Conditions: Ulcerative Colitis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 179
Healthy volunteers: no

Brief summary:
There are fewer therapeutic options for patients with active ulcerative colitis (UC) compared to patients with active Crohn's disease (CD) and the investigators are facing a persistent unmet need for additional effective and affordable therapies for patients with UC. Methotrexate (MTX) 25 mg once weekly administered subcutaneously (sq) or intramuscularly (im) is an efficient therapy to induce and maintain steroid free remission in patients with CD. To evaluate the efficacy of a similar approach in patients with active ulcerative colitis the investigators conduct a double-blind, placebo controlled, randomized, multicenter, parallel group trial to investigate the safety and efficacy of 25 mg MTX applied subcutaneously once weekly in patients with active UC, who either failed 5-ASA therapy, or are steroid dependent or are intolerant or not responding to azathioprine/6-mercaptopurine therapy or have no response/ lost response to infliximab prior to the study inclusion. The study is designed as a drug withdrawal trial and includes two periods, the Induction Period (week 0-16) and the Maintenance Period (week 17-48). In the open label Induction Period every patient will receive a steroid taper, MTX 25 mg sq once weekly + daily folic acid 1 mg tablets for the induction of clinical response or remission. Patients responding to the open label MTX therapy and being off steroids between week 12-16 will be randomized at week 16 1:1 to Placebo sq once weekly + daily folic acid 1 mg tablets + 2.4 g mesalamine or to MTX 25 mg sq once weekly + daily folic acid 1 mg tablets+ 2.4 g mesalamine. The Specific Aims of the trial are: i) To evaluate the safety and tolerability of 25 mg MTX applied sq once weekly over a time period of 48 weeks; ii) To evaluate the relapse-free survival of MTX maintenance therapy compared to placebo over a time period of 32 weeks; iii) To evaluate the efficacy of MTX over a time period of 16 weeks to induce steroid free remission; iiii) To establish a DNA, plasma and serum library to enable the evaluation of clinical and pharmacogenomic models to predict the response to MTX therapy in patients with UC. With 25-30 participating centers actively enrolling, the investigators anticipate to complete enrollment for this study in a time period of 3 years. Completion of this trial will define the therapeutic value of MTX in UC, potentially changing the current therapeutic strategy in UC.

Detailed description:

Arms:
arm 1: 25 mg MTX sq once weekly + 1 mg folic acid daily + 2.4 g mesalamine arm 2: Placebo sq once weekly + 1 mg folic acid daily + 2.4 g mesalamine

Interventions: intervention 1: Methotrexate
Intervention descriptions: intervention 1: Induction period (week 1-16) (Open label):

25 mg MTX sq once weekly + Steroid taper + 1 mg folic acid daily

Maintenance period (week 17-48) (Randomization):

25 mg MTX sq once weekly + 1 mg folic acid daily + 2.4 g mesalamine or Placebo sq once weekly + 1 mg folic acid daily + 2.4 g mesalamine

Protocol document:
Prot_000.pdf: 
Randomized, double blind, prospective trial investigating the 
efficacy of Methotrexate in induction and maintenance of steroid 
free remission in ulcerative colitis (MEthotrexate Response In 
Treatment of UC - MERIT-UC) 
 
Protocol Version 1.9, July 1, 2014 
Sponsor/Principal Investigator:  
 
Hans H. Herfarth, MD, PhD 
Division of Gastroenterology and Hepatology 
University of North Carolina 
Phone:919-966-6806 
Fax: 919-966-7592 
email: hherf@med.unc.edu 
 
Co-Investigators: 
 
Kim Isaacs, MD, PhD 
Division of Gastroenterology and Hepatology 
University of North Carolina 
 
James Lewis, MD, MSCE 
Center for Clinical Epidemiology and Biostatistics,  
University of Pennsylvania 
 
Mark Osterman, MD 
Center for Clinical Epidemiology and Biostatistics,  
University of Pennsylvania 
 
Bruce Sands, MD, MS 
Dr. Henry Janowitz Division of Gastroenterology 
The Mount Sinai Medical Center, New York 
 
Biostatistician 
Anastasia Ivanova, PhD 
Gillings School of Global Public Health 
University of North Carolina at Chapel Hill 
 
Lead Study Coordinator 
Susan Jackson 
Division of Gastroenterology and Hepatology 
University of North Carolina 
Phone: 919-843-9071 
Fax: 919-843-6633 
Email: susan_jackson@med.unc.edu 
 
Data Management Center 
Center of Gastrointestinal Biology and Disease 
Biostatistics Core 
University of North Carolina  
Christopher Martin 
Phone: 919-966-9340Fax: 919-966-7592 
Email: cmartin@med.unc.edu 
 
Funding Agent 
National Institutes of Health (NIH) 
 


Methotrexate response in treatment of UC 
 
2
1 
SUMMARY 
4 
2 
INTRODUCTION AND BACKGROUND 
10 
Clinical pharmacology of MTX 
10 
Effectiveness of MTX in inflammatory bowel diseases 
11 
JUSTIFICATION AND OBJECTIVES OF THE CLINICAL TRIAL 
12 
3 
PATIENT SELECTION 
13 
Inclusion criteria 
13 
Exclusion criteria 
13 
Concomitant medication 
15 
Overview of time sequence 
15 
4 
DESCRIPTION OF THE CLINICAL TRIAL SEQUENCE 
19 
Screening visit 
19 
Induction Period Visit week 0 
21 
Follow-up visits Induction Period = Visits Week 2, 8 
22 
Follow-up visits Induction Period = Visits Week 4, 12 
22 
Final Visit Induction Period = Week 16 / premature withdrawal Induction Period 
23 
Follow-up phone visits and blood draw Maintenance Period = Visits Week 20, 28, 36, 44 
25 
Follow-up visits Maintenance Period = Visits Week 24, 32, 40 
25 
Final Visit Maintenance Period = Week 48 / premature withdrawal Maintenance Period 
26 
Endoscopy and endoscopic assessment (week 0 and week 48/Premature Withdrawal Maintenance 
Period) 
27 
Laboratory parameters 
27 
Purpose of the DNA, plasma and serum repositories 
28 
5 
RECORDING OF COMPLIANCE 
28 
6 
ADVERSE EVENTS 
28 


Methotrexate response in treatment of UC 
 
3
Definitions 
28 
7 
DOCUMENTATION AND REPORTING OF ADVERSE EVENTS 
30 
Documentation and Reporting of Serious Adverse Events 
33 
8 
ABNORMAL LABORATORY RESULTS 
34 
Monitoring of abnormal liver enzymes and complete blood count 
34 
9 
WITHDRAWAL OR STOP OF STUDY DRUG CRITERIA 
35 
Criteria in individual cases 
35 
Treatment recommendations in case of flare of disease 
36 
Criteria for the termination of the whole study 
36 
10 
MEDICATION PREPARATION AND DISTRIBUTION 
36 
11 
DATA SAFETY MONITORING 
39 
12 
RANDOMIZATION, DATA MANAGEMENT AND DATA MONITORING 
39 
13 
STATISTICAL CONSIDERATIONS 
40 
Sample Size Calculation 
40 
Efficacy analysis population 
42 
Statistical analyses 
43 
DSMB interim analyses and early stopping rules for interim analyses 
47 
14 
REFERENCES 
49 
15 
APPENDIX 
51 
Study Flow Chart 
51 
Timeline study visits 
52 
Mayo Score 
54 
Clinical Adverse Event Reporting Form 
57 
Summary of Abnormal Lab Values and Required Actions 
59 
 


Methotrexate response in treatment of UC 
 
4
1 SUMMARY 
Title: 
Randomized, double blind, prospective trial investigating the efficacy of 
Methotrexate in induction and maintenance of steroid free remission in 
ulcerative colitis (MEthotrexate Response In Treatment of UC - Merit-
UC) 
Research 
Hypothesis: 
MTX applied subcutaneously (sq) once weekly is effective to induce and 
maintain steroid free remission or response in patients with active 
ulcerative colitis, who have either failed 5-aminosalicylic acid (5-ASA) 
therapy, or are steroid dependent or have failed or are intolerant to 
azathioprine or 6-mercaptopurine therapy or failed to respond or lost 
response to anti-TNF therapy, compared to placebo. 
Phase: 
II 
Study design: 
Double-blind, placebo controlled, randomized, multicenter, two parallel 
groups  
Study population: 
Patients with active ulcerative colitis who are either failing 5-
aminosalicylic acid (5-ASA) therapy, or are steroid dependent or have 
failed or are intolerant to azathioprine or 6-mercaptopurine therapy or 
failed to respond or lost response to anti-TNF therapy.  
Sample size: 
220 patients 
Treatment: 
Induction period (week 1-16): 
 25 mg MTX sq once weekly + Steroid taper + 1 mg folic acid daily 
Maintenance period (week 17-48) (Randomization): 
 
 25 mg MTX sq once weekly + 1 mg folic acid daily + 2.4 g 
mesalamine 
 Placebo sq once weekly + 1 mg folic acid daily + 2.4 g mesalamine 
Treatment 
duration: 
The study comprises an open label Induction Period of 16 weeks and a 
placebo controlled Maintenance Period of 32 weeks. In the Induction 
Period the patient will be treated with MTX 25 mg/once weekly + daily 
folic acid and a prednisone-tapering schedule for 10-12 weeks starting at 
either 40 mg or 20 mg depending on the need for steroids 2 weeks before 
inclusion in the study. Additionally, a reduction to MTX 15 mg once 
weekly is possible in case of MTX associated side effects. 


Methotrexate response in treatment of UC 
 
5
If the patient has responded (as defined by a reduction from baseline of a 
partial Mayo score ≥ 2 points and at least 25% with an accompanying 
decrease in the rectal bleeding subscore of > 1 point or an absolute rectal 
bleeding subscore of 0-1 point and has a clinical Mayo score ≤ 5 or is in 
remission (as defined as a partial Mayo score of 2 points or lower with no 
individual subscore exceeding 1 point) and is off steroids after 16 weeks 
of therapy with MTX 25 mg/weekly, the patient will be randomized to 
either MTX 25 mg/weekly1 + 2.4 g 5-ASA + folic acid or placebo + 2.4 g 
5-ASA + folic acid until week 48 
1in case of previous dose reduction in the induction phase the patient will be treated with 
15 mg MTX weekly. Also a dose reduction in the Maintenance Period in case of suspected 
MTX associated side effects is possible. 
Main criteria for 
inclusion: 
 Signed informed consent. 
 Man or woman between 18 and 70 years of age. 
 UC diagnosed by routine clinical, radiographic, endoscopic, and 
pathological criteria. 
 Active UC with a Mayo score of 6 to 12 points and moderate-to severe 
active disease on sigmoidoscopy (Mayo endoscopic subscore of at 
least 2) and at least one of the following criteria 
o Steroid dependent UC as defined by the Clinical Trials Task Force 
of the International Organization of Inflammatory Bowel Disease 
(IOIBD) 2  
o Primary failure or loss of response to an anti-TNF therapy in the 
past 
o Primary failure or loss of response to vedolizumab in the past 
o Intolerance/failure of azathioprine/6-MP therapy in the past 
o Failure of 5-ASA therapy 
(2Steroid dependence is defined as a clinical response to treatment with 
prednisone 40 to 60 mg/day and relapse within 30 days after prednisone 
treatment was completed or as a requirement for a daily dosage of not less 
than 10 mg of prednisone and impossibility of weaning the patient off 
steroid without clinical relapses {two attempts to discontinue the medication 
within the preceding six months of the start of the study}). 
Main criteria for 
exclusion: 
 
Failure to respond to 40 mg of prednisone or higher/day in the last 2 
weeks before inclusion (Week 0 Visit) 
 
Azathioprine (AZA) or 6-mercaptopurine (6-MP) must be 
discontinued at least 2 weeks before inclusion into the study)  
(Week 0 Visit) 


Methotrexate response in treatment of UC 
 
6
 
Anti-TNF therapy (Infliximab (Remicade), Adalimumab (Humira),  
Golimumab (Simponi)),  in the last 4 weeks before the Week 0 
Visit 
 
 Vedolizumab (Entyvio) in the last 4 weeks before the Week 0 Visit 
 
Failure of cyclosporine therapy in the previous 6 months prior to 
Screening visit 
 
Patients with serum albumin < 2.5 g/dl at baseline 
 
Low serum folate defined as decrease of >10% below normal range 
 
Patients with WBC< 3.0 x109th/L at baseline 
 
Patients with platelet count < 100 x109th/L 
 
Patients with initial elevation of AST or ALT > 1.5 times above 
normal limit at baseline 
 
Patients with an underlying infection with C. difficile at Screening 
visit 
 
Patients with pre-existing hepatic disease 
 
Patients with known non-alcoholic fatty liver disease (NAFLD) 
 
Patients with known Hepatitis B or Hepatitis C 
 
Patients with pre-existing renal dysfunction (creatinine >1.5 mg/dl). 
 
Patients with a pre-existing chronic lung disease other than well 
controlled asthma 
 
Patients with interstitial lung disease of unknown cause  
 
Patients with a BMI >35 
 
Known previous or concurrent malignancy (other than that 
considered surgically cured, with no evidence for recurrence for 5 
years).  A recent history of basal cell or squamous cell carcinoma, 
which is considered surgically cured, does not exclude the subject. 
 
