT study site. Investigators will have a checklist form 
for each potential subject to confirm eligibility. A diagnosis of UC for this study will require 
[145]: 


PROTECT Study Protocol 2FEB2015 
 
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1) Clinical history consistent with colonic inflammation (e.g. any combination of diarrhea, 
bleeding, abdominal pain) 
2) Negative culture for enteric pathogens as noted in Inclusion/Exclusion criteria 
3) Endoscopic findings of diffuse continuous mucosal inflammation involving the rectum and 
extending to a variable degree proximally.  Features may include granularity, loss of 
vascular pattern, friability, small superficial ulcers, mucopurulent exudate, and a line of 
demarcation between abnormal and normal colon in a patient whose colitis does not extend 
to the cecum. Histologic features of chronicity must be present. There will be cryptitis 
and/or crypt abscesses to demonstrate activity. Chronicity will be demonstrated by mucin 
depletion, crypt distortion, crypt branching, crypt atrophy, basal lymphocytosis (not all 
features need be present). 
4) Assessment of the small bowel is not required to establish an initial  diagnosis of UC. 
However imaging of the small bowel via barium contrast, magnetic resonance 
enterography, CT enterography, or capsule endoscopy is generally part of standard 
evaluation of all newly diagnosed children with inflammatory bowel disease and will be 
performed during the initial 3 months after diagnosis. Visualization of the terminal ileum 
during colonoscopy may substitute for small bowel imaging at the discretion of the 
investigative site but the site will be encouraged to do additional imaging as noted above.   
 
Non classic finding of UC that do not exclude a diagnosis of UC include[145]: 
 Gastritis without distinct aphthous lesions (non specific gastritis)  
 Backwash ileitis in the presence of pancolitis (ileal erythema without ulceration)  
 Periappendiceal inflammation in a patient without pancolitis (cecal patch)  
 Rectal inflammation less severe than more proximal colon (relative rectal sparing)  
 Microscopic ileitis without granuloma 
 Microscopic gastritis without granuloma 
 Macroscopic patchiness (macroscopically normal colonic mucosa between two areas of 
colonic inflammation)  
 Serological reactivity to anti-microbial antigens (ASCA, CBir, OmpC, I2). 
 
5) A diagnosis of UC will be EXCLUDED if any of the following are present: 
 Absolute histologic rectal sparing 
 Perianal fistula, atypically located fissures, or significant skin tags 
 Stenosis, cobble stoning, or significant linear ulceration in the terminal ileum 
 Segmental colitis i.e. discontinuous disease: macroscopic and microscopic skip lesions 
 Radiologic evidence of stricture or fistula 
 Histopathology showing transmural inflammatory cell infiltrate (noted at colectomy 
and in the absence of fulminant disease) or epithelial granulomas not related to crypt 
rupture (at any time) and absence of identifiable infectious agents.  Transmural 
inflammation might be established at a time after diagnosis and would subsequently 
establish a diagnosis of CD 


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Capsule endoscopic lesions that are considered indicative of CD by the treating physician or 
MRI/CT enterography demonstrating disease in the small bowel consistent with CD or  barium 
contrast series indicating CD. 
 
6.2.2.  Consent 
The parents or legal guardians of children suspected of having ulcerative colitis will be 
approached for consent for enrollment into the study.  Assent will also be obtained from the 
patient when appropriate based on age and institutional IRB requirements. 
 
6.2.3.  Ethical Considerations 
Prior to initiating enrollment at each clinical site, the informed consent document will be 
reviewed by the Steering Committee, NIDDK Project Office, Data and Safety Monitoring 
Board (DSMB), and the Institutional Review Board (IRB) at each clinical site to ensure that 
the document is consistent with study protocol, federal regulations, and individual institutions’ 
policies.  Enrollment will not commence in the study as a whole until this document has been 
approved by the Steering Committee, DSMB, and the NIDDK Project Office.  In addition, 
enrollment will not commence at a site until approval is granted by that site’s IRB. 
 
6.2.3.1.  Special Ethical Issues 
Genetics 
PROTECT will be collecting specimens for and conducting analyses of genetic information on 
all participants.  Strict confidentially standards are in place and will be maintained to protect 
the privacy of PROTECT participants.  Biospecimen samples will be labeled with a unique 
identifier that does not contain any protected health information or otherwise identifies a 
participant. 
In the event a new genetic marker is found during the course of PROTECT, participants with 
this hypothetical marker will not be notified.  The analyses conducted as part of PROTECT are 
not intended to be diagnostic, and may not take place in a Clinical Laboratory Improvement 
Amendments (CLIA) certified lab. 
Consenting for use of genetic material will be over and above consenting to participate in the 
study.  A patient may participate in the study without consent being given to analyze genetic 
information. 
 
Specimen Storage for Future Studies 
Some of the biospecimens collected as part of this study will be stored for future analyses.  
These specimens will not be individually identifiable by the specimen repository, laboratory or 
Data Coordinating Center (DCC) personnel.  The DCC will develop and maintain a tracking 
system whereby study participants can modify their level of consent.  Participants can ask that 
any specimens still in storage be destroyed and not included in future analyses.  The request to 
remove stored specimens will be made at the clinical sites to preserve participant 
confidentiality. 
 
Week 52 Flexible Sigmoidoscopy 


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All patients will undergo a diagnostic colonoscopy prior to study entry.  This is part of the 
standard work-up for diagnosing ulcerative colitis and is not considered to be a study 
procedure.  An optional repeat flexible sigmoidoscopy will be offered to study subjects at week 
52 who are in clinical remission and receiving mesalamine only to assess for mucosal healing.  
Consent for the week 52 sigmoidoscopy will be separate from consent for the study and 
patients may refuse the week 52 sigmoidoscopy without jeopardizing either their continued 
participation in the study or the medical care they receive. 
 
Additional clinically indicated colonoscopy 
If a patient undergoes a colonoscopy during the study course that the attending clinician deems  
needed for clinical care decisions then consent will be sought to obtain 4 additional rectal 
mucosal biopsies for study purposes. Consent for this additional biopsy material will be 
separate from consent for the study. 
 
6.2.4.  Risks and Benefits to Participants 
6.2.4.1.  Potential Risks at Clinical Sites 
Blood Draws:  Phlebotomy can cause discomfort, bruising, a small risk of infection, or 
thrombosis.  Standard aseptic technique will be employed to prevent infection. All blood draws 
will take place at the time of routine blood test monitoring. 
Stool Sample:  There is no specific risk to the subject in obtaining a stool sample though 
bringing a stool sample from home may be inconvenient. 
Rectal biopsy:  The procurement of four additional rectal biopsies from the proximal to mid  
rectum by grasp forceps at the time of diagnostic colonoscopy or during a follow-up clinically 
indicated colonoscopy engenders minimal additional risk to the patient. The risk of perforation 
is largely related to the colonoscopy itself, not mucosal biopsies [146].  
Flexible sigmoidoscopy at 52 weeks:  Risks of flexible sigmoidoscopy include making a hole 
in the lining of the colon which would need to be surgically repaired (perforation); bleeding; 
and infection.  Each of these risks is quite low.  The risk of colon perforation is approximately 
1 out of every 1000 patients and may not be related to the biopsies. If subjects agree to 
additional biopsy samples for research, obtaining the additional   colon biopsy samples may not 
significantly increase the risk of perforation, bleeding, or infection associated with the 
colonoscopy.  The subject would not ordinarily have the week 52 sigmoidoscopy as part of 
regular medical care unless there were a medical indication or in the view of the attending 
physician it was needed.  
Questionnaire data collection:  All measures have been used in previous research, without any 
apparent significant risk.  It is feasible that subjects may experience some anxiety or distress 
associated with completion of the questionnaires; however this is unlikely to result in severe or 
ongoing effects.  All project staff will be extensively trained on administration of instruments, 
with emphasis on preventing and dealing with anxiety or discomfort.  If, as a function of the 
assessment, a significant mental health risk is identified, parents will be informed and an 
appropriate referral for treatment will be made with parental consent. If imminent danger is 
noted the patient will be escorted to a local emergency room or mental health facility for 
additional evaluation. More information about the questionnaires is provided later in sections 
6.5-6.7. 


PROTECT Study Protocol 2FEB2015 
 
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Genetic Testing:  A breech in confidentiality could have potential unforeseen effects, and 
potentially could affect insurability.  Every effort will be made to maintain confidentiality by 
identifying samples by a laboratory sample number rather than by personal identifier, and 
keeping all study data in locked files as noted above. 
Treatment protocols:  The treatment protocols have been determined by consensus of pediatric 
inflammatory bowel disease experts and are considered to represent the standard of care for 
children and adolescents with ulcerative colitis.  This standard of care therapy would therefore 
not be expected to engender any risks of therapy that would exceed normal clinical practice at 
any of the investigative sites. The potential risks and benefits of mesalamine and prednisone or 
liquid equivalent prednisolone will be discussed with each patient and family in the customary 
fashion. For patients requiring escalation of therapy including a rescue medication or surgery, 
each investigative site will use its customary explanation of risks and benefits of each therapy, 
and the determination of which rescue therapy to be used will be at the discretion of the 
attending physician per their normal standard of care. 
Breach of confidentiality:  There is a risk that someone not authorized to view participant 
information, including identifiable information, will gain access to this information.  Clinical 
sites will take measures to prevent this from happening and will comply with local regulations. 
 
6.2.4.2.  Adequacy of Protection Against Risks at Clinical Sites 
Recruitment and Informed Consent:  Study participants will be identified based on a suspected 
new diagnosis of ulcerative colitis, and will be approached by research staff.  Research staff 
will explain the study, answer questions, and obtain informed consent and assent as indicated.  
Informed consent/assent will be documented by the parents’ and child’s signature on the 
appropriate form and a signature of the person obtaining the consent.  A copy of the signed 
form will be returned to the family. 
Protection Against Risk:  Every effort will be made to maintain confidentiality.  Study records 
and samples will be recorded by a numerical identifier.  The list of names associated with the 
identifying numbers will be kept separate from the questionnaires in a folder labeled in such a 
way that its meaning or significance will not be evident to anyone not involved in the study.  
Subjects will be informed in advance of the voluntary nature of the study. Previously consented 
individuals have the option to opt out of this study at any time. 
We have devised several layers of protection for Protected Health Information (PHI).  All 
information entered into the PROTECT study database is stored on a secure server and the 
database is password protected.  Access to the database is restricted to those directly involved 
in data collection or analysis.  Each individual is given a unique identifier in the database.  The 
PHI is stored separately from the clinical information, laboratory values, and genotype data, 
and is not available to laboratory personnel. 
 
6.2.4.3.  Potential Risks at the Data Coordinating Center 
The only direct risk to participants originating from the DCC is the risk of breach of 
confidentiality.  The DCC has many safeguards against this, including staff training, controlled 
access to computer and paper files, back-up systems for electronic data, and protection against 
malicious code.  More detail regarding the DCC’s security plans and measures is available in 
Section 10 of this protocol: Data Management. 


PROTECT Study Protocol 2FEB2015 
 
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6.2.4.4.  Change in or Loss of Insurance Coverage 
There is a risk that a participant may experience change in coverage, change in cost, or loss of 
insurance as a result of incidental findings during the course of the study.  Treatments provided 
and some of the tests conducted are part of routine clinical care and will be billed as such.  
Other tests that are performed specifically as part of the PROTECT study are for research 
purposes only, and are not for clinical or diagnostic use.  Therefore, the results of these tests 
will not be reported to insurance companies. 
During the course of a research-related procedure, it is possible that a previously unknown 
disease or illness will be discovered or that a change in disease severity will be found.  If one 
of these two events occurs, the participant may be referred to care outside of the PROTECT 
study.  The resultant diagnosis and care may result in changes to a participant’s insurance. 
 
6.2.4.5. Potential Benefits 
This study may not result in any direct benefit to participants.  However, results of this study 
will help determine response and remission rates to standardized treatment protocols, and 
potentially identify clinical, immunologic, or genetic factors that are associated with disease 
that requires escalation of therapy to include rescue medications or surgery facilitating future 
clinical trials in ulcerative colitis.  Subjects will be paid a small stipend for biospecimens and 
Pentasa (mesalamine) will be provided free of charge for up to one year from initiation  of 
Pentasa therapy.  
Importance of the Knowledge to be Gained:  This study is the first step in establishing  baseline 
knowledge of response and remission to standardized care in the treatment of UC in children 
and potentially help build predictive models of response.  This information will be invaluable 
in designing future clinical trials, particularly in children who are deemed to be at high risk for 
requiring escalation of therapy and use of rescue medications or possible colectomy.  The 
additional risk to participants is minimal compared to usual clinical practice as they will 
receive standardized care designed by experts in pediatric IBD and then will utilize all 
additional available therapies as their disease course warrants.  Given the minimal risk to 
participants, as well as the potential benefit to future children with UC, we believe the study is 
justified. 
 