Existing pregnancy, lactation, or planned pregnancy* (men and 
women) within the next 12 months. (*Methotrexate should not be 
used for at least 3 months before planned pregnancy for men and 
women and should not be used during pregnancy or breast feeding) 
 
High alcohol consumption (more than seven drinks per week) 
 
Non - steroidal inflammatory medications (NSAIDs) as long-term 
treatment, defined as use for at least 4 days a week each month 
 
Continuous treatment with one of the following drugs:  
o Probenecid,  


Methotrexate response in treatment of UC 
 
7
o Trimethoprim/sulfamethoxazole 
o Sulfasalazine 
o Acitrecin,  
o Streptozocin 
 
Non-use of appropriate contraceptives in females of childbearing 
potential (e.g. condoms, intrauterine device {IUD}, hormonal 
contraception, or other means considered adequate by the 
responsible investigator) or in males with a child-fathering 
potential (condoms, or other means considered adequate by the 
responsible investigator during treatment 
 
Participation in another clinical trial within the last 30 days, 
simultaneous participation in another clinical trial, or previous 
participation in this trial 
 
Well-founded doubt about the patient’s cooperation. 
Concomitant 
medication: 
Following drug groups are permitted as concomitant medication:  
 
Orally administered mesalamine containing drugs (which should be 
kept at a stable dose throughout the trial) and should be at a stable 
dose 2 weeks before inclusion in the study. Every patient (except 
those with known intolerance of 5-ASA) will receive 2.4g 
mesalamine in the Maintenance Period.  
 
Local therapies containing 5-ASA (enemas, suppositories) or 
steroid enemas should be at a stable dose 2 weeks before inclusion 
in the study. 
 
Promethazine and/or ondansetron for treatment of MTX associated 
nausea 
 
 Following drug groups are not permitted as concomitant medication:  
 
Cytostatics/immunosuppressants, cyclosporine, tacrolimus, 
mycophenolate mofetil, 6-MP, azathioprine. 
 
Anti-TNF therapies (Infliximab (Remicade), Adalimumab (Humira) 
Golimumab (Simponi)). 
 
,Vedolizumab (Entyvio) 
 
Long-term oral antibiotics as defined as use for >14 days each 
month. 
 
Trimethoprim/sulfamethoxazole 
 
Probenecid 


Methotrexate response in treatment of UC 
 
8
 
Sulfasalazine 
 
Acitrecin 
 
Streptozocin 
 
Non - steroidal anti-inflammatories (NSAIDs) as long-term 
treatment, defined as use for at least 4 days a week each month 
 
No live vaccines should be administered during the trial 
 
Oral or intravenous corticosteroids with the exception of initial 
tapering  
Criteria for 
Evaluation:  
The severity of the mucosal disease will be evaluated by two 
sigmoidoscopies (week 0 and week 48) 
The Mayo Score will be used as the disease activity index. The Mayo 
Scoring system ranges from 0 - 12 points and is a composite index 
consisting of 4 disease variables (stool frequency, rectal bleeding, 
endoscopy evaluation, and Physician’s Global Assessment). A clinical 
response in this trial is defined as a reduction from baseline in the clinical 
Mayo score of ≥ 2 points and at least 25%, with an accompanying 
decrease in the rectal bleeding subscore of ≥ 1 point or an absolute rectal 
bleeding subscore of 0-1 point and a clinical Mayo score ≤5. Clinical 
remission is defined as a Mayo score of ≤ 2 points with no individual 
subscore exceeding 1 point.  
Primary variables 
 Relapse-free survival, comprised of three components, all of which 
must be met to be categorized as relapse-free: total week 32 Mayo 
score not exceeding 2 points, with all individual subscores not 
exceeding 1 point and relapse free survival defined by a numerical 
stable Mayo score throughout 32 weeks of maintenance therapy 
without increase of 3 or more points in the partial Mayo clinic 
score (excluding sigmoidoscopy) compared to the partial Mayo 
score of the individual patient at randomization at week 16 (2) and 
no steroid use or other immunosuppressive medication (anti-TNF 
agents, thiopurines, cyclosporine, tacrolimus) to control disease 
activity throughout the 32 week maintenance period. 
Secondary 
variables: 
 Mucosal healing defined as an absolute subscore for endoscopy of 
0 or 1 at week 48. 
 Relapse of disease in the Maintenance period as defined as an 
increase of 3 or more points in the partial Mayo clinic score 
(excluding sigmoidoscopy) with an absolute clinical Mayo score ≥ 
4 or need for re-treatment with steroids. 
Statistical analysis: 
The primary aim is to test effectiveness of MTX in maintaining clinical 
remission over 32 weeks of therapy compared to placebo. Analyses of this 


Methotrexate response in treatment of UC 
 
9
aim will be conducted at two time points: (1) final analyses at the 
conclusion of the trial and (2) one interim analysis when about half of the 
patients completed follow-up in the maintenance phase. The interim 
analysis is planned to allow the DSMB to determine whether treatments 
are so convincingly different that continuation of the trial would be 
unethical, and also whether side effects of treatment are too severe to 
warrant continued therapy given the potential risk: benefit ratio.  
In addition, we plan to conduct a single analysis of open label induction 
phase remission after the first 75 patients have completed this phase of the 
trial. Accrual of sufficient numbers of patients into the randomized 
maintenance trial depends on the effectiveness of MTX in achieving 
remission during the 16 week open label induction phase. Therefore, we 
will test whether response and remission rates during induction in the first 
75 patients are sufficient to meet goals for accrual to randomization in a 
timely manner. 
Duration of study 
12 months of therapy per patient. The anticipated time of the overall study 
duration is 4 years  
Number of centres: 
25-30 centers in United States 
 


Methotrexate response in treatment of UC 
 
10
2 INTRODUCTION AND BACKGROUND 
Methotrexate (MTX), which was initially developed in 1948 for the treatment of leukemia, 
has been clinically used for nearly 60 years as low and high dose therapy in leukemias, 
certain lymphomas, Wegeners’ disease, psoriasis and rheumatoid arthritis. In inflammatory 
bowel disease the clinical efficacy has been established for steroid dependent Crohn’s 
disease (CD). 
Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disorder, which is limited 
to the colon and characterized by the involvement of the mucosa only (in contrast to the 
transmural inflammation seen in CD). UC primarily affects young adults (20 - 40 years) but 
may present also at a very early age (5-10 years) or in later in life (>60 years). The 
inflammatory process in UC is primarily localized to the rectum (proctitis) or can extend 
proximally in a contiguous manner involving the mucosa up to the splenic flexure (left sided 
colitis) or involving the entire colon (extensive colitis). A key clinical feature of UC is 
bloody diarrhea. The clinical course of UC is characterized by periods of spontaneous 
exacerbation (acute flares) and remission (50 - 80% of patients) while some patients have a 
continuous active disease course (15 - 30% of patients) and others develop severe colitis (5 - 
10% of patients), which can result in colectomy if medical therapy is not effective. Long 
standing UC is associated with an increased risk of colorectal cancer; the use of 5-ASA 
products may reduce this risk. UC can also be associated with inflammation involving extra-
intestinal sites such as the skeletal, skin, or biliary system.   
 
Clinical pharmacology of MTX 
MTX is an analogue of folic acid and of aminopterin, which is also a folic acid antagonist. 
One of the main mechanisms of action is the inhibition of dihydrofolate reductase. This 
enzyme is involved in the de novo synthetic pathway for purines and pyrimidines. The 
rationale for the use of high dose MTX in the treatment of cancer is that the fast proliferating 
malignant cells become starved of purine and pyrimidine precursors and therefore are not 
able to sufficiently maintain DNA and RNA synthesis leading to a decreased proliferation. 
In contrast, the underlying anti-inflammatory effect of low-dose MTX in inflammatory 
diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel diseases is less 
clear, since the anti-proliferative activity of low-dose MTX is minimal. Multiple 
mechanisms of actions are proposed including promotion of adenosine release, inhibition of 
production of pro-inflammatory cytokines, suppression of lymphocyte proliferation, 
neutrophil chemotaxis and adherence and reduction of serum immunoglobulin’s 1, 2.  
MTX can be administered by oral, subcutaneous, intramuscular or intravenous routes. 
Orally, MTX is absorbed in the jejunum and good bioavailability is only achieved in doses 
below 15 mg. Above this threshold absorption rates can vary up to 30% 3. The serum half-
life of MTX is approximately 7-10 hours, but some patients have a prolonged serum half-life 
for up to 26 hours. After MTX is taken up by tissues, it is converted to methotrexate 
polyglutamates, which are long-lived derivates that retain biochemical and biological 
activity. Sixty-five to eighty percent of the drug is excreted by the kidneys (the major part in 


Methotrexate response in treatment of UC 
 
11
the first 12 hours after administration) and 20-35% is secreted with the bile. Plasma MTX 
measurements are clinically not meaningful in defining an optimal plasma concentration 4.  
 
Adverse event profile and toxicity of MTX 
The most often observed adverse event of MTX is nausea. Other significant adverse events 
include hepatotoxicity, bone marrow suppression 5 and rarely hypersensitivity pneumonitis 6. 
Due to the potential teratogenicity, MTX is contraindicated in pregnancy 7. Elevations of 
liver enzymes (AST, ALT, AP) is observed in up to 25% of the patients, but the 
development of hepatic fibrosis or cirrhosis probably due to the intrahepatic accumulation of 
MTX metabolites is overall a rare event 8. In the first trial by Feagan et al, investigating the 
efficacy of 25 mg once weekly sq MTX in the induction of a steroid free remission in 
patients with steroid dependent CD, 7 patients (7.4%) had to be withdrawn due to elevated 
LFT’s, whereas no patient experienced elevations of LFT’s warranting withdrawal from the 
trial in the maintenance study (15 mg MTX once weekly sq) 9, 10. No significant toxicities of 
MTX 25 mg /weekly intramuscularly (im) applied over 50 weeks were reported in a recently 
completed study comparing the combination therapy of MTX and infliximab with infliximab 
alone in patients with CD 11. 
 
Effectiveness of MTX in inflammatory bowel diseases 
The effectiveness of intramuscularly administered MTX 25 mg once weekly in steroid 
dependent CD is well documented. In the North American Crohn’s Study Group landmark 
trial, 39.4% of the patients receiving MTX 25 mg sq/week were in clinical remission, as 
defined as a Crohn’s disease activity index (CDAI) < 150 points and the discontinuation of 
prednisone, compared to 19.1% of the patients in the placebo group 10. Of note is that two 
smaller placebo controlled studies with orally administered lower doses of MTX did not 
demonstrate a statistical significant difference to placebo, suggesting that the route of 
administration and the dose of MTX play an important role 12, 13. Feagan et al. also 
demonstrated a significant efficacy of 15 mg MTX sq once weekly in maintaining steroid 
free remission in CD patients (after 40 weeks 65% remission in the MTX group vs. 39% in 
the placebo group) 9.  
 
So far no large randomized controlled trials have investigated the efficacy of MTX in 
patients with UC. Kozarek et al. reported the successful induction of remission in five of 
seven patients with UC with MTX 25 mg given once weekly im 14. Baron et al. also 
observed a steroid sparing effect of MTX 15 mg orally once weekly in a small group of UC 
patients 15. Two small, randomized, placebo controlled trials investigated the efficacy of oral 
MTX in steroid dependent patients and did not find a significant therapeutic efficacy 
compared to placebo 16, 17. However, MTX was administered orally at relative low doses 
(12.5 or 15 mg once weekly). A clear dose response has been observed in patients with 
rheumatoid arthritis 18, suggesting that higher doses of MTX administered either sq or im 
could be clinically effective in UC. Paoluzi et al. compared azathioprine (2 mg/kg body 
weight/day) to MTX (12.5 mg/kg im once weekly) in patients with steroid dependent or 


Methotrexate response in treatment of UC 
 
12
refractory UC 19. In this prospective single center open label study 69% (22/32) and 19% 
(6/32) of the patients on AZA went into remission or experienced an improvement of 
symptoms, respectively, compared to 60% (6/10) and 40% (4/10) in the MTX group. 
Additionally, several single center observations published in the last 2-3 years employing 
higher doses of MTX suggest a high clinical efficacy of MTX in purine analog 
(azathioprine, 6-mercaptopurine) intolerant UC patients or in UC patients with steroid 
dependent disease course. Cummings et al. analyzed retrospectively the efficacy of orally 
administered MTX in 42 patients with either lack of benefit (n=11) or intolerance (n=31) to 
azathioprine and 8 patients with rheumatoid arthritis and UC with a primary MTX therapy 
for the rheumatoid arthritis 20. The group from Oxford describes response and remission 
rates of 58% and 45% in azathioprine intolerant patients, 27% and 0% in previous 
azathioprine failure patients and 80% and 62% in patients with a concurrent rheumatoid 
arthritis. The mean dose for MTX was in the range of 20-25 mg. Nathan et al. report in a 
retrospective analysis of patients treated in a single hospital in Victoria, Australia, remission 
rates of 48% (11 of 23 patients) after subcutaneous administration of 20-25 mg MTX once 
weekly 21. Wahed et al. describe a clinical response, defined as steroid withdrawal, 
normalization of previously raised C-reactive protein or physician’s clinical assessment of 
improvement) in 22/32 (68%) of UC patients 22 in a retrospective analysis of single 
University hospital in London. The analyzed group consisted mainly of patients, who were 
either previously intolerant (efficacy 14/21; 63%) or failed (efficacy 7/9; 78%) purine 
analogues. 
 
JUSTIFICATION AND OBJECTIVES OF THE CLINICAL TRIAL 
The therapeutic armamentarium for patients with active UC despite 5-aminosalycilates and 
steroids is considerably smaller than for patients with CD (therapeutic choices for steroid 
dependent UC patients: azathioprine, infliximab - therapeutic choices for steroid dependent 
CD 
patients: 
azathioprine, 
methotrexate, 
infliximab, 
adalimumab, 
certolizumab, 
natalizumab), there is clearly a need for further exploration of new therapies with a 
calculable risk profile for this patient group. Currently several new therapeutic approaches 
investigating the role of different “biologics” e.g. in the inhibition of the co-stimulation of T 
cells (Abatacept) or selective blockade of interactions between leukocytes and vascular 
endothelium (MLN 0002 antibody) are being tested in clinical trials. However, given the 
high costs of biologics, there is an additional need for affordable, easy to administer 
therapies such as MTX. Additionally, use of small molecules is favorable over biologics as 
the risk of anti-medication antibody formation is less likely. Since MTX is an old and non-
patented drug, it is highly unlikely that this drug will be ever prospectively investigated in 
patients with UC.  
 