6.2.5.  When and How to Withdraw Participants from Therapy or Assessment 
6.2.5.1.  Change in Diagnosis 
A participant will be withdrawn from the study if there is a change in his/her diagnosis to CD.. 
 
6.2.5.2.  Principal Investigator (PI) Discretion 
A participant can be withdrawn from the study if the clinical site PI determines that it is the 
best interest for the subject to stop participation in the study or that it is unsafe or unethical to 
continue in the study. 
 


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6.2.5.3.  Participant Request 
Participants may withdraw at any time for any reason.  At the time of withdrawal participants 
can either 1) decline to provide any more data or specimens to the study but allow use of 
previously collected data and/or specimens, 2) withdraw all their data from study databases and 
request that any stored samples be destroyed, or 3) withdraw some portion of the data collected 
(i.e., participants may withdraw specimens but not examination data or vice versa). 
 
6.2.5.4.  Participant Exit Interview 
If a participant chooses to withdraw from the PROTECT study, the study coordinator will 
endeavor to conduct an exit interview to determine the disposition of the participant’s study 
data.  The participant may decline to participate in the exit interview, in which case the 
consent/assent in place at the time of study withdrawal will be used to determine the status of 
the participant’s data. 
 
6.3.  Baseline and Follow-up Evaluation 
The schedule of study visits and assessments is presented in Table 6.1.  “Baseline” may consist 
of two separate visits, one for diagnosis and another to begin treatment.  A maximum of 14 
days will be allowed between diagnosis and start of treatment. The beginning of treatment 
will be regarded as day 0 for all subsequent milestones.  Follow-up clinic visits will be 
conducted for a minimum of 1 year to a maximum of 5 years following study enrollment 
depending upon the actual time of enrollment.  Demographic and contact information will be 
collected at baseline.  Several assessments will be made at baseline and at each study visit.  
These include PUCAI scoring, and completion of behavioral and quality of life instruments.  
Adherence to mesalamine therapy will be assessed at post-baseline visits.  Biospecimens will 
be collected according to the schedule in the table.  The Mayo endoscopy sub-score will be 
determined from the diagnostic colonoscopy and in those patients qualifying for and agreeing 
to the week 52 sigmoidoscopy. 
Various assessments will also be made through telephone contacts between study visits, 
including response to treatment, and adherence to medication.  
 


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Table 6.1.  Schedule of visits and assessments# 
Activity 
Baseline 
2, 6, 8, 10 
weeks –
telephone 
assessment
#  
4
α
 
weeks 
12α 
weeks 
26α 
weeks 
39α 
weeks 
52α 
weeks 
78α 
weeks 
104α 
weeks 
**Annual 
visits: 
1-5 years 
Routine Blood 
Monitoring* 
X§ 
 
X 
X 
X 
X 
X 
 
X 
X 
Study Specific 
Blood 
X 
 
X 
X 
 
 
X 
 
 
 
Urinalysis# 
X 
 
 
 
X 
 
X 
 
X 
X 
Rectal 
biopsies 
X 
 
 
 
 
 
X 
 
 
 
Stool sample 
X 
 
X 
X 
 
 
X 
 
 
 
Questionnaire$ 
X 
Up to  
X 
 
 
 
X 
 
X 
 
PUCAI 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adherence 
monitoring 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Colectomy 
free status 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Height/Weight 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
#     Visit timing is scheduled relative to start of therapy. Additional phone calls (2,6,8,10 weeks)  may 
occur during the first 12 weeks of therapy for routine patient contact and oral corticosteroid 
adjustment as necessary. It is expected that all patients will receive a week 2 phone call.  
* 
Routine blood monitoring for standard of care for children with ulcerative colitis includes: complete 
blood count with differential, erythrocyte sedimentation rate, serum albumin, C-reactive protein, 
serum aminotransferases, serum gammaglutamyl transferase (GGT) and renal function studies 
(BUN, creatinine).  Serum amylase and lipase are also monitored in patients on mesalamine therapy 
over the first 12 weeks of therapy. Patients on mesalamine will have yearly urinalysis.  
§    Baseline routine blood monitoring should be done as close to Day 0 as possible, and no longer than 
14 days previously in a clinically stable patient 
α    Follow-up routine blood monitoring should be done as close as possible to the designated time point 
when deemed necessary for clinical care by the attending physician 
** Follow-up visits beyond 1 year will depend upon time of enrollment in the study. 
$     Questionnaire will be completed between baseline and 4 weeks, and then again at 52, 104 weeks. 
   
 
6.4.  Treatment 
Details of the standardized PROTECT treatment regimen are provided in section 7 of this 
protocol. 
 
6.5.  Pediatric Ulcerative Colitis Activity Index (PUCAI) [2] 
The Pediatric Ulcerative Colitis Activity Index (PUCAI) is a key component of primary and 
secondary outcomes of PROTECT as well as guiding treatment.  PUCAI scoring for study 
outcome data will be done by a PROTECT Study physician and will be based on information 


PROTECT Study Protocol 2FEB2015 
 
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they have directly obtained during clinical evaluation of the patient that visit.  Study nurses or 
coordinators may obtain a PUCAI score by patient report in a bi-weekly telephone follow-up 
as part of steroid tapering.  The scores obtained by the Study Nurse/Coordinator will be based 
on patient self-report and may be used to adjust treatment but will not be utilized for 
determination of study outcomes. 
 
6.5.1.  PUCAI Determination 
The PUCAI is scored using Table 6.2.  The range of possible scores is 0-85 and the 
classification within this range is: 
 
PUCAI<10 
Inactive disease 
 
PUCAI 10-34 
Mild activity 
 
PUCAI 35-64 
Moderate activity 
 
PUCAI ≥65 
Severe activity 
 
6.5.2.  Justification for the Use of PUCAI 
The PUCAI is now considered the standard assessment tool for UC in children [2, 147, 148].  
It has been validated in large numbers of patients both during its initial development and 
subsequently in real world experience in the Pediatric IBD Collaborative Research Group 
Registry.  A recent sub-analysis of the data originally published previously [2] demonstrated 
no difference in validity between groups ≤12 years and >12 years as seen in Table 6.3.  
Moreover, age (entered as a continuous variable) was not associated with the PUCAI score in a 
multivariate analysis in which colonoscopic score was the dependent variable and age and the 
PUCAI score as the explanatory variables (beta=1.17, p<0.001 for the PUCAI and beta=–
0.146, p=0.26 for age). 
Data to support cut scores for defining various levels of disease activity and changes in level of 
disease activity have been validated previously [2]. The sub-domains of the PUCAI have been 
validated as appropriate, comprehensive, and able to detect change in patient condition. The 
major strength of the PUCAI lies in its high psychometric properties (high reliability, validity, 
and responsiveness). A recently published study demonstrated that a physician based index is 
required to adequately assess disease activity in children with UC [149]. The study data 
demonstrated that disease rating by physicians (using the PUCAI) more closely relates to 
measures of disease activity than did patients’ rating of symptoms, regardless of the patient’s 
age. 
 


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Table 6.2.  PUCAI Scoring [2] 
ITEM 
POINTS 
1. Abdominal pain: 
 
No pain 
Pain can be ignored 
Pain cannot be ignored 
0 
5 
10 
2. Rectal Bleeding 
 
None 
Small amount only in less than 50% of stools 
Small amount with most stools 
Large amount (>50% of the stool content) 
0 
10 
20 
30 
3. Stool consistency of most stools 
 
Formed 
Partially formed 
Completely unformed 
0 
5 
10 
4. Number of stools per 24 hours 
 
0-2 
3-5 
6-8 
>8 
0 
5 
10 
15 
5. Nocturnal stools (any episode causing awakening) 
 
No 
Yes 
0 
10 
6. Activity level 
 
No limitation of activity 
Occasional limitation of activity 
Severe restricted activity 
0 
5 
10 
SUM OF PUCAI (0-85) 
0-85 
 


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Table 6.3.  Correlations between constructs of disease activity and the PUCAI stratified 
by age groups (numbers represent rho coefficient) 
 
≤12 years 
>12 years old 
Colonoscopic score # 
0.83** 
0.72** 
Mayo score 
0.93** 
0.95** 
CRP  
0.45* 
0.54* 
Physicians’ global assessment 
of disease activity 
0.92** 
0.91** 
#Scored according to Beattie et al [150] in each part of the colonic segment, as 
previously described [151]. 
*P<0.01  **P<0.001 
6.6.  Mayo Sigmoidoscopy Score 
Endoscopic severity will be assessed at study entry in all study subjects and in the subset of 
patients who qualify for and agree to a repeat week 52 sigmoidoscopy using the Mayo 
endoscopy score [1]. Photographs corresponding to each score will be provided to investigative 
sites to serve as a guide.  This scoring system is summarized in Table 6.4. A de-identified 
photo of the most inflamed segment of the rectosigmoid will be electronically recorded and 
labeled with the patient’s unique study identification number and transmitted to the DCC 
where it will also be read by central readers. A centralized histologic evaluation of a rectal 
biopsy that assesses inflammation (acute, chronic, severity) as well as architectural changes 
(crypt distortion/atrophy, surface villiform changes, basal plasmacytosis, and Paneth cell 
metaplasia) will also be performed from each endoscopic evaluation and recorded on a case 
report form. 
A recent study in which four expert endoscopists determined the Mayo score from images 
obtained from 93 UC patients demonstrated very good intra- and inter-observer agreement for 
the Mayo score (kappa value of 0.75 and 0.74, respectively) [152]. 
 
Table 6.4.  Schroeder endoscopic scoring system (Mayo score) 
0 = Normal or inactive disease 
1 = Mild disease (erythema, decreased vascular pattern, mild friability) 
2 = Moderate disease (marked erythema, absent vascular pattern, friability, erosions) 
3 = Severe disease (spontaneous bleeding, ulceration) 
  
 
6.7.  Other Assessments 
6.7.1.  Quality of Life Assessment 
Study subjects (ages 9-17 years) will complete a standardized quality of life instrument 
(IMPACT-III) [153] between initiation of standardized therapy and their 4 week visit, at 52 
weeks, and at 104 weeks.  A score of 144 or greater will be used as indicative of a good quality 
of life.  The instrument has been validated in pediatric patients from age 9 to 17 with 
inflammatory bowel disease, and the results will serve as a secondary endpoint [153].  These 


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data will be also used in an ancillary analysis of the effects of behavioral factors, adherence, 
and clinical disease severity upon quality of life. 
 
6.7.2.  Behavioral Assessment 
In order to account for behavioral variables that may affect medication adherence and thereby 
treatment outcome, the Behavior Assessment System for Children – Second Edition (BASC-2) 
will be used in this trial [154]. The age appropriate self- or parent-report version of these 
measures will be completed by patients and parents between initiation of therapy and  the 4 
week visit and at 52 weeks, and at 104 weeks.   The BASC-2 is a widely used, reliable (α = 
0.90 – 0.94, test-retest r = 0.84 – 0.90 for parent report; α= 0.94 – 0.96 , test-retest r= 0.81 – 
0.82 for child self-report), and valid inventory to assess and identify children and adolescents 
(ages 2-18; different forms for developmental levels) with emotional disturbances and 
behavioral disorders.  The BASC-2  measures externalizing, internalizing, and school 
problems, adaptive skills, and other problems.  Both the Parent Rating Scale and Child Self-
report forms will be used.  Respondents rate the presence and frequency of behavioral 
symptoms via a four-point Likert scale and by answering several true/false questions.  
Computerized scoring provides subscale T-scores.  Elevated T-scores indicate clinically 
significant pathology in the particular domain (e.g., internalizing problems) compared to 
norms.  These data will be used in an ancillary analysis of the effects of emotional disturbances 
and behavioral disorders on adherence. 
 
6.7.3.  Adherence 
For this trial, the Medication Event Monitoring System (MEMS®) will be used for adherence 
measurement.  The MEMS® 6 Trackcap monitoring system includes a standard plastic vial and 
cap containing a microelectronic circuit that records each time the cap is removed.  Adherence 
data parameters include number and percentage of doses removed, optimal daily dosing, 
estimation of therapeutic coverage, and mean and range of interdose intervals.  Research using 
these electronic monitors has demonstrated excellent validity, with 93% predictive value, 82% 
sensitivity, and 89% specificity.  Data from these devices are transferred to computer software 
that generates graphic feedback for patients to view.  MEMS monitoring can start at any time 
up to and including the Week 4 visit. At study visits at weeks  4, 12, 26, 39, and 52 data will be 
downloaded from the cap. Pill counts and self-report adherence data will also be obtained in 
conjunction with study visits and electronic adherence data collection.  These additional 
adherence assessments will be used for supplementary data (e.g., mechanical failure of MEMS 
devices, family forgets to bring MEMS device to study visit for data download). In the (rare) 
event of missing electronic monitor data, we will use pill count data to impute an adherence 
percentage during the missing time point. We will use www.mymedschedule.com to utilize 
either text messaging or email reminders when possible 
 
6.8.  Collection of Laboratory Samples 
6.8.1.  Blood and Stool 
Blood and stool will be collected at clinically indicated specified time points during a patient’s 
disease course.  “Standard of Care” laboratory testing including complete blood count, 
erythrocyte sedimentation rate, C-reactive protein, serum albumin, blood urea nitrogen, 
creatinine, serum aminotransferases, and gammaglutamyl transferase (GGT) will be measured 


PROTECT Study Protocol 2FEB2015 
 
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at diagnosis, 4 weeks, 12 weeks, 26 weeks, 39 weeks, 52 weeks, 104 weeks and will be 
performed at the Clinical Core Laboratory facility of the enrolling institution at diagnosis. Any 
significant laboratory abnormality detected will be followed up with subsequent testing as 
clinically indicated until resolution. “Non-Standard” testing will be shipped to a central facility 
at Emory University School of Medicine, the repository for biospecimens of the PRO-KIIDS 
research group.  A summary of the biospecimens which will be collected, their specific use for 
the PROTECT study, and their potential future use for ancillary studies, is provided in Table 
6.5. 
Blood draws will be performed by qualified nurses, physicians and phlebotomists according to 
standard phlebotomy techniques.  Participants will be asked to collect a stool sample at 
baseline (i.e., prior to the commencement of induction therapy) and at week 4, week 12, and 
week 52.   
 
Table 6.5.  Blood and Stool Sample Collection and Usage 
 
Biospecimen 
 
Entry 
Week 4 
(Day 30) 
Week 
12 
Week 
26 
Week 
39 
Week 
52 
PROTECT 
Data 
 
Ancillary Studies 
Blood 
 
 
 
 
 
 
 
 
Standard of 
Care 
X 
X 
X 
X 
X 
X 
 
 
Study Specific 
X 
X 
X 
 
 
X 
Vitamin. D 
Serology 
Multiplex Cytokines 
 
Proteomics 
Metabolomics 
Genomic DNA 
X 
 
 
 
 
X 
SNP Chip 
Fine mapping of 
genes associated 
with SFR 
Patient epigenetics 
Mucosal Biopsy 
 
 
 
 
 
 
 
Colon DNA 
X 
 
 
 
 
X 
None 
Microbiome 
Patient epigenetics 
Colon RNA 
X 
 
 
 
 
X 
RNA-seq by 
week 52 SFR 
Multiplex PCR 
Stool 
 
 
 
 
 
 
 
 
Study Specific 
X 
X 
X 
 
 
X 
Calprotectin 
OPG 
Microbiome 
Metabolomics 
Timing of biospecimen collection is relative to start of initial therapy 
 
6.8.2.  Mucosal Biopsies 
Four (4) rectal biopsies will be obtained at the time of diagnostic colonoscopy from the 
proximal to mid rectum for research purposes along with routine biopsies used to establish a 
diagnosis.  This will include all subjects at entry, and the subset of subjects who qualify for and 
agree to a week 52 sigmoidoscopy, or those who consent for study biopsy during additional 
routine colonoscopy.. 
 


PROTECT Study Protocol 2FEB2015 
 
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7.  TREATMENT REGIMEN 
 
7.1.  Overview 
An overview of the treatment regimen is presented graphically in Figure 7.1. 
 
Figure 7.1.  Graphical Representation of Treatment Regimen 
 
 
7.2Mesalamine 
Mesalamine (Pentasa®) has been chosen as the aminosalicylate for this study because of ease 
of administration to children of all ages in clinical practice over the past 10-15 years, ease of 
standardization of dosage, previous adult studies showing safety and efficacy [15, 17] and lack 
of oral staining that is associated with balsalazide.  Additionally, it can be administered to 
children unable to swallow the capsule by opening up and mixing with apple sauce or yogurt-
like foods.  The investigators have obtained an IND (111863) for this study.  Mesalamine will 
be provided by Shire Pharmaceuticals in 500 mg capsules that will be distributed to study sites.  
The package insert describing properties of the drug as well as potential toxicity is in the 
Appendix  attached to this protocol. This medication will be provided free of charge to each 
patient participating in this clinical protocol until the 52 week outcome assessment or until it is 
discontinued prior to 52 weeks. 
 
7.3.  Initiation of Medical Therapy 
The dosing schedule of mesalamine is shown in Table 7.1.  Pentasa®) comes in 500mg 
capsules, and doses will need to be rounded to the nearest 500mg increment, with a maximum 
dose of 75 mg/kg/day.  The average dose for the pediatric population will be approximately 70 
mg/kg/day.  Patients will be allowed to escalate to the final dose over 4 days to minimize side-
effects such as headache. 
 
Diagnosis of UC
Mild
PUCAI 10-34
Moderate/Severe
PUCAI 35-64
Severe/Fulminant ≥65
Mesalamine
Response
No Response
Remission
Flare
PO Steroids
Response
No Response
Add
Mesalamine
Wean
Prednisone
Remission
Maintainance
Mesalamine
C.I.
Anti-TNF
Flare: Early (<6 mon)
Late (≥ 6 mon)
IM/Anti-TNF
Hospital
IV Steroids
Response
No Response
C.I.
Anti-
TNF
S
Flare1
Stop
Mesalamine
C.I. = calcineurin inhibitor
Anti-TNF = anti-TNFα
IM = Immunomodulator
S= Surgery
(1) Paradoxical worsening with    
mesalamine
IM/Anti-TNF
Maintenance
Maintainance
Mesalamine
Early
Late


PROTECT Study Protocol 2FEB2015 
 
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Table 7.1.  Reference 5-ASA Total Daily Dosing 
Weight 
Range 
5-ASA Daily Dose 
Rounded to the nearest 
500 mg, max. 4 g 
5-ASA Daily Dose Range (mg/kg) 
Up to 75 mg/kg/day 
Min 
Max 
15 – 19.9 kg 
1000 mg 
50 mg/kg 
66 mg/kg 
20 – 26.4 kg 
1500 mg 
57 mg/kg 
75 mg/kg 
26.5 – 32.9 kg 
2000 mg 
61 mg/kg 
75 mg/kg 
33 – 39.9 kg 
2500 mg 
63 mg/kg 
75 mg/kg 
40 – 46.4 kg 
3000 mg 
65 mg/kg 
75 mg/kg 
46.5 – 52.9 kg 
3500 mg 
66 mg/kg 
75 mg/kg 
53+ kg 
4000 mg 
 
75 mg/kg 
 
7.3.1.  Ambulatory patients with mild disease at diagnosis (PUCAI 10-34) 
Initiate treatment with mesalamine (Pentasa®) in two divided doses.  If PUCAI is not <10 
within 4 weeks, or if patient worsens (PUCAI ≥ 35) on mesalamine then prednisone or liquid 
equivalent prednisolone is instituted and mesalamine is stopped.  Prednisone dosing schedule 
will be as noted below.  Mesalamine will be added back to regimen after 2 weeks on 
prednisone if patient has responded (20 point decrease in PUCAI and PUCAI<34).  The 
tapering schedule of prednisone will then be conducted per protocol to allow possible 
discontinuation of prednisone by week 10. If PUCAI worsens (increases by 20 or more points) 
upon re-introduction of Pentasa then consider paradoxical response to Pentasa and need for 
rescue therapy. 
 
7.3.2.  Ambulatory patients with moderate to severe disease (PUCAI≥35) 
Initiate treatment with prednisone or prednisolone, 1-1.5 mg/kg/day, (maximum 40-60 mg in a 
single morning dose, either as tablet or liquid equivalent), rounded up to the nearest 5 mg 
value.  This dose will be continued for 2 weeks to assess response.  If the patient responds 
(PUCAI decrease of at least 20 points with resulting PUCAI ≤ 34, and steroids administered 
for at least 2 weeks),  mesalamine will be added and the initial prednisone or liquid 
prednisolone equivalent dose continued for one more week to allow for observation for 
paradoxical worsening with the mesalamine.  The mesalamine (Pentasa®) will be given as per 
Table 7.1. There are some patients in whom the PUCAI is in the 35-50 range in whom the 
clinician feels that initial CS therapy is not warranted. For those patients an initial trial of 
Pentasa is allowable. However, if a patient does not respond and then requires prednisone or 
liquid prednisolone equivalent they should enter the standardized CS pathway. 
 
7.3.3.  Hospitalized patients with severe UC (PUCAI≥65) 
Treatment with intravenous CS is instituted after stool cultures/toxin assays reveal no enteric 
pathogen.  If intravenous CS therapy is used for ≤14 days and the patient is discharged at that 
point, then discharge CS will be given in the same manner as for ambulatory patients with 
moderate to severe disease.  Addition of mesalamine would then take place as noted above. 


PROTECT Study Protocol 2FEB2015 
 
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Patients with fulminant disease at diagnosis who receive intravenous CS for more than 14 days, 
or those who require rescue with infliximab, CI, or surgery while hospitalized, will be eligible 
for recording of clinical outcomes and the biomarkers sub-study but will not enter the oral 
standardized treatment protocol.  They will be considered treatment failures with respect to the 
study primary outcome.  These patients will be considered part of the up to 475 patients 
enrolled in this study.  
 
7.3.4.  CS Tapering Schedule 
The initial oral prednisone or liquid equivalent prednisolone dose will be maintained for at 
least three weeks (including the intravenous CS phase if patient initially hospitalized for ≤7 
days) and for at least 7 days after the introduction of mesalamine.  Mesalamine will be added 
after a minimum of 2 weeks of steroid therapy and once the PUCAI is ≤34.  This will allow for 
a period of observation to ensure no paradoxical worsening by the mesalamine occurs prior to 
tapering the prednisone or liquid equivalent prednisolone .  Prednisone or liquid equivalent 
prednisolone  dose decreases will then be contingent upon adequate clinical improvement, 
defined by a decrease in PUCAI of ≥20 points from initial PUCAI with the total score being 
≤ 34 points and the clinician deeming tapering appropriate.  The goal will be to taper so that 
prednisone or liquid equivalent prednisolone is stopped by week 10 to allow evaluation of 
corticosteroid-free status at the 12 week endpoint (that is, off prednisone for 2 weeks).  
Prednisone can be weaned according to the schedule in Table 7.2.  
 
Table 7.2.  CS tapering schedule.  All doses are in mg/day prednisone equivalent 
Week 
0-1 
Week 
1-2 
Week 
2-3 
Week 
3-4 
Week 
4-5 
Week 
5-6 
Week 
6-7 
Week 
7-8 
Week 
8-9 
Week 
9-10 
Week 
10-11 
Week 
11-12 
60 
60 
60 
50 
40 
30 
20 
15 
10 
5 
0 
0 
50 
50 
50 
40 
30 
25 
20 
15 
10 
5 
0 
0 
45 
45 
45 
40 
30 
25 
20 
15 
10 
5 
0 
0 
40 
40 
40 
35 
30 
25 
20 
15 
10 
5 
0 
0 
35 
35 
35 
30 
30 
25 
20 
15 
10 
5 
0 
0 
30 
30 
30 
25 
20 
20 
15 
15 
10 
5 
0 
0 
25 
25 
25 
20 
20 
15 
15 
10 
5 
5 
0 
0 
20 
20 
20 
15 
15 
12.5 
10 
7.5 
5 
2.5 
0 
0 
15 
15 
15 
12.5 
12.5 
10 
7.5 
7.5 
5 
2.5 
0 
0 
 
Criteria for not weaning prednisone or liquid equivalent prednisolone at weekly milestone:  If 
the PUCAI increases from the previous week (but <20 points) the prednisone or liquid 
equivalent prednisolone dose will not be weaned.  Alternative criteria for not weaning based on 
text above would be: PUCAI score has increased and is no longer ≥20 points lower than 
baseline PUCAI or is no longer <34.  The attending physician will have the discretion to 
attempt to bring a patient back into the above weaning schedule if there was a temporary 
increase in PUCAI. The final decision whether to wean the prednisone or liquid equivalent 
prednisolone dose will be contingent upon the physician’s discretion that weaning is 
appropriate. 
Criteria for increasing prednisone or liquid equivalent prednisolone therapy:  If the PUCAI 
increases by ≥20 points from the previous week the dose will be increased to the last dose at 


PROTECT Study Protocol 2FEB2015 
 
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which the patient had improvement in symptoms and maintained at that dose for one week.  If 
the initial dose was <1.5 mg/kg/day (and <60 mg/day) and the patient maintains a PUCAI >34 
the clinician may increase the oral prednisone or liquid equivalent prednisolone to 1.5 
mg/kg/day (max 60 mg/day) for up to one week. Failure to respond at that point will prompt 
consideration of rescue therapy. 
Subsequently, if the PUCAI decreases by ≥20 points from the increased PUCAI that 
necessitated an increase, the dose can then be lowered by the protocol above.  If the PUCAI 
increases by <20 points from the previous week then the attending physician will have the 
discretion to leave the dose the same and re-assess in one week.  
CS weaning from 12 weeks to 24 weeks:  For those patients still receiving prednisone or liquid 
equivalent prednisolone at 12 weeks, further weaning will be at the discretion of the attending 
physician.  Initiation of rescue therapy with IM or infliximab can occur at the discretion of the 
attending physician during weeks 12 to 24.  Any patient who has failed to totally wean 
prednisone or liquid equivalent prednisolone by 24 weeks will be considered a treatment 
failure. 
 