Given the lack of large placebo controlled prospective studies investigating doses of MTX, 
which were clinically effective in inducing steroid free remission in CD patients, so far 
neither a negative nor a positive conclusion can be drawn from the published data about the 
clinical efficacy of MTX in UC. Due to an obvious dose –response relationship observed for 


Methotrexate response in treatment of UC 
 
13
MTX in the treatment of rheumatoid arthritis, there seems to be an unmet need for 
investigating prospectively MTX in higher doses in patients with UC using a subcutaneous 
or intramuscular application.  
 
3 PATIENT SELECTION 
Inclusion criteria 
 Signed informed consent. 
 Man or woman between 18 and 70 years of age. 
 UC diagnosed by routine clinical, radiographic, endoscopic, and pathological criteria. 
 Active UC with a Mayo score of 6 to 12 points and moderate-to severe active disease 
on sigmoidoscopy (Mayo endoscopic subscore of at least 2) and at least one of the 
following criteria 
o Steroid dependent UC as defined by the Clinical Trials Task Force of the International 
Organization of Inflammatory Bowel Disease (IOIBD) 2  
o Primary failure or loss of response to an anti-TNF therapy in the past 
o Primary failure or loss of response to vedolizumab in the past  
o Intolerance/failure of azathioprine/6-MP therapy in the past 
o Failure of 5-ASA therapy 
 2Steroid dependence is defined as a clinical response to treatment with prednisone 40 to 60 mg/day and 
relapse within 30 days after prednisone treatment was completed or as a requirement for a daily dosage of not 
less than 10 mg of prednisone and impossibility of weaning the patient off steroid without clinical relapses 
{two attempts to discontinue the medication within the preceding six months of the start of the study}). 
Exclusion criteria 
Patients who meet one of the following criteria are to be excluded from the trial: 
 
 
Failure to respond to 40 mg of prednisone or higher/day in the last 2 weeks before 
inclusion (Week 0 Visit) 
 
Concomitant use of AZA or 6-MP (AZA or 6-MP therapy has to be stopped at least 2 
weeks before inclusion into the study) (Week 0 Visit) 
 
Anti-TNF therapy (Infliximab (Remicade), Adalimumab (Humira) Golimumab 
(Simponi)) in the last 4 weeks before inclusion in the study (Week 0 Visit) 
 
Concomitant use of vedolizumab (Entyvio) in the last 4 weeks before the Week 0 
VisitFailure of cyclosporine therapy in the previous 6 months prior to Screening visit 
 
Patients with serum albumin < 2.5 g/dl at baseline 


Methotrexate response in treatment of UC 
 
14
 
Low serum folate defined as decrease of >10% below normal range 
 
Patients with WBC< 3.0 x109th/L at baseline 
 
Patients with platelet count < 100 x109th/L 
 
Patients with initial elevation of AST or ALT > 1.5 times above normal limit at 
baseline 
 
Patients with an underlying infection with C. difficile at Screening visit 
 
Patients with pre-existing hepatic disease 
 
Patients with known non-alcoholic fatty liver disease (NAFLD) 
 
Patients with known Hepatitis B or Hepatitis C 
 
Patients with pre-existing renal dysfunction (creatinine >1.5 mg/dl) 
 
Patients with a pre-existing chronic lung disease other than well controlled asthma 
 
Patients with interstitial lung disease of unknown cause  
 
Patients with a BMI >35 
 
Known previous or concurrent malignancy (other than that considered surgically 
cured, with no evidence for recurrence for 5 years).  A recent history of basal cell or 
squamous cell carcinoma does not exclude the subject. 
 
Existing pregnancy, lactation, or planned pregnancy* (men and women) within the 
next 12 months 
(*Methotrexate should not be used for at least 3 months before planned pregnancy 
for men and women and should not be used during pregnancy or breast feeding) 
 
High alcohol consumption (more than seven drinks per week) 
 
Non-use of appropriate contraceptives in females of childbearing potential (e.g. 
condoms, intrauterine device {IUD}, hormonal contraception, or other means 
considered adequate by the responsible investigator) or in males with a child-
fathering potential (condoms, or other means considered adequate by the responsible 
investigator during treatment 
 
Non - steroidal anti-inflammatory medications (NSAIDs) as long-term treatment, 
defined as use for at least 4 days a week each month 
 
Continuous treatment with one of the following drugs:  
o Probenecid 
o Trimethoprim/sulfamethoxazole 
o Sulfasalazine 
o Acitrecin 
o Streptozocin 


Methotrexate response in treatment of UC 
 
15
 
Participation in another clinical trial within the last 30 days, simultaneous 
participation in another clinical trial, or previous participation in this trial 
 
Well-founded doubt about the patient’s cooperation 
Concomitant medication 
Following drug groups are permitted as concomitant medication:  
 
Orally administered mesalazine containing drugs (which should be kept at a stable 
dose throughout the trial) and should be at a stable dose 2 weeks before inclusion in 
the study. In the Maintenance Period, every patient (except those with known 
intolerance to 5-ASA) will receive 2.4 g mesalamine and no additional mesalamine 
should be administered. 
 
 
Local therapies containing 5-ASA (enemas, suppositories) or steroid enemas should 
be at a stable dose 2 weeks before inclusion in the study 
 
 
Promethazine/Ondansetron for treatment of MTX associated nausea 
 
 Following drug groups are not permitted as concomitant medication:  
 
Oral or intravenous corticosteroids with the exception of initial tapering 
 
Cytostatics/immunosuppressants, cyclosporine, tacrolimus, mycophenolate mofetil, 6-
MP, azathioprine 
 
Anti-TNF therapies ((Infliximab (Remicade), Adalimumab (Humira) Golimumab 
(Simponi)) 
 
Vedolizumab (Entyvio)Trimethoprim/sulfamethoxazole 
 
Probenecid 
 
Sulfasalazine 
 
Acitrecin 
 
Streptozocin 
 
Long-term oral antibiotics as defined as use for >14 days each month. 
 
Non - steroidal anti-inflammatories (NSAIDs) as long-term treatment, defined as use 
for at least 4 days a week each month 
 
No live vaccines should be administered during the trial 
Overview of time sequence 
The trial will begin with the screening visit (days -14 to 0). The investigator will ask patients 
who are interested in participating in the study after a careful explanation, to sign an informed 
consent form prior to study specific examinations. The patient will receive a copy of the consent 


Methotrexate response in treatment of UC 
 
16
form, which outlines the sequence of events in brief and summarizes the advantages and 
disadvantages of participation in the study. 
After the patient has given his/her written consent, the screening examination will be 
performed to evaluate whether the patient is probably eligible for the study. 
At the screening visit, a complete history will be taken including concomitant medication 
and the cumulative steroid dose of the last week prior to the screening visit and the 
demographic data will be collected. A physical examination will be performed and vital 
signs will be documented. The blood samples (max. 30 ml) will be taken from the patient 
(for hematology, serum chemistry, screening for hepatitis B and hepatitis C, beta-HCG) as 
well as the samples for the DNA, plasma and serum bank. Female patients with childbearing 
potential are only eligible for the study in case of a negative serum pregnancy test result. The 
patient has to provide a stool sample to exclude for Clostridium difficile infection. Also the 
patient will be provided with a stool collection kit to provide a stool sample for calprotectin 
determination, which should be brought back by the patient at the visit at week 0. A chest x-
ray will be performed to exclude interstitial lung disease. Inclusion and exclusion criteria 
will be checked and completed.  
Once the screening visit of the patient is entered in the database, a screening number will be 
assigned and the patient will receive 1 vial of MTX 2 ml (25mg/ml), 7 tablets folic acid 1 
mg, and 2 tablets promethazine (25mg) by mail delivered to his home address from the 
investigational drug service. The patient should bring this vial and the folic acid tablets to 
the next visit at week 0. Depending on the local regulatory requirements the drug may be as 
well delivered to the investigational site.  
The patient will also be provided with a diary, which he/she will be asked to complete every 
day in the evening for the last 3 days prior the next visit (week 0). At week 0 visit a 
sigmoidoscopy should be performed, to evaluate the degree of mucosal inflammation. The 
sigmoidoscopy score should be ≥2, defined as moderate to severe active disease. Thereafter 
the full Mayo score should be calculated. 
 If the patient still fulfils all inclusion criteria and does not have an exclusion criterion the 
investigators will log onto the UNC data management center web site, and complete 
eligibility and exclusion criteria forms online. If the patient meets the criteria they will be 
assigned a unique study ID number that will appear on all future study instruments and 
medication packs.  
The sigmoidoscopy at the week 0 visit may be omitted if the following requirements are 
met:  (1) A colonoscopy was performed by a MERIT-UC investigator or sub-investigator   
within 14 days before the Screening visit of MERIT-UC. In this context the Week 0 Visit 
must be completed within 21 days of the colonoscopy. (2) The colonoscopy report must 
include the grade of inflammation in the recto-sigmoid colon so the Mayo score can be 
calculated.  (3)  Images of the recto-sigmoid colon should be obtained during the 
colonoscopy, if possible. 
 
The patients will be then allocated to the methotrexate Induction Period: 
 
25 mg MTX sq once weekly + steroid taper + 1 mg folic acid orally daily 


Methotrexate response in treatment of UC 
 
17
 
The patient will be instructed on proper injection technique, storage and disposal 
requirements prior to receiving first injection. The first injection of MTX will be 
administered at the investigational site at week 0, using the single MTX vial (2 ml, 25 
mg/ml), which was delivered to the patient after the screening visit (or to the investigational 
site depending on local regulatory requirements). Once the patient received the first MTX 
injection at the investigational site and he should also start to take folic acid tablets 1 tablet 
(1 mg) daily. Each site will be supplied with syringes and sharp disposal containers.  Patients 
with nausea after injection of MTX should be advised to take promethazine on the day of 
MTX injection.  
 
After week 0 kits containing MTX vials for future injections and folic acid tablets will be 
delivered to the patient’s home address or to the investigational site depending on the local 
regulatory requirements. 
Every enrolled patient will be treated with systemic corticosteroids for 10 to 12 weeks 
according to a taper schedule outlined below.  
Assignment to either the high or low dose prednisone taper will be made by the local 
investigator, based on the subject’s individual treatment needs.  Generally, patients will be 
grouped according to their previous steroid exposure. Patients who were receiving 20 mg or 
more of prednisone daily two weeks before randomization will be allocated to a higher 
steroid taper schedule (high dose prednisone stratum), the others to a lower steroid taper 
schedule (low-prednisone stratum). Depending on the subject’s response, the local 
investigator may alter the rate of the taper during the induction phase.  All subjects, however, 
must be off of steroids by the Week 12 Visit. 
The steroid taper will follow the sequence described below.  
Prednisone taper 
Stratum 
High prednisone 
Low prednisone 
Screening 
≤40 mg, >20 mg 
≤ 20 mg 
Week 0 
40 mg 
20 mg 
Week 2 
30 mg 
17.5 mg 
Week 3 
25 mg 
15 mg 
Week 4 
20 mg 
12.5 mg 
Week 5 
17.5 mg 
10 mg 
Week 6 
15 mg 
10 mg* 
Week 7 
12.5 mg 
7.5 mg 
Week 8 
10 mg 
5 mg 
Week 9 
7.5 mg 
2.5 mg 


Methotrexate response in treatment of UC 
 
18
Week 10 
5 mg 
0 mg 
Week 11 
2.5 mg 
 
Week 12 
0 
 
Table 1: Prednisone taper 
*Tapering is slowed due to the delayed onset of MTX 
activity, which is expected between week 6-8 
 
Induction Period 
The Induction Period lasts for up to 16 weeks and includes 2 regular follow-up visits (visits 
week 4, 12), 2 phone visits and blood draws (visits week 2, 8) and a visit at week 16. 
Study visits during the Induction Period have a +/- 3 day window. The MTX dose of 25 mg 
once weekly can be reduced to 15 mg MTX in case of non-tolerable side effects such as 
nausea, which cannot be treated with promethazine or ondansetron, at the discretion of the 
local investigators. Promethazine tablets (25 mg to be taken on the day of MTX injection) 
will be provided by the investigational pharmacy directly to the patient, if concurrent nausea 
after the injection of MTX is reported by the local investigator.  If promethazine is not 
effective in treating MTX induced nausea, ondansetron 4 mg tablets can be ordered by the 
investigator under certain precautions (see also “Medication preparation and distribution”). 
In case of elevations of liver enzymes or a drop of WBC due to MTX therapy there are 
special instructions in “Abnormal laboratory results”). 
 
Maintenance Period 
If patient responded (as defined by a reduction of the partial Mayo score ≥ 2 points and at 
least 25% with an accompanying decrease in the rectal bleeding subscore of ≥ 1 point or an 
absolute rectal bleeding subscore of 0-1 point and has a clinical Mayo score of < 5) or is in 
remission (as defined as a partial Mayo score of ≤ 2 points with no individual subscore 
exceeding 1 point) and is off steroids after 16 weeks of therapy with MTX 25 mg/weekly, 
the patient will be randomized to the following therapy groups: 
 
 
Group 1 Maintenance: 25 mg MTX* sq once weekly + 1 mg folic acid orally 
daily + 2.4 g 5-ASA 
 
Group 2 Maintenance:  Placebo sq once weekly + 1 mg folic acid orally 
daily+ 2.4 g 5-ASA 
 
*In case of previous dose reduction the patient will be treated with 15 mg MTX/once weekly 
The assigned drug regimen will be send directly to the patient or to the participating center, 
depending on local regulatory requirements. The study coordinator will call the subject to 
confirm that the medications were received. Documentation will be kept in the subject’s 
study file.  