7.4.  Definitions of CS Status 
CS-free:  Subjects will be considered to be CS-free if they are taking no CS at the assessment 
and have been CS-free for a minimum of 2 weeks at that point.  Alternate day CSwill not be 
considered CS-free. 
CS responsive:  Clinical improvement and termination of CS within 12 weeks of initiating 
therapy, and remaining off CS for the remainder of the first year for a minimum of six months 
following initial tapering before requiring re-treatment with CS.  
CS dependent:  Required CS >12 weeks to control symptoms, or at least one additional course 
of CS starting <6 months after initial successful CS weaning  
CS refractory:  Unresponsive to oral CS within four weeks of starting therapy and required 
rescue therapy with CI or anti-TNFα therapy; or required rescue therapy with CI or anti-TNFα 
therapy because of failure to respond to intravenous CS, or required colectomy because of 
failure to respond to CS. 
 
7.5.  Criteria to Institute Rescue or Second Line Therapy 
If there is a lack of sustained response/remission with use of mesalamine as a maintenance 
agent,  or if there is an adverse reaction to mesalamine preventing its use as a maintenance 
agent, or if the patient is CS refractory or dependent then rescue or second line therapy should 
be instituted.  The following will establish the need to institute rescue/second line therapy: 
 The PUCAI continues to be ≥34 despite a minimum of 2-4 weeks of ≥1 mg/kg/day 
prednisone or liquid equivalent prednisolone 
 Failure to wean prednisone or liquid equivalent prednisolone below 0.5 mg/kg/day by 
week 10 after commencing initial steroid therapy 
 Continued activity (PUCAI >10) and need for CS at weeks 12-24 
 Clinical relapse within 6 months of initial successful weaning of prednisone or liquid 
equivalent prednisolone 


PROTECT Study Protocol 2FEB2015 
 
40 of 74 
 Adverse reaction to mesalamine 
 Hospitalized patient with severe colitis (PUCAI ≥65) and failure to respond to 
intravenous CS within 14 days, or at the discretion of the attending physician rescue 
therapy with a calcineurin inhibitor or infliximab is warranted because of continued 
refractory/fulminant disease.  Specific guidelines will be provided as per recent 
consensus recommendations[12, 155]. 
The choice of rescue or second line therapy will be at the discretion of the attending physician.  
Below are guidelines for use of such therapies to establish a standard-of-care amongst 
investigative centers. 
It is possible that rescue/second line medications will be given for reasons other than continued 
active ulcerative colitis. Examples would be concomitant autoimmune hepatitis associated with 
ulcerative colitis, or arthritis. In these cases the patient would be considered a failure with 
respect to the primary outcome at one year on an intent to treat basis ((ITT) but would continue 
to be followed.  
Immunomodulator or biologic therapy:  If a thiopurine is used then dosing of azathioprine 
(2.5-3 mg/kg/day) and 6-mercaptopurine (1-1.5 mg/kg/day) will be initiated assuming normal 
baseline thiopurine methyltransferase (TPMT) activity.  If TPMT activity is intermediate the 
starting dose of azathioprine will be 1.25 mg/kg/day and the starting dose of 6-mercaptopurine 
will be 0.5 mg/kg/day. Subsequent measurement of 6-thioguanine metabolites will be at the 
discretion of the attending physician.  Patients who require thiopurine therapy will be 
considered treatment failures with respect to the study’s primary outcome.  If the clinician 
chooses to use methotrexate as the initial or subsequent immunomodulatory therapy dosing 
will be at the discretion of that physician. It is suggested that all patients receiving IM will 
have regular monitoring of the laboratory tests listed in Table 7.3.    The timing of monitoring 
noted below is will respect to the first day the IM is administered.  
 
Table 7.3.  Suggested laboratory tests to be monitored following institution of 
immunomodulator therapy 
Lab Test 
Baseline 
(Week 0) 
Week 
1 
Week 
2 
Week 
4 
Week 
8 
Every 8-12 
weeks 
TPMT activity* 
For thiopurine 
treated patients 
only 
 
 
 
 
 
Amylase/Lipase* 
X 
 
X 
X 
X 
X 
CBC, differential 
X 
X 
X 
X 
X 
X 
AST/ALT 
X 
 
X 
X 
X 
X 
BUN/CR† 
X 
 
X 
X 
X 
X 
Drug levels 
(cyclosporine, 
tacrolimus) † 
 
X 
X 
X 
 
X 
*  For those on immunomodulator therapy 
†  For those on cyclosporine, tacrolimus 


PROTECT Study Protocol 2FEB2015 
 
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Calcineurin inhibitor (cyclosporine, tacrolimus) or anti-TNFa therapy:  These therapies 
may also be instituted if in the judgment of the attending physician immediate rescue therapy is 
warranted.  Additionally, anti-TNFα therapy can also be used for non-fulminant disease in lieu 
of IM for CS-dependency or CS-refractory disease.  The dosage of these medications will be 
determined by the attending physician and recorded on the CRF.  Patients who require 
cyclosporine, tacrolimus, or anti-TNFα will be considered treatment failures with respect to the 
study’s primary outcome but will continue to be followed in the study for clinical outcomes as 
well as safety observations.  It is suggested that patients receiving  these medications will have 
regular monitoring of the laboratory tests listed in Table 7.3.   Prior to anti-TNFα therapy all 
patients will be screened for tuberculosis with either a PPD (5 units) or QuantiFERON-Tb test, 
and a chest radiograph. 
 
7.6.  Concomitant therapy 
Rectal therapy:  Rectal therapy with either 5-ASA suppositories/enemas/foam or 
hydrocortisone suppositories/enemas will be allowed at the discretion of the physician for 
either severity cohort and will be recorded on the CRF. 
Probiotics/Fish oil:  Probiotic therapy or fish oil will not be given  per protocol.  The use of 
these preparations will be discouraged.  If a patient is reported to be receiving either probiotics 
or fish oil that will be noted on the CRF but the patient will continue to be followed. 
Antibiotics: If a patient is receiving an antibiotic for a presumed enteric infection at the time 
of diagnosis of ulcerative colitis, and investigation has shown no enteric infection, then the 
antibiotic will be stopped at the time the patient starts standardized therapy with either 
mesalamine or prednisone or liquid equivalent prednisolone. If a patient during the study 
receives an antibiotic for a gastrointestinal or extra-gastrointestinal illness the use of the 
antibiotic will be recorded on a CRF.  
Other medications:  Anti-diarrheal medications (loperamide, diphenoxylate HCl) will not be 
allowed for 48 hours prior to each determination of a PUCAI score.  Their use at other times 
will be recorded on the CRF.  Other preparations such as iron, multivitamins, vitamin D 
supplements, and calcium will be used at the discretion of the prescribing physician and 
recorded on the CRF.  Patients will be discouraged from taking non-steroidal anti-
inflammatory agents (NSAIDs). If taken their use will be recorded on a CRF.  Patients will be 
prohibited from taking aspirin or other products containing, or metabolized to mesalamine 
(e.g., sulfasalazine, olsalazine, balsalazide). Vitamin D supplementation can be given at the 
discretion of the site investigator based on locally determined serum Vitamin D levels and 
customary practice.  The use of supplemental Vitamin D and dose will be recorded on CRFs. 
 
7.7.  Intolerance to mesalamine 
Intolerance to mesalamine:  Severe intolerance to mesalamine (paradoxical disease 
worsening, pancreatitis, hepatitis, or other significant side-effects) that preclude its use as a 
maintenance agent will constitute a treatment failure with respect to the primary study outcome 
and will prompt the use of IM or anti-TNFα for maintenance therapy.  These patients will 
continue to be followed and have clinical outcomes recorded. The reasons for mesalamine 
intolerance will be recorded in the CRF. It is anticipated that some patients may develop more 


PROTECT Study Protocol 2FEB2015 
 
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mild signs of intolerance such as headache or nausea. In that case an attempt will be made to 
initially lower the mesalamine dose by 50% and then if now tolerated the site will gradually 
increase the dose back to the standardized dose if possible. If there is loss of efficacy of the 
reduced mesalamine dose, and the patient cannot tolerate an increased dose, this will be 
considered failure and will prompt the use of other second line agents. 


PROTECT Study Protocol 2FEB2015 
 
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8.  ADVERSE EVENTS 
 
PROTECT involves the study of a treatment protocol for ulcerative colitis.  The treatment plan 
was arrived at by consensus among the investigators and is considered standard-of-care rather 
than investigative.  The study does not involve comparisons between treatments nor is there 
any use of medications not yet licensed for use in humans.  The investigators have obtained an 
IND (111863)  for Pentasa (mesalamine) use in the pediatric population. 
During telephone contacts and follow up visits, study participants will be asked to report on 
any hospital or other medical visits since their last study contact, and on any possible adverse 
reactions.  All potential serious adverse events or reported side effects will be recorded as 
study data. 
 
8.1.  Definitions of Adverse Event and Serious Adverse Event 
An adverse event is any untoward or dangerous reaction to a study intervention. 
A serious adverse event is an adverse event that: 
 Results in the participant’s death; 
 Is life-threatening; 
 Results in hospitalization (initial or prolonged); 
 Results in significant, persistent, or permanent change, impairment, damage, disruption, 
or disability in the participant’s body function/structure, physical activities or quality of 
life; 
 Results in a congenital anomaly; or 
 Requires intervention to prevent permanent impairment or damage. 
Non-serious adverse events are all adverse events that do not meet the above criteria for 
“serious.” 
 
8.2.  Reporting of Serious and Other Adverse Events 
All serious adverse events will be reported to the DCC within 24 hours of the clinical site 
personnel becoming aware of the occurrence of the event.  Clinical sites are responsible for 
reporting adverse events (serious or non-serious) to their local IRB in accordance with the local 
IRB requirements.  The medical director at the site will determine the proper response per the 
research protocol, i.e., changing therapy or initiating new therapy. 
The DCC will report serious adverse events to the study safety officer and the independent 
Data Safety Monitoring Board (DSMB) according to the guidelines and schedule established 
by that group.  All adverse events and safety data will also be reported to the DSMB in 
regularly scheduled reports. 
Because the study will be conducted under an IND, the DCC will also be responsible for 
reporting adverse events and serious adverse to the FDA. Anticipated adverse events based 
upon previous experience in adult patients occurring with a frequency of ≥1% include diarrhea, 
headache, nausea, abdominal pain, dyspepsia, vomiting, and rash. 
 


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9.  CENTRAL LABORATORIES 
 
9.1.  Biospecimen Collection and Shipping 
As noted in Table 6.5, biospecimens will be collected at several specified time points in the 
study.  Standard of care laboratory testing including complete blood count, serum chemistries, 
serum aminotransferases, renal function studies, erythrocyte sedimentation rate, C-reactive 
protein, and urinalysis will be performed at the local clinical laboratories of each participating 
institution and will be monitored as part of routine clinical care.  TPMT testing will be sent to a 
reference laboratory determined by the clinical site. Results will be recorded on CRFs. 
Harmonization of reference units will be made so data will be comparable between sites.  
Biospecimens for research purposes will be shipped to the PROTECT biospecimen repository 
at Emory University School of Medicine.   
 
9.2.  Biospecimen Analysis 
The biospecimen repository will send biospecimens to various facilities for analysis as noted in 
the list below.   
 
Cincinnati Children’s Hospital and Medical Center:  Serum vitamin D ; colon RNA-seq; 
fecal calprotectin. 
Emory University:  Genotyping. 
Connecticut Children’s Medical Center:  Fecal osteoprotegerin (OPG). 
Cedars-Sinai:  IBD serology. 
Broad Institute: Microbiome. 
 
 


PROTECT Study Protocol 2FEB2015 
 
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10.  DATA MANAGEMENT 
 
10.1.  Data Management System 
A web-based data management system (DMS) will be used for this study.  The data 
management system will provide all of the capabilities required for research data management, 
including: data transfer, data entry, data validation, database updating, database closure, data 
retrieval, data inventory, security and confidentiality, and archiving, and in addition will 
support eligibility determination. Each clinical site will be responsible for entering the data it 
collects.  The clinical site staff will use the DMS to enter screening and eligibility data and run 
an algorithm to check eligibility.  Follow-up data will also be entered into the DMS at the 
clinical sites. 
The server and main database reside at the DCC at the Collaborative Studies Coordinating 
Center (CSCC) at the University of North Carolina at Chapel Hill. 
 