Methotrexate response in treatment of UC 
 
19
If the patient failed to respond or still has a partial Mayo score ≥ 6 or needed steroid therapy 
after week 12, the patient cannot be randomized and therefore will be excluded from 
entering the Maintenance Period of the study. For these patients, the week 16 visit will be 
considered an early termination visit. 
The Maintenance Period lasts 32  1 week and includes 3 regular follow-up visits (visits 
week 24, 32, 40), 4 phone visits and blood draws (week 20, 28, 36, 44) and the final visit 
at week 48 which includes a sigmoidoscopy. Study visits during the Maintenance Period 
have a +/- 7 day window. Again the MTX dose of 25 mg once weekly can be reduced to 15 
mg MTX in case of non-tolerable side effects such as nausea, which cannot be treated with 
promethazine or ondansetron, at the discretion of the local investigators. Promethazine 25 
mg tablets or, if necessary, ondansetron 4 mg tablets (1 tablet to be taken on the day of MTX 
injection) will be provided by the investigational pharmacy directly to the patient, if 
concurrent nausea after the injection of MTX or placebo is reported by the local investigator 
(see also “Medication preparation and distribution”). In case of elevations of liver enzymes 
or a drop of WBC due to MTX therapy there are special instructions in “Abnormal 
laboratory results”). The mesalamine medications will be provided by the investigational 
pharmacy directly to the patient (see medication preparation and distribution). Patients not 
tolerating mesalamine should stop the drug, but will be continued in the study. 
 
At each regular follow-up visit in the Induction Period and in the Maintenance Period (week 
4, 12, 16, 24, 32, 40) a partial Mayo Score will be calculated, the concomitant medication 
(CM) and adverse events (AEs) will be assessed. The visit at week 48 will include a 
sigmoidoscopy for the calculation of the endoscopic Mayo score. Additionally at visit 2, 4, 
8, 12, 16, 24, 32, 40 and 48 clinical laboratory examinations (hematology, serum chemistry) 
and at visit 4, 8, 12, 16, 24, 32, 40 and 48 urine samples for pregnancy tests will be 
performed and the vital signs will be measured (see timeline of study visit in appendix). 
Patients should bring stool samples for calprotectin evaluation at the screening visit and at 
week 16.  
 
At each visit in week 4, 12, 16, 24, 32, 40 and 48 the partially used vials of the medication 
will be collected. The investigator shall compare whether the amount of actually 
administered vials concurs with the days in the study, and will enter his/her estimation 
concerning the patient’s compliance and the number of returned vials in the electronic CRF. 
 
4 DESCRIPTION OF THE CLINICAL TRIAL SEQUENCE 
Screening visit 
Data to be recorded: 
General parameters: 
 
Initials 


Methotrexate response in treatment of UC 
 
20
 
Date of birth 
 
Sex 
 
Smoker (yes/no/ex-smoker) 
 
Race (NIH categories) 
 
Ethnicity (NIH categories) 
 
Case history 
 
Date of confirmation of the diagnosis 
 
Classification of UC (proctitis, left sided colitis, pancolitis) 
 
Extraintestinal symptoms: type, duration of present symptoms 
 
Treatment of UC within the last year 
– type of medication 
 
Treatment of UC within the last 4 weeks 
– type of medication, dose 
 
Examinations 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg), calculation of 
BMI 
 
Height (cm) 
 
Physical examination:  
- Head (including ENT and eyes) 
 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
 
- Urogenital system 
- Endocrine system 
 
- Nervous system 
- Lungs and respiratory tract 
 
- Muscular and skeletal system 
- Peripheral vascular system 
 
- Skin and connective tissue 
- Cardiovascular system 
 
- Other 
 Each: actual status: Present finding / normal condition / not examined 
 
Blood draw DNA, serum and plasma bank (to be sent to the central laboratory). Vials 
are provided by the central laboratory. These samples should be sent on dry ice to the 
central laboratory on the same day. 
 
 
Blood sampling and measurement of laboratory parameters 
- Hematology  


Methotrexate response in treatment of UC 
 
21
- Serum chemistry + serum folate 
- Screening for hepatitis B and C (HBsAg, anti-HBs, anti-HBc, Hep-C AB) 
- Serum pregnancy test (only female patients with childbearing potential) 
 
- Stool sample for C. diff. toxin and calprotectin measurement 
 
 Chest x-ray (if a chest x-ray was obtained within 1 year before screening, there is no 
need for a new chest x-ray, but the results and the date of this x-ray should be recorded 
in the CRF) 
Formal aspects: 
Informed consent procedure 
Verification of inclusion / exclusion criteria (eligibility check), as far as possible 
 
At the end of this visit all data should be immediately entered in the database, so that the 
Investigational Drug Service can send 1 vial MTX (2 mg/ml), 7 tablets folic acid, and 2 
tablets of promethazine to the patient (or to the investigational site depending on local 
regulatory requirements) before the visit at week 0. The patient should bring the vial and the 
tablets to the visit at week 0.  
Induction Period Visit week 0 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg) 
 
Height (cm) 
 
Physical examination:  
- Head (including ENT and eyes) 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
- Urogenital system 
- Endocrine system 
- Nervous system 
- Lungs and respiratory tract 
- Muscular and skeletal system 
- Peripheral vascular system 
- Skin and connective tissue 
- Cardiovascular system 
- Other 
 
Each: actual status: Present finding / normal condition / not examined 
 
 
Collect venous blood sample for hematology and serum chemistry for laboratory 
analyses (only if conducted > 14 days after screening)  
 
Sigmoidoscopy 
 
Training of injection technique with sodium chloride and injection of first MTX 
dose. Patient should start taking folic acid 1 tablet (1 mg) once daily.   


Methotrexate response in treatment of UC 
 
22
 
The patient should be advised to take 1 25 mg tablet promethazine in case of nausea 
in the next 24-48 hours after the injection. In case of nausea the patient should also 
be advised to call the local coordinator for further supply of promethazine, which 
could be ordered by weborder from the investigational drug services (IDS). The IDS 
will then supply the patient with one promethazine tablet for each weekly MTX 
injection. In case promethazine is not effective, ondansetron can be directly ordered 
from the IDS (see “Medication and Distribution”)  
 
The supply of further drug and the injection utensils (see “Medication preparation 
and distribution”) will be shipped to the patient or handed to the patient at the 
investigational site depending on local regulatory requirements.  
 
Formal Aspects: 
Calculation of the Mayo score including sigmoidoscopy 
Verification of inclusion / exclusion criteria (eligibility check) 
 
Follow-up visits Induction Period = Visits Week 2, 8 
 
Measurement of laboratory parameters  
- Hematology  
- Serum chemistry 
- Urine pregnancy test (week 8 only) 
 
Phone interview with patient about adverse events (interview can be as well 
conducted in study center if feasible) 
 
Follow-up visits Induction Period = Visits Week 4, 12 
Examinations 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg) 
 Physical examination:  
- Head (including ENT and eyes) 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
- Urogenital system 
- Endocrine system 
- Nervous system 
- Lungs and respiratory tract 
- Muscular and skeletal system 
- Peripheral vascular system 
- Skin and connective tissue 
- Cardiovascular system 
- Other 
 Each: actual status: Present finding / normal condition / not examined 


Methotrexate response in treatment of UC 
 
23
 
 
Measurement of laboratory parameters  
- Hematology  
- Serum chemistry 
- Urine pregnancy test 
Formal aspects: 
 
Return of study medication (empty vials) 
 
Calculation of Mayo score excluding the sigmoidoscopy score 
 
Compliance assessment (count of empty vials returned) 
 
Concomitant medication 
 
Adverse events 
 
Evaluate the progress of the steroid taper; patients should be off steroids by latest in 
week 12. 
 
Appointment for next visit 
 
At week 12 remind the patient to bring stool sample for next visit (container provided 
by central laboratory). 
 
Final Visit Induction Period = Week 16 / premature withdrawal Induction 
Period 
Examinations 
 
Physical examination:  
- Head (including ENT and eyes) 
 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
 
- Urogenital system 
- Endocrine system 
 
- Nervous system 
- Lungs and respiratory tract 
 
- Muscular and skeletal system 
- Peripheral vascular system 
 
- Skin and connective tissue 
- Cardiovascular system 
 
- Other 
 Each: actual status: Present finding / normal condition / not examined 
 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg) 
 Measurement of laboratory parameters  
- Hematology  


Methotrexate response in treatment of UC 
 
24
- Serum chemistry 
- Urine pregnancy test 
- Stool sample for calprotectin stool levels. 
-Plasma and serum collection for study repository 
 
Formal aspects: 
 
Return of study medication (empty syringes) 
 
Compliance assessment (count of empty vials returned) 
 
Concomitant medication 
 
Adverse events 
 
Calculation of the partial Mayo score and evaluation if patient meets criteria for 
response or remission. 
 
If patient meets criteria for response or remission, perform randomization for 
Maintenance Period. If the patient failed to respond or still needed steroid 
therapy after week 12, the patient cannot be randomized and must be excluded 
from the trial. In this case week 16 is the last study visit for the patient. 
 
In case of premature withdrawal from the study (yes, no)  
 
If yes, reason: 
 Intolerance of medication 
 Lack of efficacy (need for steroids e.g. increase of steroids or re-introduction of 
steroids after previously successful taper) 
 Severe adverse event 
 Lack of patient’s cooperation 
 Inclusion criterion not fulfilled or exclusion criterion fulfilled coming to knowledge 
after recruitment  
 Need for steroid therapy after week 12 
 Other reason 
Follow Up after premature withdrawal 
If a subject withdraws before the completion of the study, follow up data will be collected 
one and three months after withdrawal.  Information about which treatment options were 
started after withdrawal (checklist) and whether the patient is in remission (yes/no) will be 
collected.  


Methotrexate response in treatment of UC 
 
25
Follow-up phone visits and blood draw Maintenance Period = Visits Week 
20, 28, 36, 44 
 Measurement of laboratory parameters  
- Hematology  
 Phone interview:  Phone interview with patient about adverse events (interview can 
be as well conducted in study center if feasible) 
 
Follow-up visits Maintenance Period = Visits Week 24, 32, 40 
Examinations 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg) 
 Physical examination:  
- Head (including ENT and eyes) 
 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
 
- Urogenital system 
- Endocrine system 
 
- Nervous system 
- Lungs and respiratory tract 
 
- Muscular and skeletal system 
- Peripheral vascular system 
 
- Skin and connective tissue 
- Cardiovascular system 
 
- Other 
 Each: actual status: Present finding / normal condition / not examined 
 
Measurement of laboratory parameters  
- Hematology  
- Serum chemistry 
- Urine pregnancy test 
Formal aspects: 
 
Return of study medication (empty vials) 
 
Calculation of Mayo score excluding the sigmoidoscopy score 
 
Compliance assessment (count of empty vials returned) 
 
Concomitant medication 
 
Adverse events 
 
Appointment for next visit 
 


Methotrexate response in treatment of UC 
 
26
Final Visit Maintenance Period = Week 48 / premature withdrawal 
Maintenance Period 
Examinations 
 
Physical examination:  
- Head (including ENT and eyes) 
 
- Gastrointestinal tract, liver, spleen 
- Lymphatic system 
 
- Urogenital system 
- Endocrine system 
 
- Nervous system 
- Lungs and respiratory tract 
 
- Muscular and skeletal system 
- Peripheral vascular system 
 
- Skin and connective tissue 
- Cardiovascular system 
 
- Other 
 Each: actual status: Present finding / normal condition / not examined 
 
Sigmoidoscopy 
 
Vital signs: blood pressure (mm Hg), heart rate (min-1), weight (kg) 
 
Measurement of laboratory parameters  
- Hematology  
- Serum chemistry 
- Stool sample for calprotectin stool levels 
- Urine pregnancy test 
 
Formal aspects: 
 
Return of study medication (empty vials) 
 
Calculation of Mayo score with the sigmoidoscopy score 
 
Compliance assessment (count of empty vials returned) 
 
Concomitant medication 
 
Adverse events 
 
In case of premature withdrawal from the study (yes, no)  
 
If yes, reason: 
 Intolerance of medication 
 Lack of efficacy (need for steroids e.g. increase of steroids or re-introduction of 
steroids after previously successful taper) 
 Severe adverse event 


Methotrexate response in treatment of UC 
 
27
 Lack of patient’s cooperation 
 Inclusion criterion not fulfilled or exclusion criterion fulfilled coming to knowledge 
after recruitment  
 Need for steroid therapy 
 Other reason 
 
 
Follow Up after premature withdrawal 
If a subject withdraws before the completion of the study, follow up data will be collected 
one and three months after withdrawal.  Information about which treatment options were 
started after withdrawal (checklist) and whether the patient is in remission (yes/no) will be 
collected.  
Endoscopy and endoscopic assessment (week 0 and week 48/Premature 
Withdrawal Maintenance Period) 
The endoscopy performed at week 0 should be a colonoscopy for patients that have a 
diagnosis of UC for ≥ 8 years and have not had a screening colonoscopy for ≥1 year. This 
colonoscopy would be considered as standard of care. For all other patients and all other 
visits, a sigmoidoscopy should be performed. Regardless of the type of endoscopy 
performed, only the 30 cm on retraction of the scope should be used for Mayo scoring 
purposes (range from 0 to 3 points). Photographs depicting examples of each of the 4 
endoscopic subscores will be provided to sites for standardization purpose and the 
endoscopist should refer to these photos during the procedure to guide assignment of the 
endoscopy subscore. The endoscopy subscore at week 0 should be at least ≥2. The 
endoscopies should be photo documented the pictures either digital or as print sent to the 
data management center. 
Laboratory parameters 
All laboratory analyses will be carried out in a central laboratory. All samples including C. 
diff toxin will be sent by overnight courier to this laboratory. Dry ice will be delivered to the 
participating sites if necessary.  
Acute phase reactants (nonspecific inflammatory markers) 
• 
C-reactive protein (CRP) 
Hematology: 
• 
Blood count (erythrocytes, hemoglobin, hematocrit, leukocytes, platelets and 
differential WBC) 
Serum chemistry: 
• 
Kidney function: serum urea and creatinine 


Methotrexate response in treatment of UC 
 
28
• 
Liver function (alanine aminotransferase {ALT}, aspartate aminotransferase {AST}, 
γ-glutamyl transferase {GGT}, alkaline phosphatase, bilirubin), albumin 
• 
Other serum parameters (total protein, Na+, K+, Mg) 
• 
Folate serum level at screening visit only 
Hepatitis B and C screening 
HBsAg, anti-HBs, anti-HBc, Hep-C AB 
 
Stool sample for C. diff .toxin  
Purpose of the DNA, plasma and serum repositories  
The purpose of the repositories for DNA, serum and plasma is the evaluation of MTX 
pharmacogenetics and drug metabolite levels in the participating patients to evaluate criteria for 
MTX efficacy. MTX has a complex intracellular pathway and several genes have been associated 
with toxicity and response to MTX. Inter-individual variation in response and toxic side effects of 
UC patients to treatment with MTX may be influenced by inter-individual variation in candidate 
genes. We will also obtain DNA extracted from the buffy coat of blood samples drawn at inclusion 
into the study. Aliquots of serum and plasma will be collected from blood drawn at the same time. 
All specimens will be stored at -80C at the central laboratory for the duration of the trial and after 
the successful completion of the study at the NIDDK Central repository indefinitely. All samples and 
data information will be de-identified.  
5 RECORDING OF COMPLIANCE 
Compliance will be assessed by checking the study medication (vials) returned at the follow-
up visits and the final visit by the investigator.  
During the Induction and Maintenance Period all returned vials will be counted by the 
investigator and the number will be documented in the electronic CRF.  
 
6 ADVERSE EVENTS 
Definitions 
Adverse events (AEs) 
Adverse events (AEs) will be recorded at each regular scheduled study visit or study phone 
contact in the patient record (source document) as well as on a specific AE form on the 
electronic CRF. 
 


Methotrexate response in treatment of UC 
 
29
An AE is any untoward medical occurrence in a patient or clinical investigation patient 
administered a pharmaceutical product and which does not necessarily have a causal 
relationship with this treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom, or disease temporally associated 
with the use of a medicinal product, whether or not related to the medicinal product, e.g.: 
 
 
any new diagnosis 
 
any symptom that requires medical clarification or leads to in-patient admission 
(surgery or accident) 
 
any suspected adverse drug reaction (ADR) 
 
any symptom that appears on the patient’s medical records 
 
any event related in time with the application of the study medication and affecting 
the health of the patient (including laboratory value changes) 
Serious Adverse Events (SAE) 
A serious adverse event (experience) is any untoward medical occurrence that at any dose: 
 
results in death 
 
is life-threatening 
 
requires inpatient hospitalization or prolongation of existing hospitalization  
 
results in persistent or significant disability/incapacity 
 
Non-serious adverse events are all AEs that do not fall into any of the above categories. 
Unexpected Adverse Drug Reactions  
An unexpected adverse drug reaction (ADR) is an adverse reaction, the nature or severity of 
which is not consistent with the applicable product information. 
Expected Adverse Drug Reactions 
Common ADRs caused by methotrexate include the following: 
 
 anorexia, nausea, vomiting 
 elevation of liver enzymes (AST, ALT , AP or GGT) 
 stomatitis 
 malaise 
 fatigue 
 abdominal discomfort 


Methotrexate response in treatment of UC 
 
30
 chills 
 fever 
 dizziness 
 diarrhea 
 anemia 
 thrombocytopenia 
 leukopenia 
 rash 
 pruritus 
 alopecia 
 photosensitivity 
 abortifacient, teratogenic 
 
7 DOCUMENTATION AND REPORTING OF ADVERSE EVENTS 
The patients will be instructed to contact the investigator, if any serious or unexpected AE 
occurs, so that appropriate action can be taken. 
Moreover, the investigator must ask at each follow-up visit a generally worded question 
without searching for any special symptoms, e.g. “Has your state of health worsened since 
we last met?” If the answer to this question is “no”, no further questions will be asked. If the 
answer to the question is "yes”, the investigator will document the nature, time, severity, 
seriousness and duration as well as the causality of the AE. For each AE a specific AE 
documentation form will be provided to the investigators. In case of an AE, this should be 
completed by the physician as an initial report, and sent (via fax) to the principal investigator 
within 72 hours. The report must contain a detailed description of the symptoms observed 
and the concomitant treatment administered. Furthermore, the investigator must comment on 
a possible causative relationship between the AE and the trial medication. Each AE must be 
followed until it is resolved or can be explained satisfactory. 
 
The following has to be documented for each AE: 
 Nature of the event 
 Time of onset: date, time 
 Concomitant treatment: product (generic name), indication, dosage, dosage interval, 
presentation, mode of administration, administration regimen 
 Duration of the AE 


Methotrexate response in treatment of UC 
 
31
 Severity 
 Seriousness 
 Causality 
 Outcome 
 
Severity 
The severity is evaluated as follows: 
1. Mild: 
- event/symptom does not interfere with normal daily activities 
2. Moderate: 
- event/symptom interferes with normal daily activities 
3. Severe: 
- event/symptom prevents normal daily activities 
 
Causality 
The relationship between an AE and the study medication is classified according to the 
WHO classification: 
Certain 
A clinical event, including laboratory test abnormality, is occurring in a plausible time 
relationship to drug administration, and which concurrent disease or other drugs or 
chemicals cannot explain. The response of the patient to withdrawal of the drug should be 
clinically plausible. The event must be definite pharmacologically or phenomenologically, 
using a satisfactory rechallenge procedure if necessary. 
Probable/Likely 
A clinical event, including laboratory test abnormality, with a reasonable time sequence to 
the administration of the drug, unlikely to be attributed to concurrent disease or other drugs 
or chemicals, and which follows a clinically reasonable response on withdrawal (de 
challenge). Rechallenge information is not required to fulfil this definition. 


Methotrexate response in treatment of UC 
 
32
Possible  
A clinical event, including laboratory test abnormality, with a reasonable time sequence to 
administration of the drug, but which could also be explained by concurrent disease or other 
drugs or chemicals. Information on drug withdrawal may be lacking or unclear. 
Unlikely  
A clinical event, including laboratory test abnormality, with a temporal relationship to drug 
administration which makes a causal relationship improbable, and in which other drugs, 
chemicals or underlying disease provide plausible explanations. 
Conditional/Unclassified 
A clinical event, including laboratory test abnormality, reported as an adverse reaction, 
about which more data are essential for a proper assessment or the additional data are under 
examination. 
 Not assessable / Unclassifiable 
A report suggesting an adverse reaction, which cannot be judged because information is 
insufficient or contradictory, and which cannot be supplemented or verified. 
 
As an alternative to the above-mentioned WHO assessments, the following assessment can 
be made: 
 
Not related 
There is sufficient information available to show that the etiology is unrelated to the study 
medication. 
 
Measures at the onset of adverse events  
Measures at the onset of adverse events are classified and described as follows: 
1) 
None, i.e. the study medication was not changed 
2) 
The dose of the study medication was reduced 
3) 
The study medication was withdrawn and/or 
4) 
Other measures (clear text) 
The course and outcome of the adverse event will be commented on as follows: 
1) 
Recovered without sequelae 
2) 
Not yet recovered 
3) 
Recovered with sequelae 
4) 
Fatal 
 


Methotrexate response in treatment of UC 
 
33
Documentation and Reporting of Serious Adverse Events 
On enrolment in the study, the patients will be instructed to contact the investigator if a 
serious or unexpected AE occurs, so that appropriate measures can be taken. 
Any SAE (including death, irrespective of the cause) occurring during or for up to 
14 days after the end of the study must be reported without delay, i.e. within 24 hours, 
by telephone and by fax to the principal investigator of the study organization or his 
designee, irrespective of its relationship with the administration of the study 
medication (minimum information required: investigator’s name/study center, patient 
number, patient initials, date of first dose, date of last dose, date of event, description of 
event, causality assessment, and countermeasures). 
 
Address: 
Dep. of Internal Medicine 
130 Mason Farm Rd. 
Chapel Hill, NC, 27599-
7080 
Principal investigator 
Hans Herfarth, MD, PhD 
Phone:  919-843-9071 
 
Fax: 919-843-6633 
 
A specific AE documentation form will be provided to the investigators (see appendix). In 
case of an SAE, this should be completed by the physician as an initial report, and sent (via 
fax) to the principal investigator. The report must contain a detailed description of the 
symptoms observed and the concomitant treatment administered. Furthermore, the 
investigator must comment on a possible causative relationship between the AE and the trial 
medication. Each SAE must be followed until it is resolved or can be explained 
satisfactorily. 
The general procedure for the observation, collection and analysis of drug risks (regulatory 
affairs) in conformity with the appropriate national Drug Law shall apply without 
qualification. 
In accordance with drug safety and national requirements, the study PI will inform the Data 
and Safety Monitoring Board (DSMB) of the study and will make sure that the involved 
persons will obtain adequate information. Also the local principal investigator will inform 
the local Ethics Committee.  
The following instructions must be heeded: 
In the case of an intolerable SAE, the patient must, at the decision of the investigator, be 
withdrawn from the clinical trial, and symptomatic treatment must be administered. The 
measures taken must be recorded on the electronic CRF. 
In accordance with local legislation, the investigators will submit copies of the final SAE-
report to the Regulatory Authorities concerned, if necessary. 
 


Methotrexate response in treatment of UC 
 
34
8 ABNORMAL LABORATORY RESULTS  
All laboratory values outside of the normal range will be repeated if judged appropriate by 
the investigator to ensure the validity of the abnormal result. The investigator will document 
all laboratory values on the relevant page of the electronic CRF and will assess the etiology 
of the clinically relevant abnormal laboratory values.  
Clinically relevant abnormal laboratory values are only documented on the AE form of the 
electronic CRF, if the patient is discontinued, hospitalized, or, in the investigator’s opinion, 
it should be considered as an AE. 
Abnormal laboratory results caused by ulcerative colitis should not be reported on the AE 
form. 
Monitoring of abnormal liver enzymes and complete blood count 
- 
See appendix for summary of actions for abnormal lab values 
- 
If the AST or ALT (LFT’s) increase ≥ 2 times the upper limit of normal after the 
start of the study medication, the investigator has to repeat the measurements of 
LFTs after 2-3 weeks. If LFTs normalize the patient will continue with current 
dose of MTX, however, if LFTs remain elevated ≥ 2 times the upper limit at 
second draw, the dose of methotrexate should be reduced to 15 mg/week. Another 
check of LFT’s should be performed after 4 weeks at the lower dose. If the LFT’s 
normalizes or remain stable, then further laboratory controls of the LFT’s should 
be performed in the setting of the regular lab draws in the context of the regular 
study visits.  
- 
If WBC below 3.0 x109th/L, the investigator should stop therapy with MTX and 
perform another blood draw in the setting of an unscheduled visit 14 days +/- 3 
days later. If WBC at this unscheduled visit is ≥ 4.0 x109th/L then patient may 
resume MTX therapy at the lower dose of 15 mg MTX/week. The next laboratory 
control should be then performed in the setting of a regular scheduled visit or at 
least 4 weeks +/- 2weeks after the dose adaption of MTX in the maintenance. If 
WBC should drop again below 3.0 x109th/L stop therapy with MTX.  
Stop of study medication in case of following pathologic laboratory values: 
- 
renal dysfunction (serum creatinine levels exceeding 1.5 mg/dL or 130 mol/L or 
increase of 0.5 mg/dL or 43 mol/L compared to baseline value) 
- 
hepatic dysfunction as defined as an increase of AST or ALT ≥ 3 times upper limit 
of normal after the start of study medication. 
- 
Drop of WBC below 3.0 x109th/L despite dose reduction of MTX after the start of 
study medication. 