10.2.  Data Collection, Entry, Editing and Reporting 
Data are collected at clinic visits and by telephone interviews.  Direct data entry, where data 
initially are entered on the screen without having completed a paper form first, will be 
available at each center.  Direct data entry eliminates the time-consuming and error prone 
process of keying from paper forms.  Paper versions of each data collection instrument will be 
available as backup in situations in which the computer systems are inaccessible for any 
reason.  In addition, if there are forms that are routinely collected on paper for convenience or 
another reason, then the data on these forms will subsequently be keyed at the clinical sites 
using the web-based DMS. 
The data entry system will display data entry screens that closely resemble the paper data 
collection forms.  The system will be menu driven, with context-sensitive help available at any 
time.  Each data field will be edited during entry. 
The DMS will include the ability for each center to generate locally a variety of summary 
reports concerning the data completeness, outstanding questionable values, etc.  This capability 
is valuable in permitting study coordinators to monitor the quality of their site’s performance.  
This facilitates timely identification and resolution of problems in data collection and 
processing. 
 
10.3.  Central Laboratory Management System 
Laboratories typically have their own data management systems.  The central laboratories will 
prepare data files from their local data management systems in a standardized format and 
transfer these to the DCC on a regular schedule.  Upon receipt at the DCC, these data files will 
be processed for incorporation into the study’s consolidated database. 
 
10.4.  Confidentiality and Security 
The PROTECT clinical database is housed in a secure, climate controlled server facility at 
UNC.  The CSCC has in place an IT Security Plan as required by NIH contracts and grants.  
The plan documents standard operating procedures required to secure the CSCC network and 
databases, including management, operational and technical controls.  As part of the plan, the 


PROTECT Study Protocol 2FEB2015 
 
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principle of least access privilege for study files is implemented.  Included in the plan are a risk 
assessment, a system continuity plan, and a disaster recovery plan. 
Data confidentiality and security measures are applied at all levels of PROTECT data 
acquisition, transfer and storage, and applied to all study agencies, including the clinical 
centers, the DCC, central laboratories, and any reading center that may be engaged for the 
study.  The PROTECT DMS meets exacting data management standards of confidentiality, as 
well as HIPAA requirements.  Beyond the password-controlled access to the study equipment 
and the DMS, data collected at the clinical centers are encrypted by the system and can only be 
decrypted for display on-screen by authorized study personnel.  Personal identifiers are 
collected on separate CRFs as an additional safeguard against linking of identifiers and medical 
information.  The DCC is responsive to data confidentiality requirements originating from 
providers of medical care or IRBs, as needed to enable the work of the clinical centers.  It is a 
goal of the PROTECT study to collect all data electronically however, should paper data 
collection forms be used they will be retained at secure locations at the clinical centers until the 
Steering Committee acts on recommendations from the DCC to dispose of such records (e.g., 
after incremental data closure).  The secure storage and disposition of hard copy records at 
clinical centers will follow institutional procedures at each site. 
As standard practice, output mailed or otherwise sent to a clinical center identifies participants 
only by ID number.  At the CSCC, printed material containing confidential information is 
discarded through supervised loading, transportation, and storage using a chain of custody 
control process, until the material can be recycled into paper pulp. 
Any personally identifying information such as dates of medical procedures that are entered 
into the central DMS will be stored on a secure server at UNC following the procedures 
described above. 
 
10.5.  Records Retention 
10.5.1.  Data Coordinating Center 
The DCC will comply with all local and federal regulations in maintaining study related 
documentation.  This documentation includes financial records, supporting documents, and all 
records related to the award.  NIH policy states that these must be kept for at least three years 
after study closure[156]. 
 
10.5.2.  Central Laboratories and Biospecimen Repositories 
The biospecimens stored at central facilities remain the property of the PROTECT Study.  The 
samples are available only to approved investigators for approved uses.  For any use beyond 
what is stated in this protocol, investigators must submit an application to the Ancillary Studies 
Committee.  Any unused biospecimens will become the property of the NIH at a time to be 
agreed between the PROTECT Steering Committee and the NIDDK Project Office. 
Samples sent to the laboratories will be identified only by a laboratory sample ID label, distinct 
even from the unique study ID assigned to each participant.  In particular, no PHI (Protected 
Health Information) identifiers will be sent to the laboratory.  The DCC will maintain the link 
between the participant ID and the laboratory sample ID and this link will not be shared 
outside the DCC. 


PROTECT Study Protocol 2FEB2015 
 
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Regarding the destruction of samples, it is PROTECT Study policy that participants retain their 
rights to have their samples removed from the laboratory inventories at any time and have no 
further analytical disbursements performed.  The withdrawal request must be made to the 
originating PI.  This individual PI will transmit a request to the DCC, which will identify the 
link between the participant ID and the laboratory sample ID and generate a 
removal/destruction request for all samples and records associated with a specific laboratory 
sample ID.  Samples already released to approved requesting investigators cannot be returned 
or destroyed.  In addition data generated prior to a participant’s request for sample removal 
will not be destroyed. 
The only other time biospecimen sample will be destroyed is at the request of a site PI when a 
participant turns out to be ineligible, in accordance with the IRB documentation.  At this point 
both the sample and any data associated with it are destroyed in the same manner as described 
above. 
 
10.5.3.  Clinical Sites 
The secure storage and disposition of hard copy records at clinical centers will comply with 
local institutional procedures as well as federal regulations. 
 


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11.  QUALITY ASSURANCE 
 
11.1.  Training and Certification 
All staff involved with data collection will be required to have appropriate training and 
certifications.  Each clinical site will have a study coordinator or project manager who has 
received formal training from the DCC about the PROTECT protocol and procedures.  This 
individual may then train other local staff in study procedures.  A copy of the PROTECT 
Manual of Procedures will be given to all investigative sites and will be used for training and 
standardization of procedures.  This manual will be submitted to the FDA and investigative 
sites prior to initiation of this study. 
 
11.2.  Data Reporting for Quality Assurance 
Monthly data reports of study status and data quality are prepared by the DCC and posted on 
the secure study website.  Data will be summarized overall and by clinical site.  These reports 
include information such as the number of patients screened, number enrolled, percent of 
follow-up contacts completed, and the number and percent of missing forms.  In addition, the 
DCC will generate site-specific reports for data quality, such as missing and overdue forms, 
missing or suspicious data items, outstanding data queries, etc., and facilitate the timely review 
and resolution of data quality issues within the study. 
 
11.3.  Site Monitoring Visits 
The DCC will conduct periodic monitoring visits to each participating clinical site, and central 
laboratory.  DCC monitoring visits will be made annually to each site. In addition to evaluating 
the quality with which the study is being conducted at individual sites, monitors will assess 
specific implementation methods, compare implementation strategies across sites, and make as 
well as receive suggestions for improving study performance.  Monitors will review research 
and medical records of PROTECT participants for accuracy of case report forms.  In addition, 
if enrollment at a site falls below a certain level or other problems with study conduct arise, a 
more diverse site visit team may be initiated.  This larger site visit team would include 
personnel designated by the Executive Committee, such as a team consisting of a clinician 
from a highly productive center, and representatives from the NIDDK Project Office and DCC. 
 
11.4.  Replicate Measures Program 
Some procedures or data collection may be repeated for quality assurance purposes.  When this 
involves additional participant burden, informed consent will be obtained.  A subset of 
participants will be asked to provide additional biospecimens for blinded laboratory analysis 
and quality assurance comparison with the study values.  These additional biospecimens will 
be processed, shipped, stored and assayed or examined in exactly the same way as the regular 
study samples.  The “blinding” involves labeling the samples in such a way that laboratory 
personnel are not aware which samples are regular study samples and which are quality control 
replicates.  The samples which may potentially be most affected by variability in shipping and 
storage processes will be the blood samples used to prepare plasma.  In order to test the 
reliability of our sample shipping, processing, and storage processes, duplicate blood samples 
will be collected into BD P100 tubes from 40 subjects.  The BD P100 tubes will be shipped 
from the study sites to the biorepository on different days, and processed and stored separately.  


PROTECT Study Protocol 2FEB2015 
 
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Plasma aliquots from these duplicate samples will be used to measure 25OHD at the CCHMC 
site, and results will be tested for variance.  The DCC has standard programs for analyzing 
results from these blinded replicate pairs. 
 
 
 


PROTECT Study Protocol 2FEB2015 
 
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12.  STATISTICAL ANALYSIS 
 
12.1.  Analysis of the Primary Endpoint 
The primary endpoint is SFR at 52 weeks.  To define rates of SFR at week 52 on mesalamine 
the analysis will be restricted to patients who are able to initiate standardized induction therapy 
and do not require rescue therapy within 4 weeks.  The primary hypothesis is that the 
proportion of patients in SFR at 52 weeks will be higher among those who have PUCAI <10 at 
week 4.  Because patients may receive rescue therapy at any time and some may be lost to 
follow-up, we will analyze the primary endpoint using time-to-event (survival analysis) 
methods, with the event being initiation of corticosteroids or rescue therapy.  Patients will be 
censored at the time of loss to follow-up or the end of the period of interest, if still in SFR 
remission at that time.  The initial analysis will use the log-rank test to compare time-to-event 
between those with PUCAI < 10 at week 4 and those with PUCAI ≥ 10, first using all patients 
who did not require rescue therapy within 4 weeks and then stratified by the patients' disease 
severity at diagnosis (Mild  (PUCAI < 34) vs. Moderate/Severe (PUCAI ≥ 34)) and mean 
adherence to prescribed mesalamine therapy (≥80% or <80%).  Additional analyses will use 
proportional hazards models to adjust for covariates including disease severity at diagnosis 
(Mild vs. Moderate/Severe).  We will use the proportional hazards models to estimate the 
proportion (and associated 95% confidence interval) in each group who are in SFR at 
remission at 52 weeks.  
We will not consider the effect of different induction therapies explicitly as the induction 
therapies will be prescribed based upon the disease severity at diagnosis and their effects may 
be confounded with the effects of the disease severity. Calculation of adherence is described 
below. 
 
12.2.  Analysis of the Secondary Endpoints 
Some of the secondary endpoints are the same as the primary endpoint except that they are 
over different time intervals (12, 26 or 104 weeks).  For these and for time to colectomy (or 
censoring) we will use the same approach as for the primary endpoint. 
Other endpoints are dichotomous and assessed at a specified time rather than occurring at 
arbitrary times (for example, PUCAI < 10 at 4 weeks).  For these and as a sensitivity analysis 
for the primary endpoint, we will calculate the proportions at the specified time points among 
those with PUCAI < 10 at week 4 and among those with PUCAI ≥ 10 at week 4, first using all 
patients who did not require rescue therapy within 4 weeks and then stratified by the patients' 
disease severity at diagnosis (Mild  (PUCAI < 34) vs. Moderate/Severe (PUCAI≥ 34)) and 
mean adherence to prescribed mesalamine therapy (≥80% or <80%).  We will use a two-
sample test of proportions to compare the pooled (or unadjusted) proportions.  We will then 
use a Cochran-Mantel-Haenszel (CMH) test to compare the stratified proportions (adjusted for 
the initial disease severity and adherence to prescribed mesalamine therapy).  The proportion in 
SFR, whether unadjusted or adjusted, may vary across clinical centers and we will use a 
generalized linear mixed model (GLMM) to accommodate the center-to-center variability in 
comparing the proportions. 
 


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12.3.  Prediction Models 
We will use the study sample as an index set and logistic regression to build a prediction model 
for the probability of the SFR outcome at week 52.  We will first include the following 
variables to test their hypothesized predictive powers, adjusted for adherence to prescribed 5-
ASA medication: (1) age > 12, (2) female gender, (3) mild clinical disease activity at diagnosis 
(PUCAI <34), (4) serum 25-OH vitamin D > 15 ng/mL at diagnosis. 6) serum GM-CSF Ab < 
0.6 mcg/mL at diagnosis, 7) Mayo score of 1 at diagnosis (mild endoscopic severity), 8) fecal 
calprotectin at diagnosis, and 9) fecal OPG at diagnosis.  We will then include additional 
clinical, genetic/genomic, and immune variables via a forward stepwise variable selection 
procedure.  This will include testing the ability of candidate genetic variants associated with 
week 52 SFR identified by our genotyping array results to improve the predictive power of the 
model.  On a subset of the patients where data are available for the following factors: (10) 
PUCAI < 10 at week 4, 11) change in fecal calprotectin between entry and week 4, (12) fecal 
calprotectin ≤ 150 mcg/gm at week 4 or 12, 13) change in fecal OPG between entry and week 
4, and/or (14) fecal OPG ≤ 50 pmol/L at week 4 or 12, we will test their predictive powers in 
addition to the variables (1)-(9) above. In each case we will use the area under the (receiver 
operating characteristic) curve (AUC) to summarize the operating characteristics of the 
prediction model, and determine sensitivity, specificity, positive predictive value (PPV) and 
negative predictive value (NPV). We will use bootstrap method to calculate the standard errors 
of each statistics. To check the fit of the logistic regression model, we will use the Hosmer-
Lemeshow goodness of fit test.  We will also generate a plot of the absolute risk for week 52 
SFR as a function of the risk score. 
 