Methotrexate response in treatment of UC 
 
35
9 WITHDRAWAL OR STOP OF STUDY DRUG CRITERIA 
Criteria in individual cases 
Any patient can withdraw from the study at any time without personal disadvantages and 
without having to give a reason. The time of withdrawal, the results available up to that time, 
and, if known, the reason for withdrawal must be documented on the electronic CRF. Of 
note,  that patients not tolerating mesalamine (5-ASA) in the Maintenance Period should 
stop the drug, but will be continued in the study. The stop of the mesalamine) 5-ASA should 
be documented in the CRF. 
The investigator can also discontinue the study after considering the risk-to-benefit ratio, if 
he/she no longer considers the further participation of the patient justifiable. The date of and 
the primary reason for the withdrawal as well as the observations available at the time of 
withdrawal are to be documented on the electronic CRF. 
Reasons leading to the withdrawal of a patient can include the following (primary reason 
must be determined): 
 
 inclusion criterion not fulfilled or exclusion criterion fulfilled; coming to knowledge 
after recruitment  
 patient’s request 
 technical/logistical reasons (e.g. a change in place of residence, not referred by family 
physician, etc.)  
 other reasons (noting reason) 
 
Reasons leading to the stop of study drug but follow-up according protocol can include 
the following (primary reason must be determined): 
 lack of efficacy of the study medication, e.g. 
- 
need for a prohibited concomitant medication (e.g. need for steroids in the 
maintenance period) 
 intolerable adverse events, e.g. 
- 
renal dysfunction (serum creatinine levels exceeding 1.5 mg/dL or 130 mol/L or 
increase of 0.5 mg/dL or 43 mol/L compared to baseline value) 
- 
hepatic dysfunction as defined as an increase of AST or ALT ≥ 3 times upper limit 
of normal after the start of study medication. 
- 
Drop of WBC below 3.0 x109th/L despite dose reduction of MTX to 15 
mg/weekly after the start of study medication. 
 lack of patient’s cooperation, e.g. 
- 
patient’s request 


Methotrexate response in treatment of UC 
 
36
- 
lack of compliance (fails to attend the follow-up visits as agreed)  
- 
existing pregnancy or intended pregnancy* (females, men), lactation (females) 
(*Methotrexate should not be used for at least 3 months before planned pregnancy for men 
and women and should not be used during pregnancy or breast feeding 
In all patients who finish the study prematurely, a withdrawal examination should be carried 
out within 2 weeks after the last application of the study medication. The withdrawal 
examination must be conducted as a final examination and documented in the electronic 
CRF. It is most important to calculate at least the final Mayo score for each patient. If 
possible, an endoscopy should be performed and the blood samples should be taken. The 
investigator will continue to observe patients withdrawn from the study because of 
intolerable AEs until the findings have been clarified. Also the investigator should follow the 
patients, in whom the study drug was stopped according to the regular scheduled study 
visits.  
 
Treatment recommendations in case of flare of disease 
In the event of a flare up in the Induction or Maintenance Period, the patient should be 
treated according standard of care and at the discretion of the investigator. The treatment 
may include the application of steroids, treatment of an underlying GI-infection or 
hospitalization for closer observation and intravenous hydration and/or intravenous or oral 
immunosuppressive therapies (e.g. steroids). Also a premature withdrawal visit should be 
conducted, but even after withdrawal of the study drug, the patient should be followed up 
until completion of the Induction Period or Maintenance Period, depending on in which 
Period of the trial the flare up occurred.  
 
Criteria for the termination of the whole study 
If serious safety concerns arise, the coordinating investigator can terminate or interrupt the 
study by agreement with the sponsor. If new information on the risk-to-benefit ratio of the 
drug or on the treatment methods used in the study is obtained in the meantime, the 
coordinating investigator reserves the right to interrupt or terminate the project by agreement 
with the sponsor. Premature termination is also possible if the coordinating investigator, or 
the investigators and the sponsor if patient recruitment is insufficient and cannot be 
expedited by appropriate measures. Criteria for early termination of the study related to 
results of interim analyses are discussed in the section “Statistical Considerations”. 
 
 
10 MEDICATION PREPARATION AND DISTRIBUTION 
Methotrexate 


Methotrexate response in treatment of UC 
 
37
The drug kits will be sent by the University of Pennsylvania Investigational Drug Service 
(IDS) directly to the patient unless due to local regulatory requirements the site would prefer 
to distribute the drug kits.  In this case, IDS will send the drug kit to the site and the site will 
send the kit to the patient.  All kits will have a unique number and will contain at least 1 
month supply of medication or placebo and syringes for administration. When a patient 
qualifies for randomization, the IDS will implement the randomization according to the 
Group the patient is in.  Sites will be notified by fax and email regarding the date that the 
blinded study medication pack was shipped. The form that identifies the treatment 
assignment code will be retained in the source documents.  The coordinator will contact the 
patient to ensure they received the drug kit and to note the date the patient began study drug.  
In the case where the drug is shipped to the site, the site will distribute the treatment pack to 
the patient and then contact the patient to be sure they received it and to note the date they 
began study drug.  All drug kits will contain dosing instructions.  
Once the screening visit of the patient is entered in the database, a screening number will be 
assigned and the patient will receive 1 vial of MTX 2 ml (25mg/ml) and 7 tablets folic acid 
(1 mg) and 2 tablets promethazine (25 mg) by mail delivered to his home address from the 
IDS. The patient should bring the MTX vial and the folic acid tablets to the next visit at 
week 0. Depending on the local regulatory requirements the drug may be as well delivered to 
the investigational site. Should the patient not qualify for the inclusion into the trial at week 
0, the investigational site should collect and discard the single MTX vial and the folic acid 
tablets them.  
Each patient will be trained at week 0 in the Induction Period in administration of the study 
medication by a nurse using sodium chloride at the participating study sites. An instruction 
page will be provided and the patients will be observed doing the first subcutaneous 
administration themselves. Patients will be instructed to administer the medication 
subcutaneously in either the abdomen or thigh; whichever is easier for the patient. The first 
administration of MTX will take place at week 0 (visit 1) at the participating center. After 
the first injection of MTX the patient should also start to take folic acid tablets 1 mg tablets 
once daily. 
After the first injection is applied at week 0 of the Induction Period the patient will receive, 
in regular time intervals, study drug kits containing (1)unblinded 2 ml vials MTX 
(concentration 25 mg/ml); (2) 1 ml syringes (U100 27g ½ inch syringes ); (3) extra needles; 
(4) alcohol swabs; and (5) containers for sharps disposal. The disposal boxes can close to 
prevent sharps from falling out. The boxes will be returned to the study site for disposal 
during study visits. Also additional folic acid tablets for the remaining 16 weeks in the 
Induction Period will be sent to the patient.  The patient will inject each week on the same 
day 1 ml (1 syringe) of MTX subcutaneously. The 1 ml syringes will be an extra caution to 
prevent MTX overdosing.  
 
In the Maintenance Period the patient will receive in a double-blinded fashion either one 2ml 
vial MTX (concentration 25 mg/ml) or one 2 ml vial placebo. Due to the yellow colour of 
MTX, placebo will be prepared and packaged into matching 2 ml vials for subcutaneous 
injection using vial 2 of Infuvit® Adult (multiple vitamins for infusion), which is a FDA 
approved formulation for multiple vitamins for infusion. The vitamins will be diluted with 
sodium chloride to match the yellow color of the MTX and have maximal the following 


Methotrexate response in treatment of UC 
 
38
concentrations (values given for undiluted solution): Biotin 12 microgram/ ml, folic acid 120 
microgram/ml and vitamin B12 1 microgram/ml. In addition both vials will be covered with 
an opaque foil-backed label with strong adhesive.  
 
Folic acid tablets 
Patients will be supplied with seven 1 mg folic acid tablets after the screening visit and once 
the patients are dosed with the first injection of MTX additionally folic acid tablets for the 
remaining 16 weeks will be sent to the patients. Patients entering the Maintenance Period 
will be supplied with 1 mg folic acid tablets for 48 weeks.  
Promethazine 
2 promethazine 25 mg tablets will be provided by the investigational drug service (IDS) 
directly to the patient after the screening visit. If the patient experiences nausea after the first 
injection the patient should be advised to take 1 promethazine tablet. In case of nausea the 
patient should also be advised to call the local coordinator for further supply of 
promethazine, which could be ordered by weborder from the investigational drug services 
(IDS). The IDS will then supply the patient with one promethazine tablet for each weekly 
MTX injection. 
 
Ondansetron 
If promethazine is not sufficient to suppress the MTX induced nausea, the patient should call 
the local coordinator. The coordinator should query the subject about a personal or family 
history (first degree relatives only) of long QT syndrome.  If a history is present, 
ondansetron will not be ordered and the local investigator may prescribe an alternate anti-
nausea therapy.  Additionally, the investigator should review the subject’s most recent 
laboratory results.  If significant electrolyte abnormalities are present on the last lab draw  
(potassium < 2.8 mEQ/L, magnesium <1.0 mEQ/L), the patient should then undergo a 
portable EKG at the investigator site to exclude a long QT-syndrome. The QTc interval 
should not be longer than 440 msec.  Once the patient has been excluded to have significant 
electrolyte abnormalities (potassium, magnesium) or a history or newly diagnosed long QT-
syndrome, the investigator can directly order ondansetron 4mg tablets from the IDS for the 
patient. The IDS will then supply the patient with one ondansetron tablet for each weekly 
MTX injection. 
 
The above precautions are necessary; since on 9/15/2011 the FDA issued a warning that 
ondansetron may increase the risk of developing abnormal changes in the electrical activity 
of the heart, which can result in a potentially fatal abnormal heart rhythm. Patients at 
particular risk include those with underlying heart conditions, such as congenital long QT-
syndrome and those who are predisposed to low levels of potassium and magnesium in the 
blood. 
 
 


Methotrexate response in treatment of UC 
 
39
Mesalamine (Asacol HD) 
16 weeks supply of mesalamine tablets will be provided by the investigational drug service 
(IDS) directly to the patient once the patient has been randomized in the Maintenance Period 
and after 15 weeks in the Maintenance Period.  If subjects have a known intolerance of 5-
ASA, they will not receive Asacol HD from the IDS during the Maintenance Period. 
11  DATA SAFETY MONITORING  
 On a monthly basis, the DCC will issue standard safety reports (enumerated by event and 
category, both by total and by type) as well as a cumulative report of all Adverse Events 
(AEs) to include Event Code, Dates of Onset/Resolution, Grade, and Outcome to the  an 
independent safety monitor. In addition, these standard safety reports will be presented to the 
DSMB at each conference.  The DSMB will review these data and make appropriate safety 
decisions based on these events. Participants will be monitored for potential AEs, including 
signs or symptoms related or unrelated to the condition under study, any clinically 
significant laboratory abnormality, or any abnormality detected during physical examination. 
  
 
12 RANDOMIZATION, DATA MANAGEMENT AND DATA 
MONITORING 
The randomization of the patients and the processing and analysis of the data will be carried 
out by the Biostatistics core of the Center for Gastrointestinal Biology and Disease at UNC 
Chapel Hill.  
Study data for this study will be collected and stored using electronic records.  Data captured 
will be entered in real time at each clinical site using web forms developed to replicate paper 
case report forms.  All data will be created, modified, maintained, archived, retrieved and 
distributed by a computer system.  The use of electronic records will increase the speed of 
data collection and exchange.  This will reduce the manpower necessary to perform double-
data entry from paper forms and transcription error.  In addition, electronic records permit 
economical storage of study data and ease of accessibility and analysis. Data management 
and data quality systems will be built into the system.   
Data quality using electronic records will ensure that data are attributable, legible, 
contemporaneous and original.   
The Data Management Center (DMC) at the CGIBD at the University of North Carolina will 
track the data collection, provide data security, control for confidentiality of study data, 
maintain computer backups to protect data until study closure and archive study data 
according to FDA requirements (21 CFR 11).  Electronic signatures will be linked to each 
entry.   


Methotrexate response in treatment of UC 
 
40
All computer systems and programs will be password protected, and all electronic 
communications of study and other confidential information will be encrypted. Personnel at 
the CGIBD have extensive training and experience using electronic data systems.  Good 
computer security practice (restricting physical access to machines, prohibition of password 
sharing, and logging off computers after work hours or when away from the machine) will 
be required of all study personnel.  
Standard Operating Procedures exist for users of the DMC.  Only authorized persons are 
authorized for data entry and access.  Data security systems require password protected 
identification codes for data entry and provide protection against data manipulation.  The 
database is located on a server protected by firewalls.  Access to the database server will not 
be allowed by users on computers outside of the firewall-protected zone.  Virus protection 
software is installed on each study machine.  System access to computer systems will be 
audited.  Redundant backups and off-site backup storage will allow for quick restoration of 
data in the unlikely event that a hardware failure, disaster, or security breach should occur.  
Servers and backups will be located in a secured location with access limited to authorized 
personnel. 
Data cleaning will include range and edit checks, cross form edit checks, query generating 
and tracking and periodic data status reports.  Any data errors or inconsistencies detected 
after data entry will be automatically tracked, communicated and resolved using a web-
based application.  An audit trail of all data changes over the life of the study will be 
maintained.  All study raw data, forms, documents, software programs, software applications 
and computer data files will be indexed and archived routinely.  Strict version control of 
documents and software applications will be instituted.  Retention of study documentation 
after study completion will conform to FDA and NIH requirements. 
Standardized study management reports will be generated monthly during the recruitment 
phase of the study.  These reports will be used to track study progress including patient 
enrollment, randomization, compliance, patient status changes, and study events.  The data 
will be reported for each Study Center individually and summarized for the study as a whole.  
Every six months, a standardized report will also be generated for the DSMB meeting.  This 
report will include additional information on clinical events and adverse events that is coded 
by blinded treatment group.  Other than the study statistician and statistical analyst, no study 
personnel will see this report.  
 
13 STATISTICAL CONSIDERATIONS 
Sample Size Calculation 
The primary aim is to test whether the relapse rate of patients randomized to MTX 
maintenance therapy following 16 weeks of successful MTX treatment is clinically superior 
to the rate of similar patients randomized to placebo maintenance. To test this, we will 
compare relapse-free survival rates in each treatment group over the 32 week follow-up 
period using the log-rank test for a difference in survivor functions 23-25. We estimate rates of 
relapse-free survival in the placebo group will range from 38% to 42%. We also estimate 


Methotrexate response in treatment of UC 
 
41
that improvements in survival rates of 25% or greater are of clinical significance given the 
widespread availability and low cost of this intervention compared to alternatives. This is 
equivalent to 52-54% reduction in hazard over the expected range of placebo group relapse-
free survival rates.  
Since there are no known data of the clinical efficacy of MTX in prospective placebo 
controlled trials, we assumed a success rate (clinical remission and off steroids or clinical 
response and off steroids as defined by the partial Mayo score) of 55-70% with open label 
MTX and steroid taper after 16 weeks (see table A). This estimate is based on previous 
publications with open label administered MTX as described in the Background section of 
the protocol 14, 19-22. We postulate an absolute difference of 25% between the MTX (65% 
maintenance of remission and off steroids until week 48) and placebo group (40% 
maintenance of remission and off steroids until week 48).  
These assumptions were based on the following data:  
 A 26% difference in successful maintenance was observed between MTX 15mg/week 
vs. placebo in the landmark study investigating the efficacy of MTX in maintaining 
remission in CD patients by Feagan et al. 9. In this study maintenance therapy with MTX 
resulted in 65 % remission and no steroids at week 40 compared to 39% in the placebo 
group. 
 Previous trials in steroid dependent UC patients despite 5-ASA therapy demonstrated 
remission rates of 53% on azathioprine and 21% on 5-ASA after 6 months 26 and in 
patients on long term azathioprine a 61% relapse rate after withdrawing this drug 
compared to 31% in patients continuing on azathioprine 27. 
 