12.4.  Adherence Data 
We will calculate the adherence percentage at each assessment time point using the Medication 
Event Monitoring System (MEMS®) measured electronic data.  Initially adherence will be 
dichotomized into high versus low, defined as above or below 80% of the prescribed dose.  In 
secondary analyses we will investigate using other cut-points to categorize adherence, as the 
80% cut-point which is used by convention has not been empirically tested for association with 
clinical outcomes in this setting.  Missing electronically measured adherence data will be 
imputed based on pill count data by standard imputation methods [157] and imputed values 
will be used in the analysis.   
As well as using adherence as a predictor of other outcomes, we will also analyze adherence as 
an outcome.  We will use data collected using the BASC-2 as a potential predictor of 
adherence, as well as of week 52 SFR.  We also will investigate whether there are any 
systematic longitudinal patterns (e.g., constant “high” or “low” adherence, or tapering off 
adherence).  If such patterns exist, we will conduct association analysis to see whether the 
systematic patterns are significantly associated with the week 52 SFR rate.  After classifying 
patients into the systematic patterns we will use indicators of the patters in a logistic regression 
model with week 52 SFR as dependent variable.   
 
12.5.  Endoscopic Response and Remission 
Endoscopic response (Mayo endoscopy sub-score reduced by ≥1 and being 0 or 1) and 
remission (Mayo score 0) at week 52 are dichotomous secondary outcomes  However, these 
outcomes may require a more sophisticated analysis than the other dichotomous outcomes.  


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Because sigmoidoscopy at week 52 is not standard of care the costs of the procedure will have 
to be borne by the study.  Patients who have already failed the primary outcome prior to 52 
weeks (i.e., have received rescue therapy, are still on corticosteroids, or have had colectomy) 
will not be eligible for study sigmoidoscopy at week 52.  Only those subjects who are in SFR 
will be eligible for week 52 study sigmoidoscopy.  The way in which these patients are 
selected and factors influencing whether they consent to the procedure, means that the 
mechanism determining who has missing data may be non-ignorable.  The missing mechanism, 
if non-ignorable and unaddressed, may introduce bias into the statistical analysis.  We will 
perform two analyses, one using complete cases only by assuming the missingness is non-
informative and the other using both complete and missing cases by the estimation 
maximization (EM) method.  If the two analyses differ little, it will indicate the missingness is 
ignorable.  We will assume the missing data depend only on a group of clinical variables.   
 
12.6  Genotyping Method, Quality Assurance, and Analysis 
Genotyping: We will use the genotyping core established by Dr. Kugathasan at the Emory 
University Center for Medical Genomics (CMG) for the CCFA-sponsored CD RISK study. We 
will genotype 475 cases using the ImmunoChip (Illumina Human Immuno DNA Analysis 
BeadChip). The ImmunoChip has ~196,000 SNPs, including ~6400 that cover the extended 
MHC region, ~5000 ancestry informative markers (AIMS), and ~760 SNPs across each of 
~180 loci that have been associated in 12 immune mediated diseases including UC with 
statistical support that reaches genome wide significance. We have chosen to use the 
ImmunoChip instead of a custom made iPLEX SNP array for variety of reasons; 1) very cost 
effective for testing a large number of SNPs, 2) no need for genotyping controls as well over 
10,000 disease free controls with ImmunoChip genotyping data are available for analysis, 3) 
genotyping of common and rare variants, 4) fine mapping of all known IBD loci including UC, 
and 5) inclusion of AIMs allows us to perform accurate population stratification between cases 
and controls. Individual SNPs with 5-ASA pharmacogenomic significance including genes 
such as NAT1, NAT2, ABCC1(MRP1), ABCC2(MRP2), ABCC3(MRP3), ABCC4(MRP4), 
UGT1A6, UGT1A9, UGT1A7, UGT1A1 ABCB1(MDR1), ABCG2(BCRP), 
SLCO1A1(OATP1A1), SLCO1A3(OATP1A3), GSTM1, GSTT1, GSTP1, GSTA1, 
SULT1A1, SULT1A2, SULT1A3, Nrf2 that are not covered by the ImmunoChip will be 
performed by Taqman genotyping. SNPs encoding these genes will be selected using 
haploview.  
Quality control: Rigorous QC procedures will be applied to genotyping and analysis. 
Genotyping will be performed using the Illumina Infinium assay protocol. The florescent 
activity of the chip is detected using the Illumina HiScanSQ instrument. Data captured by the 
reader are normalized and transferred into the software package BeadStudio (Illumina) to 
convert the fluorescent information into SNP genotypes. Replicate genotyping across 
experiments will be performed to ensure concordance. Genotypic data will be used only from 
samples with call rates >95% and SNPs with a call frequency >95%. SNPs out of HWE in 
controls with p < 0.001 will be discarded. Cases and controls will be randomly distributed on 
the plates for genotyping. We will also genotype individuals from the Coriell HapMap 
collection with known genotypes as another QC check for our genotyping.  
Data analysis: For our analysis, cases will be subjects who achieve week 52 SFR, and controls 
will be subjects who do not achieve week 52 SFR.  Population stratification is always a 


PROTECT Study Protocol 2FEB2015 
 
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concern in association studies. We will address the effects of population stratification. We will 
only include samples of individuals of European ancestry in the initial analysis as the number 
of non-European samples is expected to be underpowered to detect any differences, and we 
will further ensure homogeneity by using principal component analysis (PCA) to identify and 
potentially eliminate individuals that disproportionally contribute to stratification. We will 
evaluate data for missing data proportions between cases and controls, as well as departures 
from Hardy-Weinberg equilibrium (HWE). We will estimate marker-marker LD and test for 
allelic and genotypic cases-control associations. We will calculate OR, 95% CIs, and 
probabilities. We will also evaluate haplotypes in the fine-mapping regions of the ImmunoChip 
for associations. EM-algorithm-based haplotype frequencies will be calculated and tested for 
differences between cases and controls. We will use likelihood ratio statistics and permutation 
based estimation of p values, using PLINK.  
Anticipated results and potential pitfalls: The ImmunoChip has hundreds of variants at loci 
associated with UC and other autoimmune disorders at genome-wide significance. However, 
because our sample size is modest by GWAS standards, we will only be powered to detect 
large effect loci (ORs > 1.3 if we correct for the number of independent regions, and > 2.0 if 
we correct for all ~196,000 markers) for association with week 52 SFR. Since the RISK 
samples would have already undergone ImmunoChip analysis, we will use 400 RISK study UC 
subjects as a replication cohort or cross validation with the PROTECT cohort for the outcome 
of week 52 SFR. However, UC patients in RISK will not have received standardized therapy, 
or adherence monitoring.  If we detect such an association, due to the dense coverage of each 
of these loci in the ImmunoChip, we expect to have a relatively finely mapped region 
containing that association. Based upon the demographics of pediatric UC in North America, 
we anticipate that 91% of the cohort will be white, 8% African American, and 1% other 
race/ethnicity.  Even with RISK and PROTECT in combination, we are underpowered to 
investigate subjects of non-European ancestry for association. However, we will test individual 
SNPs and haplotypes for association in all ethnicities if statistically significant association is 
found even with small numbers. 
 
12.7.  Sample Size and Statistical Power 
As noted above, the primary efficacy endpoint is being in SFR with mesalamine only as 
maintenance therapy at week 52.  Our proposed sample size of n = 475 is based on 
observations from a cohort of  353 pediatric UC patients currently enrolled in the Pediatric IBD 
Collaborative Research Group Registry (unpublished data).  In this Registry clinical remission 
was defined as a Physician Global Assessment (PGA) of inactive.  The PUCAI measurement 
of disease activity has recently been added to PGA for disease activity assessment. 
In our Registry data we observed that 20% of the UC patients required rescue therapy with 
surgery or medication escalation prior to week 4 post-diagnosis.  Week 52 SFR was 59% in 
those in remission (PGA inactive) at week 4, compared to 29% in those with PGA active at 
week 4 (OR = 3.3, 95% confidence interval (1.9, 5.6), p<0.0001), among the remaining 80% 
who had not required rescue therapy.  We conservatively anticipate two-fold higher odds of 
week 52 SFR rate in pediatric UC patients who achieve remission (as defined by PUCAI < 10) 
at week 4 after initiation of therapy.  We also assume conservatively that 10% will drop out of 
the study.  For the analyses we will initially assume that patients who drop out will not be in 
remission at 52 weeks.  (In sensitivity analyses we will investigate how this assumption 


PROTECT Study Protocol 2FEB2015 
 
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influences results.)  The proposed sample of n=475 will provide >80% power to detect a two-
fold higher odds of week 52 SFR, accounting for the 20% that will require rescue therapy prior 
to week 4, at a significance level of 0.05.  The power calculation was conducted using POWER 
V3.0.  The power calculations are based on proportions at 52 weeks rather than on time-to-
event analyses because of the lack of adequate data on which to base time-to-event models.  
We consider it to be unlikely that substantially more than 20% of patients will require rescue 
therapy by week 4.  However, if this does occur it will reduce study power.  For each DSMB 
report while recruitment is ongoing, the DCC statisticians will calculate the percentage 
requiring rescue therapy by week 4, for consideration of the potential effect on study power.  
This will be done without conducting a test of the primary outcome and so does not constitute 
an interim analysis for efficacy or futility. 
 
Power calculations were based on a planned sample size of n=430 participants. A sample size 
of 475 will facilitate enrichment of complete biospecimen ascertainment at critical study visits.  
 
Table 12.1.  Anticipated power assuming 30% SFR percentage at week 52 in those 
without the predictor, adjusted to 27% by counting drop-outs as not being in remission 
(n=430 participants) 
Prevalence of Predictor 
OR = 2 
OR = 2.5 
40% 
85% 
98% 
30% 
80% 
96% 
20% 
70% 
90% 
 
To identify other clinical, genetic, and immune predictors of SFR (PUCAI<10) at week 52, we 
will utilize the pre-specified variables summarized in Table 12.2 and additional variables as 
identified in our genetic, genomic, immune, and serologic studies in a multivariate logistic 
regression.  If the prevalence of potential dichotomous predictors is in the range of 20%-40% 
with OR ≥ 2, we anticipate power of at least 70% for each of the predictors (see Table 12.1).  
Utilizing the rule of thumb of 10 events per degree of freedom for an adequately powered and 
not overly fitted logistic model, our sample should allow the inclusion of 10 variables.   
 
Table 12.2.  Variables to predict SFR rate at week 52 
Variable 
OR (95% CI) 
p-value 
Age ≥ 12 
1.02 (0.60, 1.74) 
0.94 
Female gender 
0.78 (0.46, 1.32) 
0.35 
White race 
1.04 (0.50, 2.19) 
0.91 
Pan-colitis involvement 
0.91 (0.47, 1.80) 
0.80 
Mild colitis at diagnosis 
1.65 (1.03, 2.64) 
0.04 
Week 4 PGA inactive 
3.29 (1.93, 5.61) 
<0.0001 
OR: odds ratio for week 52 SFR from preliminary studies. 


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13.  DATA AND SAFETY MONITORING PLAN 
 
13.1.  Monitoring Data and Safety 
In any study involving humans, the safety of the participants is paramount.  The treatment 
protocol in PROTECT is regarded as standard of care by the investigators rather than being 
experimental.  There is minimal additional risk specific to study procedures.  The primary 
study-specific procedures are biospecimen collection and, in a subset of patients, a 
sigmoidoscopy at week 52.  Nevertheless, information on all adverse events will be collected 
and reported appropriately to IRBs, a study safety officer, the Data and Safety Monitoring 
Board (DSMB), and the FDA.  Details about what constitutes an adverse event and reporting 
requirements are provided in section 8 of this protocol.  Risks and benefits to participants are 
addressed in section 6.2.4. 
It is unethical to conduct a study with a sample size that is too small to provide adequate power 
to test the primary hypothesis.  In addition, substantial missing data, whether by loss to follow-
up, missed visits, or inadequate procedures, may bias analyses.  Thus it is important to monitor 
enrollment in order to keep the study on track to meet its enrollment target and also to monitor 
the completeness and quality of the data collected.  The DCC will provide the Steering 
Committee with monthly reports containing data on enrollment, completion of study visits, 
completion of data collection and quality of data.   
 
13.2 Study Safety Officer 
Sites are asked to notify the DCC of any serious adverse events within 24 hours of becoming 
aware of the event. Once the DCC is notified, the PROTECT independent study safety officer 
will be informed of the serious adverse event within one business day. The study safety officer 
will review the site investigator’s assessment of the relationship of the study drug to the serious 
adverse event, the expectedness of the event, and to make sure that the narrative of the event is 
well and completely documented for the DSMB, NIDDK, and FDA.  
 
13.3.  Data and Safety Monitoring Board (DSMB) 
13.3.1.  Role and Composition of the Data and Safety Monitoring Board 
The DSMB will be appointed by and report to the NIDDK.  The DSMB is comprised of 
independent scientists who monitor the progress of the study and the safety of the participants.  
The attention of the DSMB will focus on patient accrual, appropriate follow-up, compliance, 
data acquisition, undue complications, and whether the study as it is currently being conducted 
will be able to answer the hypotheses it addresses.  The responsibility for participant safety is 
particularly important.  At each meeting while the study is in the field, the Board will review 
all adverse events. 
The membership and frequency of meeting are at the discretion of NIDDK.  The DSMB is 
likely to consist of at least 5 members including a biostatistician and 3 clinical investigators.  It 
is anticipated that the Board will meet twice a year, with one of these being an in-person 
meeting and the other a teleconference. 
 


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13.3.2.  DSMB Reports 
The  DSMB reports will be prepared twice a year (or as specified by the DSMB).  Although the 
DSMB will determine the format of the report, we anticipate that each report will consist of six 
sections: 1) recruitment, 2) treatment, 3) adverse events, 4) patient adherence, 5) data quality,  
and 6) sub-studies.  The recruitment section will present overall recruitment, as well as by UC 
severity at baseline, and for other subgroups of interest (e.g., by gender).  The treatment section 
will contain information about the treatment status of patients.  The section on adverse events 
will provide a summary of all adverse events and serious adverse events (SAE) along with 
details about each SAE.  Patient adherence data will display adherence patterns over time and 
compare adherence between groups of patients, such as by baseline UC severity.  The quality 
control sections will include summaries of the quality control data collected by the DCC to 
monitor and correct operational data collection.  Any sub-studies will be monitored to ensure 
that they do not adversely affect recruitment or adherence. 
Because this study is not comparing treatments, there will not be any interim analyses for 
efficacy or futility. 
The DCC will provide data management and statistical computing to support the monitoring of 
the study.  Recommendations concerning the continuation or cessation of the trial will be made 
by the DSMB to the NIDDK. 
 


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14.  STUDY AND COMMITTEE ORGANIZATION 
 
14.1.  Participating Entities 
14.1.1.  Clinical Treatment Centers 
Clinical treatment centers will be selected from the roster of those currently participating in the 
Crohn’s and Colitis Foundation of America (CCFA) sponsored study entitled “ Risk 
Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease 
Progression in Children with Crohn's Disease” (www.prokiids.com). This is a 4 year inception 
cohort study of 1200 pediatric patients with Crohn’s disease which will define clinical, genetic, 
genomic, and serologic predictors for aggressive disease behavior. Successful clinical 
monitoring and biospecimen procurement have been demonstrated in the RISK study by all 
study sites who will participate in The PROTECT Study. Sites that have not participated in 
RISK will be eligible if they see an adequate number of new patients with ulcerative colitis 
each  year and have a dedicated research coordinator and principal investigator. 
. 
 
14.1.2.  Clinical Coordinating Center 
Location 
Principal Investigator 
Connecticut Children’s Medical Center 
Jeffrey Hyams, MD 
 
14.1.3.  Data Coordinating Center 
Location 
Principal Investigator 
University of North Carolina at Chapel Hill 
Sonia Davis DrPH 
 
14.1.4.  Central Laboratories and Biospecimen Repositories 
Location 
Principal Investigator 
Emory University (biorepository) 
Subra Kugathasan, MD 
Cincinnati Children’s Hospital and Medical Center 
Lee Denson, MD 
Cedars Sinai Medical Center 
Shervin Rabizadeh, MD 
UCONN Health Center 
Francisco Sylvester, MD 
 
14.1.5.  National Institutes of Health 
Institute 
Project Officers 
NIDDK 
Frank Hamilton, MD, MPH 
 
 
Jose Serrano, MD, PhD 
 
Dana Andersen, MD 
 
 
 
 
 
 
 


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14.2.  Committees 
14.2.1.  InvestigatorsCommittee 
The Investigators Committee consists of the Principal Investigators of each of the clinical sites 
and central agencies and a representative from the NIDDK.  The committee is chaired by Dr.  
Jeffrey Hyams.  Dr. Lee “Ted” Denson is the Co-Chair and will serve as Chair if Dr. Hyams is 
unable to fulfill his role. Each of the clinical centers has one vote on the Investigators 
Committee.  The representatives from the central agencies and NIDDK are ex-officio without 
vote.  
The Investigators Committee meetings bring together investigators and clinical coordinators 
from the various participating centers for discussion regarding implementation of the protocol, 
study logistics, progress of the trial, possible changes in the protocol or methodology, new 
developments in the field, revitalization of interest, and other matters of concern to participants 
in the study.  These meetings may also provide an opportunity for staff training and education.  
The Investigators Committee will establish subcommittees (see below) to develop and monitor 
aspects of the study, reporting recommendations to the Executive Committee for approval. 
 
14.2.2.  Executive Committee 
The Executive Committee provides clinical and scientific direction for the study at the 
operational level.  This committee consists of the investigators who took the lead in writing the 
application for funding of the cooperative agreement, namely Drs.  Hyams, Denson, Walters 
and Davis, and the NIDDK Project Officer. Additional members of the Executive Committee 
will be Dr. Subra Kugathasan who will serve as Director of the central laboratory at Emory, 
and two site Principal Investigators who will serve for a one year term. Criteria for designation 
of these latter two members of the Executive Committee will be determined by the 
Investigators Committee. 
The major responsibilities of the Executive Committee are reviewing overall progress of the 
study with particular emphasis on programmatic issues, including operational, budgetary, 
safety, compliance, and quality issues.  In addition to formulating the Investigators Committee 
agendas, this Executive Committee will provide planning and organization for DSMB 
meetings, including review of preliminary, non-confidential data in preparation for these 
meetings. The Executive Committee in conjunction with the Publication Committee will 
oversee all aspects of the execution and publications of the study.  The Executive Committee 
will meet as necessary with regular conference calls throughout the study, including the period 
for final analysis and writing activities that follow the conclusion of patient follow-up.   
 
14.2.3.  Ancillary Studies Committee 
It is anticipated that both intramural and extramural investigators will wish to capitalize on the 
potential for collaborative ancillary investigations afforded by the implementation of the 
PROTECT study.  The Ancillary Studies Committee will formally review and recommend 
approval or disapproval of all proposed ancillary studies, considering both their impact on the 
conduct of the PROTECT study, and their scientific merit. 
 


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14.2.4.  Publications Committee 
The Publications Committee will formulate publication policy for this collaborative research 
and review all abstracts, papers and scientific presentations which utilize study data.  The 
Publications Committee will be responsible for identifying topics for publication as well as 
making writing group assignments.  The committee will review and recommend approval or 
disapproval of all scientific abstracts and papers or presentations using unpublished study data, 
as well as every paper using published data that purports to represent official study views or 
policy.  Another major responsibility of the Publications Committee is in the development of 
plans for the dissemination of study findings and incorporation of the findings into medical 
care policy.  This will involve not only reports in medical journals but consideration of 
continuing education courses, conferences and seminars and special efforts such as press 
conferences, editorials, physician newsletters and presentations at local medical association 
meetings. 
 
14.2.5.  Study Coordinators Committee 
This committee consists of the study coordinators from each of the clinical sites and central 
agencies.  The committee will have regular conference calls to share information about best 
practices.  A primary function of the committee is to facilitate communication among the sites 
and hence to ensure that study procedures are being applied uniformly at all sites. 
 
14.2.6.  Quality Control Committee 
The Quality Control Committee will be responsible for ensuring high quality data by 
monitoring clinic, laboratory and other central agency performance and initiating corrective 
action when needed.  Quality control reports provided by the DCC will be reviewed.  A system 
for sending blinded replicate samples to the central laboratories will be developed by this 
committee and implemented by the DCC, with the results monitored by this committee. 
 
14.2.7.  Outcome Adjudication Committee 
The Outcome Adjudication Committee will oversee questions concerning study outcomes as 
well as any concerns on patient eligibility for inclusion in the study and for consideration of 
that patient’s eligibility to be part of the final study outcome.  
 
14.2.8.  Data and Safety Monitoring Board (DSMB) 
The composition and roles of the Data and Safety Monitoring Board are described in section 
13. 


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Castro-Santos, P., et al., TNFalpha and IL-10 gene polymorphisms in inflammatory 
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Rutgeerts, P., S. Vermeire, and G. Van Assche, Mucosal healing in inflammatory bowel 
disease: impossible ideal or therapeutic target? Gut, 2007. 56(4): p. 453-5. 
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Rutgeerts, P., et al., Scheduled maintenance treatment with infliximab is superior to 
episodic treatment for the healing of mucosal ulceration associated with Crohn's 
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Rutgeerts, P., et al., Comparison of scheduled and episodic treatment strategies of 
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Mahadevan, U., Mucosal healing in Crohn's disease: what you see is what you get? 
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Su, C., et al., A meta-analysis of the placebo rates of remission and response in clinical 
trials of active ulcerative colitis. Gastroenterology, 2007. 132(2): p. 516-26. 
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Vermeire, S., G. van Assche, and P. Rutgeerts, Review article: Altering the natural 
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Mack, D.R., et al., Laboratory values for children with newly diagnosed inflammatory 
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Roseth, A.G., et al., Assessment of disease activity in ulcerative colitis by faecal 
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APPENDIX A – Pentasa Package Insert 
 
PENTASA® 
(mesalamine) 
Controlled-Release Capsules 250 mg and 500 mg 
Prescribing Information as of June 2008 Rx only 
DESCRIPTION 
PENTASA (mesalamine) for oral administration is a controlled-release formulation of 
mesalamine, an amino-salicylate anti-inflammatory agent for gastrointestinal use. 
Chemically, mesalamine is 5-amino-2-hydroxybenzoic acid. It has a molecular weight of 
153.14. 
Each 500 mg capsule contains 500 mg of mesalamine. It also contains the following inactive 
ingredients: acetylated monoglyceride, castor oil, colloidal silicon dioxide, ethylcellulose, 
hydroxypropyl methylcellulose, starch, stearic acid, sugar, talc, and white wax. The capsule 
shell contains FD&C Blue #1, gelatin, titanium dioxide, and other ingredients. 
CLINICAL PHARMACOLOGY 
Sulfasalazine is split by bacterial action in the colon into sulfapyridine (SP) and mesalamine 
(5-ASA). It is thought that the mesalamine component is therapeutically active in ulcerative 
colitis. The usual oral dose of sulfasalazine for active ulcerative colitis in adults is 2 to 4 g per 
day in divided doses. Four grams of sulfasalazine provide 1.6 g of free mesalamine to the     
colon. 
The mechanism of action of mesalamine (and sulfasalazine) is unknown, but appears to be 
topical rather than systemic. Mucosal production of arachidonic acid (AA) metabolites, both 
through the cyclooxygenase pathways, ie, prostanoids, and through the lipoxygenase pathways, 
ie, leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs), is increased in patients with 
chronic inflammatory bowel disease, and it is possible that mesalamine diminishes 
inflammation by blocking cyclooxygenase and inhibiting prostaglandin (PG) production in the 
colon. 
Human Pharmacokinetics and Metabolism 
Absorption. PENTASA is an ethylcellulose-coated, controlled-release formulation of 
mesalamine designed to release therapeutic quantities of mesalamine throughout the 
gastrointestinal tract. Based on urinary excretion data, 20% to 30% of the mesalamine in 
PENTASA is absorbed. In contrast, when mesalamine is administered orally as an 
unformulated 1-g aqueous suspension, mesalamine is approximately 80% absorbed. 
Plasma mesalamine concentration peaked at approximately 1 mcg/mL 3 hours following a 1-g 
PENTASA dose and declined in a biphasic manner. The literature describes a mean terminal 
half-life of 42 minutes for mesalamine following intravenous administration. Because of the 
continuous release and absorption of mesalamine from PENTASA throughout the 
gastrointestinal tract, the true elimination half-life cannot be determined after oral 
administration. N-acetylmesalamine, the major metabolite of mesalamine, peaked at 
approximately 3 hours at 1.8 mcg/mL, and its concentration followed a biphasic decline. 
Pharmacological activities of N-acetylmesalamine are unknown, and other metabolites have 
not been identified. 
Oral mesalamine pharmacokinetics were nonlinear when PENTASA capsules were dosed from 
250 mg to 1 g four times daily, with steady-state mesalamine plasma concentrations 