The number of patients randomized depends on the proportion of those enrolled in the 
induction phase, who achieve MTX-induced remission or response and of the proportion 
who agree to continue with the 32 week blinded maintenance phase. Table A shows the 
numbers of patients (both arms) who would be randomized based on estimated induction 
phase remission rates of 55%-70%, under scenarios of 180-220 patients being enrolled into 
the induction phase, allowing for a loss of 5% of patients in remission or responding to 
MTX, who do not continue with the maintenance phase. For instance, if 200 patients are 
enrolled in induction phase, and remission is 65%, and 5% do not continue, 124 will be 
assigned to MTX or placebo and available for analysis. 
 
Table A. Number of patients randomized to maintenance trial under varying remission rates, 
for different numbers screened and enrolled into induction phase. 
 
Number needed 
to screen 
 
Induction Period 
Screen failure rates 
Number 
Remission/response week 16 
30% 
25% 
Enrolled 
0.55 
0.60 
0.65 
0.70 


Methotrexate response in treatment of UC 
 
42
257 
240 
180 
94 
103 
111 
120 
286 
267 
200 
105 
114 
124 
133 
314 
293 
220 
115 
125 
136 
146 
 
The middle range of response or remission rates (60-65%) leads to randomized study 
population sizes of 103-136 patients if 180-220 are enrolled in induction phase. We will 
monitor remission rates during the induction phase to determine the number needed to enrol 
to reach our required sample size for the randomized maintenance phase. If remission rates 
are higher, fewer patients will be needed overall; if lower, we will continue to enrol until we 
reach our target study size.  
 
We calculated statistical power for log-rank tests of differences in survival using Stata 
version 11 (Austin TX), based on Schoenfeld et al. 25. The power available to detect a 25% 
improvement in survival (40% on placebo versus 65% on MTX) is equal to 75% if 103 
patients are randomized to MTX or placebo with 50/50 allocation ratio, and is equal to 86% 
if 136 patients are randomized. This power calculations are based on the assumption that 
time to even is exponentially distributed and take into account an interim analysis for 
efficacy using O’Brien-Fleming boundary.  
 
136 patients randomized equally to MTX or placebo ranges between 75% and 86%. 
 
Using a two-sided type 1 error rate of 0.05, and assuming a screen failure rate of 30% 
(which is based on the recent trials using infliximab and abatacept for the same patient 
group), we calculated for our current protocol that we would need to screen 314 patients to 
be able to enrol 220 patients in the Induction Period to be able to randomize a total of at least 
119 patients, which will give the study a statistical power of at least 80% percent This will 
provide sufficient power to detect a clinically significant 25% improvement in survival 
based on the range of expected survival rates in the placebo group. Depending on actual 
induction remission rates, we will need to enrol between 180-220 patients in the open label 
induction phase, which implies screening between 240-324 patients at screening failure rates 
of 25-30%. 
 
Efficacy analysis population 
The efficacy analyses will be performed using the intention-to treat (ITT) principle. That is, 
all patients receiving at least one injection of study medication after randomization at week 
16. 
The statistical analyses of the primary and secondary endpoints will be performed with the 
statistical support of Joseph Galanko, PhD, who is the biostatistician at the Biostatistics Core 
of the Center for Gastrointestinal Biology and Disease at the University of North Carolina 
and Christopher Martin, MSPH, who is the director of the Biostatistics Core of the Center 


Methotrexate response in treatment of UC 
 
43
for Gastrointestinal Biology and Disease at the University of North Carolina. SAS software 
(SAS Institute, Cary NC) will be used to perform all analyses. 
 
Statistical analyses 
The primary aim is to test effectiveness of MTX in maintaining clinical remission over 32 
weeks of therapy compared to placebo. Analyses of this aim will be conducted at two time 
points: (1) final analysis at the conclusion of the trial and (2) one interim analysis when 
about half of the patients completed follow-up in the maintenance phase. The interim 
analysis is planned to allow the DSMB to determine whether treatments are so convincingly 
different that continuation of the trial would be unethical, and also whether side effects of 
treatment are too severe to warrant continued therapy given the potential risk: benefit ratio. 
Details and methods of these final and interim analyses are described below. 
 
In addition, we plan to conduct a single test of open label induction phase response rates 
after the first 75 patients have completed this phase of the trial. Accrual of sufficient 
numbers of patients into the randomized maintenance trial depends on the effectiveness of 
MTX in achieving remission during the 16 week open label induction phase. Therefore, after 
the first 75 patients have completed the induction phase, we will assess whether observed 
response rates are sufficient to meet goals for accrual to randomization in a timely manner, 
and also to determine whether inclusion/exclusion criteria should be modified to target 
subgroups of patients most likely to benefit from induction treatment. 
 
Analysis of induction phase response rates 
Each patient will be categorized as a success or failure with respect to response at 16 weeks. 
Response at week 16 is defined as either clinical remission or clinical response:  (1). Clinical 
remission is a partial Mayo score of no more than 2 points with no individual subscore 
exceeding 1 point, and no use of steroids at week 16; (2) clinical response, defined by a 
reduction of the partial Mayo score ≥ 2 points and at least 25% with an accompanying 
decrease in the subscore for rectal bleeding of at least 1 point or an absolute subscore for 
rectal bleeding of 0 or 1 and has a clinical Mayo score of < 5, and no use of steroids at week 
16 and a partial Mayo score ≤5. 
 
Because only patients who achieve response during the induction phase are randomized into 
the maintenance therapy, successful accrual into the randomized maintenance trial depends 
on the minimal proportion achieving response during induction. Based on expected numbers 
of patients available for enrollment into induction phase, and the number required to meet 
minimum power in the randomized trial, we have determined that induction rates need to a 
minimum of 30% or more to ensure sufficient accrual into the trial during the time allowed. 
 


Methotrexate response in treatment of UC 
 
44
Patients who discontinue treatment prior to 16 weeks for any reason will be categorized as 
failures. As soon as 75 patients have completed the 16-week induction phase, we will 
compute the point estimate and Clopper-Pearson exact 95% confidence interval of the 
proportion of successful induction therapy.  
 
Patients who discontinue treatment prior to 16 weeks for any reason will be categorized as 
failures. To achieve our enrollment goals in the planned time frame, we estimate that the rate 
of remission/response during induction should be at least 30%. Therefore, we will assess the 
success rate as soon as the first 75 patients have completed the 16-week induction phase. If 
the proportion achieving remission/response does not exceed 30% (23 or more patients 
achieving this outcome), the sponsor and the DSMB will review the data and decide if the 
trial should be stopped or modified and continued.  
If the proportion achieving remission/response exceeds 30%, we will continue enrollment as 
planned. However, we will utilize this assessment of remission/response rates to investigate 
possible revision of inclusion criteria to improve accrual. Specifically, we will compare 
remission/response rates across strata of patients defined by pre-enrollment therapy: steroid-
dependent only, azathioprine/6-MP failure, azathioprine/6-MP intolerance, anti-TNF failure, 
or anti-TNF loss of response. If one or more of these subgroups has significantly higher 
remission/response rates, we will explore whether revision of inclusion criteria to 
preferentially recruit patients most likely to respond to induction therapy will enhance 
accrual rates into the randomized phase. 
 
We recognize that there is a degree of uncertainty around the remission/response rate yet our 
decision to stop the trial would be based on a point estimate of response of at least 30%. 
With 75 evaluable subjects, the confidence interval around the 30% response rate would be 
20% to 42%. Thus, with 75 subjects, we will have relatively precise estimates of 
response/remission rates. Even were the response/remission rate as high as 42%, the 
medication would be viewed as relatively modestly effective. We would reserve the right to 
discuss this stopping rule with the DSMB if the proportion of the responders who have 
achieved remission is high, understanding that greater value may be placed on remission 
than response by patients and treating physicians.   
 
Primary analyses 
The primary aim of the trial is to test whether primary and secondary outcomes after 32 
weeks of maintenance therapy differ between patients randomized to MTX versus those 
randomized to placebo. As described previously, one interim analysis of primary outcomes 
is planned for DSMB review during the course of the trial. Both the primary and interim 
analyses will be based on intent-to-treat population. That is, patients will be classified 
according to the treatment arm to which they were randomized, without respect to 
compliance, drop out, or other drugs taken. We also plan secondary per-protocol analyses 
after completion of primary intent-to-treat analyses. 
 


Methotrexate response in treatment of UC 
 
45
The total period of enrollment is 48 weeks, consisting of 16 weeks open label induction, 
followed by 32 weeks of randomized maintenance therapy for patients who achieve 
remission during the induction phase. The follow-up time for planned primary comparisons 
of treatment-specific treatment survivor functions is restricted to the 32-week maintenance 
phase. The colonoscopy exam at the end of follow-up occurs at week 48 of enrollment, 
which is week 32 of follow-up. Thus, we refer to outcome assessment below as occurring at 
week 32.  
 
Definition of primary and secondary outcomes 
The primary outcome is relapse-free survival, comprised of three components, all of which 
must be met to be categorized as relapse-free: total week 32 Mayo score not exceeding 2 
points, with all individual subscores not exceeding 1 point and relapse free survival defined 
by a numerical stable Mayo score throughout 32 weeks of maintenance therapy without 
increase of 3 or more points in the partial Mayo clinic score (excluding sigmoidoscopy) 
compared to the partial Mayo score of the individual patient at randomization at week 16 (2) 
and no steroid use or other immunosuppressive medication (anti-TNF agents, thiopurines, 
cyclosporine, tacrolimus) to control disease activity throughout the 32 week maintenance 
period.For most patients, time-to-failure or censoring will occur at the final visit (32 weeks) 
when the Mayo score is determined, however for patients who relapse due to initiation of 
steroid treatment before 32 weeks, time-to-failure will be the week in which steroid therapy 
was started. 
 
Two secondary outcomes are also defined: (1) mucosal healing, defined as an absolute 
subscore for endoscopy no more than 1 at week 32, and (2) relapse of disease, defined as an 
increase of 3 or more points in the partial Mayo clinic score (excluding sigmoidoscopy) with 
an absolute clinical Mayo score ≥4, or re-treatment with steroids during maintenance. Any 
patients for whom outcomes cannot be assessed for any reason will be classified as failures 
(relapses) with the event time assigned as the date that the patient was withdrawn from the 
study. 
Patient characteristics of interest 
For assessment of randomization success, and for assessment of possible confounding of 
treatment effects with respect to primary and secondary outcomes, we will use the following 
list of patient factors: age, sex, previous medication use (5-ASA only, azathioprine/-MP, 
anti-TNF agents), extent of disease (proctitis, left-sided disease, or pan colitis) steroid 
dependent disease at the time of enrolment, entry Mayo disease activity score and smoking 
status. 
 


Methotrexate response in treatment of UC 
 
46
Analysis plan 
Descriptive Statistics 
We will also compute the treatment-arm-specific proportions and exact 95% confidence 
intervals of patients achieving each primary and secondary outcome of interest. 
 
We will describe and summarize the bivariate distributions of patient characteristics (age, 
sex, duration of disease, previous therapies, smoking status, disease extent, disease severity) 
within treatment arms. We will compute medians, interquartile ranges, means and standard 
errors of continuous variables. Categorical variables will be summarized using proportions 
in each level. We will compare distributions across treatment arms as follows: (1) for 
categorical variables, Fisher's exact chi-square tests of association in 2-by-X tables, or (2) 
for continuous variables, either Student's t-tests of differences in means (for normally 
distributed variables), or Wilcoxon rank-sum tests for non-normally distributed variables. 
Because any imbalance in the two randomized groups is by definition a chance occurrence, 
these descriptive analyses will be used to highlight potential areas of substantial unbalance 
between the study arms and to inform adjusted analyses of treatment effect. 
 
Analysis of primary and secondary outcomes 
All statistical tests for the primary and secondary outcomes will be 2-sided using 
alpha=0.05, except as described below with regard to the pre-specified interim analyses of 
the primary outcome for the DSMB.  
The primary aim is to test whether the relapse-free remission in patients randomized to MTX 
maintenance therapy following MTX-induced remission is superior to that in similar patients 
randomized to placebo maintenance. This primary outcome will be determined for each 
patient, along with the time-to-failure or, for patients who do not relapse, time-to-censoring 
of 32 weeks. These data will be used to compute survivor functions within treatment groups. 
We will then compare treatment-group-specific survivor functions using the non-parametric 
logrank test. This tests the null hypothesis  
 
 
H0 : S1(t) = S2(t) for all t ≤ 32 
 
where Sj (t) is the survival function in group j at time t. For this test, we will use alpha=.05 to 
account for the interim analysis (see below).  
 
We fully expect randomization to result in balanced distribution of potential confounders 
across treatment groups. Further, as randomization will be by permuted blocks within site, 
we also do not expect confounding by study site. We will nonetheless assess possible 
confounding by any variables found to differ across treatment groups, and, also by site if 
treatment distribution differs across sites.  