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increasing about nine times, from 0.14 mcg/mL to 1.21 mcg/mL, suggesting saturable first-
pass 
metabolism. N-acetylmesalamine pharmacokinetics were linear. 
Elimination. About 130 mg free mesalamine was recovered in the feces following a single 1-g 
PENTASA dose, which was comparable to the 140 mg of mesalamine recovered from the 
molar equivalent sulfasalazine tablet dose of 2.5 g. Elimination of free mesalamine and 
salicylates in feces increased proportionately with PENTASA dose. N-acetylmesalamine was 
the primary compound excreted in the urine (19% to 30%) following PENTASA dosing. 
CLINICAL TRIALS 
In two randomized, double-blind, placebo-controlled, dose-response trials (UC-1 and UC-2) of 
625 patients with active mild to moderate ulcerative colitis, PENTASA, at an oral dose of 4 
g/day given 1 g four times daily, produced consistent improvement in prospectively identified 
primary efficacy parameters. 
The 4-g dose of PENTASA also gave consistent improvement in secondary efficacy 
parameters, namely the frequency of trips to the toilet, stool consistency, rectal bleeding, 
abdominal/rectal pain, and urgency. The 4-g dose of PENTASA induced remission as 
assessed by endoscopic and symptomatic endpoints. 
In some patients, the 2-g dose of PENTASA was observed to improve efficacy parameters 
measured. However, the 2-g dose gave inconsistent results in primary efficacy parameters 
across the two adequate and well-controlled trials. 
INDICATIONS AND USAGE 
PENTASA is indicated for the induction of remission and for the treatment of patients with 
mildly to moderately active ulcerative colitis. 
CONTRAINDICATIONS 
PENTASA is contraindicated in patients who have demonstrated hypersensitivity to 
mesalamine, any other components of this medication, or salicylates. 
PRECAUTIONS 
General 
Caution should be exercised if PENTASA is administered to patients with impaired hepatic 
function. 
Mesalamine has been associated with an acute intolerance syndrome that may be difficult to 
distinguish from a flare of inflammatory bowel disease. Although the exact frequency of 
occurrence cannot be ascertained, it has occurred in 3% of patients in controlled clinical 
trials of mesalamine or sulfasalazine. Symptoms include cramping, acute abdominal pain and 
bloody diarrhea, sometimes fever, headache, and rash. If acute intolerance syndrome is 
suspected, prompt withdrawal is required. If a rechallenge is performed later in order to 
validate the hypersensitivity, it should be carried out under close medical supervision at 
reduced dose and only if clearly needed. 
Renal 
Caution should be exercised if PENTASA is administered to patients with impaired renal 
function. Single reports of nephrotic syndrome and interstitial nephritis associated with 
mesalamine therapy have been described in the foreign literature. There have been rare 
reports of interstitial nephritis in patients receiving PENTASA. In animal studies, a 13-week 
oral toxicity study in mice and 13-week and 52-week oral toxicity studies in rats and 
cynomolgus monkeys have shown the kidney to be the major target organ of mesalamine 
toxicity. Oral daily doses of 2400 mg/kg in mice and 1150 mg/kg in rats produced renal 


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lesions including granular and hyaline casts, tubular degeneration, tubular dilation, renal 
infarct, papillary necrosis, tubular necrosis, and interstitial nephritis. In cynomolgus 
monkeys, oral daily doses of 250 mg/kg or higher produced nephrosis, papillary edema, and 
interstitial fibrosis. Patients with preexisting renal disease, increased BUN or serum 
creatinine, or proteinuria should be carefully monitored, especially during the initial phase of 
treatment. Mesalamine-induced nephrotoxicity should be suspected in patients developing 
renal dysfunction during treatment. 
Drug Interactions 
There are no data on interactions between PENTASA and other drugs. 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
In a 104-week dietary carcinogenicity study of mesalamine, CD-1 mice were treated with 
doses up to 2500 mg/kg/day and it was not tumorigenic. For a 50 kg person of average height 
(1.46 m2 body surface area), this represents 2.5 times the recommended human dose on a 
body surface area basis (2960 mg/m2/day). In a 104-week dietary carcinogenicity study in 
Wistar rats, mesalamine up to a dose of 800 mg/kg/day was not tumorigenic. This dose 
represents 1.5 times the recommended human dose on a body surface area basis. 
No evidence of mutagenicity was observed in an in vitro Ames test and an in vivo mouse 
micronucleus test. 
No effects on fertility or reproductive performance were observed in male or female rats at oral 
doses of mesalamine up to 400 mg/kg/day (0.8 times the recommended human dose based on 
body surface area). 
Semen abnormalities and infertility in men, which have been reported in association with 
sulfasalazine, have not been seen with PENTASA capsules during controlled clinical trials. 
Pregnancy 
Category B. Reproduction studies have been performed in rats at doses up to 1000 mg/kg/day 
(5900 mg/M2) and rabbits at doses of 800 mg/kg/day (6856 mg/M2) and have revealed no 
evidence of teratogenic effects or harm to the fetus due to mesalamine. There are, however, 
no adequate and well-controlled studies in pregnant women. Because animal reproduction 
studies are not always predictive of human response, PENTASA should be used during 
pregnancy only if clearly needed. 
Mesalamine is known to cross the placental barrier. 
Nursing Mothers 
Minute quantities of mesalamine were distributed to breast milk and amniotic fluid of 
pregnant women following sulfasalazine therapy. When treated with sulfasalazine at a dose 
equivalent to 1.25 g/day of mesalamine, 0.02 mcg/mL to 0.08 mcg/mL and trace amounts of 
mesalamine were measured in amniotic fluid and breast milk, respectively. 
N-acetylmesalamine, in quantities of 0.07 mcg/mL to 0.77 mcg/mL and 1.13 mcg/mL to 
3.44 mcg/mL, was identified in the same fluids, respectively. 
Caution should be exercised when PENTASA is administered to a nursing woman. 
No controlled studies with PENTASA during breast-feeding have been carried out. 
Hypersensitivity reactions like diarrhea in the infant cannot be excluded. 
Pediatric Use 
Safety and efficacy of PENTASA in pediatric patients have not been established. 
ADVERSE REACTIONS 
In combined domestic and foreign clinical trials, more than 2100 patients with ulcerative 
colitis or Crohn's disease received PENTASA therapy. Generally, PENTASA therapy was well 


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tolerated. The most common events (ie, greater than or equal to 1%) were diarrhea (3.4%), 
headache (2.0%), nausea (1.8%), abdominal pain (1.7%), dyspepsia (1.6%), 
vomiting (1.5%), and rash (1.0%). 
In two domestic placebo-controlled trials involving over 600 ulcerative colitis patients, 
adverse events were fewer in PENTASA-treated patients than in the placebo group 
(PENTASA 
14% vs. placebo 18%) and were not dose-related. Events occurring at 1% or more are shown 
in the table below. Of these, only nausea and vomiting were more frequent in the PENTASA 
group. Withdrawal from therapy due to adverse events was more common on placebo than 
PENTASA (7% vs. 4%). 
Nervous System: depression, dizziness, insomnia, somnolence, paresthesia 
Cardiovascular: palpitations, pericarditis, vasodilation 
Other: albuminuria, amenorrhea, amylase increase, arthralgia, asthenia, breast pain, 
conjunctivitis, ecchymosis, edema, fever, hematuria, hypomenorrhea, Kawasaki-like 
syndrome, leg cramps, lichen planus, lipase increase, malaise, menorrhagia, metrorrhagia, 
myalgia, pulmonary infiltrates, thrombocythemia, thrombocytopenia, urinary frequency 
One week after completion of an 8-week ulcerative colitis study, a 72-year-old male, with no 
previous history of pulmonary problems, developed dyspnea. The patient was subsequently 
diagnosed with interstitial pulmonary fibrosis without eosinophilia by one physician and 
bronchiolitis obliterans with organizing pneumonitis by a second physician. A causal 
relationship 
between this event and mesalamine therapy has not been established. 
Published case reports and/or spontaneous postmarketing surveillance have described 
infrequent instances of pericarditis, fatal myocarditis, chest pain and T-wave abnormalities, 
hypersensitivity pneumonitis, pancreatitis, nephrotic syndrome, interstitial nephritis, 
hepatitis, aplastic anemia, pancytopenia, leukopenia, agranulocytosis, or anemia while 
receiving 
mesalamine therapy. Anemia can be a part of the clinical presentation of inflammatory 
bowel disease. Allergic reactions, which could involve eosinophilia, can be seen in connection 
with PENTASA therapy. 
Postmarketing Reports 
The following events have been identified during post-approval use of the PENTASA brand of 
mesalamine in clinical practice. Because they are reported voluntarily from a population of 
unknown size, estimates of frequency cannot be made. These events have been chosen for 
inclusion due to a combination of seriousness, frequency of reporting, or potential causal 
connection to mesalamine: 
Gastrointestinal: Reports of hepatotoxicity, including elevated liver enzymes (SGOT/AST, 
SGPT/ALT, GGT, LDH, alkaline phosphatase, bilirubin), hepatitis, jaundice, cholestatic 
jaundice, cirrhosis, and possible hepatocellular damage including liver necrosis and liver 
failure. Some of these cases were fatal. One case of Kawasaki-like syndrome which included 
hepatic function changes was also reported. 
Other: Postmarketing reports of pneumonitis, granulocytopenia, systemic lupus 
erythematosis, acute renal failure, chronic renal failure and angioedema have been received 
in patients taking PENTASA. 
OVERDOSAGE 
Single oral doses of mesalamine up to 5 g/kg in pigs or a single intravenous dose of 


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mesalamine at 920 mg/kg in rats were not lethal. 
There is no clinical experience with PENTASA overdosage. PENTASA is an aminosalicylate, 
and symptoms of salicylate toxicity may be possible, such as: tinnitus, vertigo, headache, 
confusion, drowsiness, sweating, hyperventilation, vomiting, and diarrhea. Severe 
intoxication with salicylates can lead to disruption of electrolyte balance and blood-pH, 
hyperthermia, and dehydration. 
Treatment of Overdosage. Since PENTASA is an aminosalicylate, conventional therapy for 
salicylate toxicity may be beneficial in the event of acute overdosage. This includes 
prevention of further gastrointestinal tract absorption by emesis and, if necessary, by gastric 
lavage. Fluid and electrolyte imbalance should be corrected by the administration of 
appropriate intravenous therapy. Adequate renal function should be maintained. 
DOSAGE AND ADMINISTRATION 
The recommended dosage for the induction of remission and the symptomatic treatment of 
mildly to moderately active ulcerative colitis is 1g (4 PENTASA 250 mg capsules or 2 
PENTASA 500 mg capsules) 4 times a day for a total daily dosage of 4g. Treatment duration 
in controlled trials was up to 8 weeks. 
HOW SUPPLIED 
PENTASA controlled-release 250 mg capsules are supplied in bottles of 240 capsules 
(NDC 54092-189-81). Each green and blue capsule contains 250 mg of mesalamine in 
controlled-release beads. PENTASA controlled-release capsules are identified with a 
pentagonal starburst logo and the number 2010 on the green portion and PENTASA 250 mg 
or S429 250 mg on the blue portion of the capsules. 
PENTASA controlled-release 500 mg capsules are supplied in bottles of 120 capsules 
(NDC 54092-191-12). Each blue capsule contains 500 mg of mesalamine in controlled release 
beads. PENTASA controlled-release capsules are identified with a pentagonal 
starburst logo and PENTASA 500 mg or S429 500 mg on the capsules. 
Store at 25°C (77°F) excursions permitted to 15-30°C (59-86°F) [see USP Controlled Room 
Temperature]. 
Manufactured for Shire US Inc. 725 Chesterbrook Blvd., Wayne, PA 19087, USA 
Licensed U.S. Patent Nos. B1 4,496,553 and 4,980,173 189 0107 009 
Licensed from Ferring A/S, Denmark 
© 2008 Shire US Inc. 
Rev. 06/2008 
PEN-00042 
Table 1. Adverse Events Occurring in More Than 1% of Either Placebo or 
PENTASA Patients in Domestic Placebo-controlled Ulcerative Colitis 
Trials. (PENTASA Comparison to Placebo) 
PENTASA Placebo 
Event n=451 n=173 
Diarrhea 16 (3.5%) 13 (7.5%) 
Headache 10 (2.2%) 6 (3.5%) 
Nausea 14 (3.1%) --- 
Abdominal Pain 5 (1.1%) 7 (4.0%) 
Melena (Bloody Diarrhea) 4 (0.9%) 6 (3.5%) 
Rash 6 (1.3%) 2 (1.2%) 
Anorexia 5 (1.1%) 2 (1.2%) 


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Fever 4 (0.9%) 2 (1.2%) 
Rectal Urgency 1 (0.2%) 4 (2.3%) 
Nausea and Vomiting 5 (1.1%) --- 
Worsening of Ulcerative Colitis 2 (0.4%) 2 (1.2%) 
Acne 1 (0.2%) 2 (1.2%)