Methotrexate response in treatment of UC 
 
47
We will also assess heterogeneity of effect by covariates of interest, and by site, using 
Mantel-Haenzel methods to compute and compare crude and summary incidence rate ratios, 
with accompanying tests for heterogeneity. If any heterogeneity is observed, we will report 
stratum-specific treatment effects. 
We will repeat these analyses for the secondary outcomes. 
 
Exploratory analyses of factors associated with remission at week 32 of Maintenance Period 
If a treatment effect on relapse-free survival is observed, we will also conduct exploratory 
analyses of patient characteristics predicting remission versus failure among MTX-treated 
patients. We will use bivariate methods to assess associations of each patient factor of 
interest (described above) with relapse-free remission at week 32, followed by multivariable 
logistic regression modeling having relapse-free remission as the dependent variable. To 
assess relationships of each factor to this outcome while adjusting for other potential 
confounders, we will use a 10% change-in-estimate criterion to assess whether individual 
covariates should be retained.  
 
Exploratory analyses of factors associated with successful induction therapy 
We will conduct exploratory analyses of patient factors predicting achievement of remission 
and clinical response following 16 week of open label MTX treatment (Induction Period). 
The methods used will parallel those just described used for exploratory analyses of factors 
associated with relapse-free remission at week 32 of the Maintenance Period, except the 
outcomes of interest will be remission and clinical response at 16 weeks induction therapy. 
 
DSMB interim analyses and early stopping rules for interim analyses 
Interim analyses of treatment effect will be conducted when 50% of patients will have 
completed the Maintenance Period of the study  (which will be approx. at the end of year 2). 
Results of the interim analysis will only be provided only to the Data Safety Monitoring 
Board, not to the investigators. This interim analysis will use the methods described above 
using logrank tests to detect treatment group differences in survivor functions with respect to 
the primary outcome, relapse-free maintenance of remission. The absolute and relative 
difference in survivor functions, if any, will also be summarized. Accompanying these 
results will be summaries of the distributions of covariates of interest by treatment group, as 
explained in Descriptive Analyses. In addition, summaries of adverse events will be 
calculated by treatment group with accompanying chi-square tests to indicate statistically 
different incidence of adverse events by treatment group. 
 
By definition, interim analyses require multiple analyses of the data. To avoid inflating the 
overall type I error rate for the principal analysis of efficacy, we will use the O'Brien-
Fleming method of adjusting type I error rate for planned interim analyses. This design 


Methotrexate response in treatment of UC 
 
48
utilizes very low type I error rates early in the trial, to preserve a level at or near the 
traditional 0.05 for the final analysis. In addition, an inflating type I error rate is used for 
sequential interim analyses, increasing the probability of stopping the trial in later analyses 
when more data are available. As this trial accrues patients for over three years, there will be 
a total of two analyses (one interim analysis and one final analysis). The suggested stopping 
boundaries for the analysis of primary treatment effect are shown in table 1.  
 
Table 1. Proposed stopping boundaries 
Analysis 
Z 
P 
1 
2.800 
.0050 
2 
1.977 
.0480 
 
 
Since the DSMB might not meet when exactly half of the patients completed the study, it 
might be preferable to use the error spending function f(t) = min{2-2(0.025/t),0.05}, 
where  is the cumulative distribution function of standard normal and t is the information 
fraction observed in the trial at the time of interim analysis.  
 
Any decision to stop the trial early due reasons of efficacy or safety will be made solely by 
the Data Safety Monitoring Board; these a priori stopping boundaries are merely intended to 
serve as a guideline to those decisions. 
 
 


Methotrexate response in treatment of UC 
 
49
 
14 REFERENCES 
1. 
Swierkot J, Szechinski J. Methotrexate in rheumatoid arthritis. Pharmacol Rep 2006;58:473-92. 
2. 
Wessels JA, Huizinga TW, Guchelaar HJ. Recent insights in the pharmacological actions of 
methotrexate in the treatment of rheumatoid arthritis. Rheumatology (Oxford) 2008;47:249-55. 
3. 
Hamilton RA, Kremer JM. Why intramuscular methotrexate may be more efficacious than oral dosing 
in patients with rheumatoid arthritis. Br J Rheumatol 1997;36:86-90. 
4. 
Lafforgue P, Monjanel-Mouterde S, Durand A, Catalin J, Acquaviva PC. Lack of correlation between 
pharmacokinetics and efficacy of low dose methotrexate in patients with rheumatoid arthritis. J 
Rheumatol 1995;22:844-9. 
5. 
Stein RB, Hanauer SB. Comparative tolerability of treatments for inflammatory bowel disease. Drug Saf 
2000;23:429-48. 
6. 
Searles G, McKendry RJ. Methotrexate pneumonitis in rheumatoid arthritis: potential risk factors. Four 
case reports and a review of the literature. J Rheumatol 1987;14:1164-71. 
7. 
Lewden B, Vial T, Elefant E, Nelva A, Carlier P, Descotes J. Low dose methotrexate in the first 
trimester of pregnancy: results of a French collaborative study. J Rheumatol 2004;31:2360-5. 
8. 
Kremer JM, Alarcon GS, Lightfoot RW, Jr., Willkens RF, Furst DE, Williams HJ, Dent PB, Weinblatt 
ME. Methotrexate for rheumatoid arthritis. Suggested guidelines for monitoring liver toxicity. 
American College of Rheumatology. Arthritis Rheum 1994;37:316-28. 
9. 
Feagan BG, Fedorak RN, Irvine EJ, Wild G, Sutherland L, Steinhart AH, Greenberg GR, Koval J, 
Wong CJ, Hopkins M, Hanauer SB, McDonald JW. A comparison of methotrexate with placebo for 
the maintenance of remission in Crohn's disease. North American Crohn's Study Group Investigators. 
N Engl J Med 2000;342:1627-32. 
10. 
Feagan BG, Rochon J, Fedorak RN, Irvine EJ, Wild G, Sutherland L, Steinhart AH, Greenberg GR, 
Gillies R, Hopkins M, et al. Methotrexate for the treatment of Crohn's disease. The North American 
Crohn's Study Group Investigators. N Engl J Med 1995;332:292-7. 
11. 
Feagan B, McDonald J, Panaccione R, Enns RA, Bernstein CN, Ponich TP, Bourdages R, MacIntosh 
DG, Dallaire C, Cohen A, Fedorak RN, Pare P, Bitton A, Saibil F, Anderson F, Donner A, Wong CJ, 
Zou GJ, Vandervoort M, Hopkins M, Greenberg GR. A randomized trial of methotrexate in 
combination with infliximab for the treatment of Crohn's Disease. Gastroenterology 2008;135:294-
295 (abstract). 
12. 
Oren R, Moshkowitz M, Odes S, Becker S, Keter D, Pomeranz I, Shirin C, Reisfeld I, Broide E, Lavy 
A, Fich A, Eliakim R, Patz J, Villa Y, Arber N, Gilat T. Methotrexate in chronic active Crohn's 
disease: a double-blind, randomized, Israeli multicenter trial. Am J Gastroenterol 1997;92:2203-9. 
13. 
Arora S, Katkov W, Cooley J, Kemp JA, Johnston DE, Schapiro RH, Podolsky D. Methotrexate in 
Crohn's 
disease: 
results 
of 
a 
randomized, 
double-blind, 
placebo-controlled 
trial. 
Hepatogastroenterology 1999;46:1724-9. 
14. 
Kozarek RA, Patterson DJ, Gelfand MD, Botoman VA, Ball TJ, Wilske KR. Methotrexate induces 
clinical and histologic remission in patients with refractory inflammatory bowel disease. Ann Intern 
Med 1989;110:353-6. 
15. 
Baron TH, Truss CD, Elson CO. Low-dose oral methotrexate in refractory inflammatory bowel disease. 
Dig Dis Sci 1993;38:1851-6. 
16. 
Oren R, Arber N, Odes S, Moshkowitz M, Keter D, Pomeranz I, Ron Y, Reisfeld I, Broide E, Lavy A, 
Fich A, Eliakim R, Patz J, Bardan E, Villa Y, Gilat T. Methotrexate in chronic active ulcerative 
colitis: a double-blind, randomized, Israeli multicenter trial. Gastroenterology 1996;110:1416-21. 
17. 
Mate-Jimenez J, Hermida C, Cantero-Perona J, Moreno-Otero R. 6-mercaptopurine or methotrexate 
added to prednisone induces and maintains remission in steroid-dependent inflammatory bowel 
disease. Eur J Gastroenterol Hepatol 2000;12:1227-33. 
18. 
Hamdy H, McKendry RJ, Mierins E, Liver JA. Low-dose methotrexate compared with azathioprine in 
the treatment of rheumatoid arthritis. A twenty-four-week controlled clinical trial. Arthritis Rheum 
1987;30:361-8. 


Methotrexate response in treatment of UC 
 
50
19. 
Paoluzi OA, Pica R, Marcheggiano A, Crispino P, Iacopini F, Iannoni C, Rivera M, Paoluzi P. 
Azathioprine or methotrexate in the treatment of patients with steroid-dependent or steroid-resistant 
ulcerative colitis: results of an open-label study on efficacy and tolerability in inducing and 
maintaining remission. Aliment Pharmacol Ther 2002;16:1751-9. 
20. 
Cummings JR, Herrlinger KR, Travis SP, Gorard DA, McIntyre AS, Jewell DP. Oral methotrexate in 
ulcerative colitis. Aliment Pharmacol Ther 2005;21:385-9. 
21. 
Nathan DM, Iser JH, Gibson PR. A single center experience of methotrexate in the treatment of Crohn's 
disease and ulcerative colitis: A case for subcutaneous administration. J Gastroenterol Hepatol 
2008;23:954-8. 
22. 
Wahed M, Louis-Auguste JR, Baxter LM, Limdi JK, McCartney S, Bloom SL, Lindsay JO. Efficacy of 
methotrexate in ulcerative colitis. Gastroenterology 2008;134:A662-A662. 
23. 
Collett D. Modelling Binary Data. Chapman & Hall/CRC, 2003. 
24. 
Freedman L. Tables of the number of patients required in clinical trials using the logrank test. Statistics 
in Medicine 1, 1982:121–129. 
25. 
Schoenfeld D. The asymptotic properties of nonparametric tests for comparing survival distributions. 
Biometrika 1981;68:316–319. 
26. 
Ardizzone S, Maconi G, Russo A, Imbesi V, Colombo E, Bianchi Porro G. Randomised controlled trial 
of azathioprine and 5-aminosalicylic acid for treatment of steroid dependent ulcerative colitis. Gut 
2006;55:47-53. 
27. 
Hawthorne AB, Logan RF, Hawkey CJ, Foster PN, Axon AT, Swarbrick ET, Scott BB, Lennard-Jones 
JE. Randomised controlled trial of azathioprine withdrawal in ulcerative colitis. Bmj 1992;305:20-2. 
 
 


Methotrexate response in treatment of UC 
 
51
15 APPENDIX 
 
Study Flow Chart 
 
 
 


 
Timeline study visits 
 
Screening 
Week  
0 
Week  
2 
Week  
4 
Week  
8 
Week  
12 
Week  
16 
Week 
20 
Week  
24 
Week 
28 
Week  
32 
Week 
36 
Week  
40 
Week 
44 
Week  
48 
MTX 25 mg/ week 
* 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MTX 25 mg* or 
placebo/week 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Steroid Taper 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mesalamine 
2.4g 
daily 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug Dispensation 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
Mayo score 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Partial Mayo Score 
a 
 
 
 
X 
 
X 
X 
 
X 
 
X 
 
X 
 
 
Phone interview 
(AE) 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AE, CM 
 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
Physical Exam 
X 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
Chest x-rayf 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood Tests b 
X c 
Xe 
X 
X 
X 
X 
Xc 
X h 
X 
X h 
X 
X h 
X 
X h 
X 
Urine 
pregnancy 
test 
Xg 
 
 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
Stool sampled 
X d 
 
 
 
 
 
X d 
 
 
 
 
 
 
 
X d 
Sigmoidoscopy 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 


AE, adverse event; CM, concomitant medication 
 
* or 15 mg/weekly in case of dose reduction due to MTX associated side effects. 
a Diary entries from the 3 days preceding the study visit will be used to calculate the Mayo 
score 
b CBC, AST, ALT, GGT, alkaline phosphatase, bilirubin, albumin, total protein, creatinine, 
CRP, serum folate, magnesium, sodium, potassium, and hepatitis (screening visit only) 
c additional DNA sample for DNA, serum and plasma library. At week 16, plasma and serum 
sample only. 
d Stool sample for C. diff. at screening (screening visit) and stool samples for calprotectin 
(central laboratory) at screening, Week 16 and Week 48.  
e laboratory evaluation not necessary if inclusion in a time period of 14 days after screening 
visit 
f if a chest x-ray was obtained within 1 year before screening, there is no need for a new chest 
x-ray, but the results and the date of this x-ray should be recorded in the CRF 
g serum beta-HCG send to central laboratory 
h CBC only 
 


Methotrexate response in treatment of UC 
 
54
 
Mayo Score 
MAYO SCORING FOR ASSESSMENT OF  
ULCERATIVE COLITIS ACTIVITY 
 
Stool frequency*  
0 = Normal no. stools for this for this patient  
1 = 1-2 stools more than normal  
2 = 3-4 stools more than normal  
3 = 5 or more stools more than normal  
* Each patient served as his or her own control to establish the degree of abnormality of the 
stool  
frequency.  
 
Rectal bleeding  
0 = No blood seen  
1 = Streaks of blood with stool less than half the time  
2 = Obvious blood with stool most of the time  
3 = Blood alone passed  
 
Findings of flexible proctosigmoidoscopy  
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)  
 
Physician’s global assessment**  
0 = Normal (there are no symptoms of colitis, the patient feels well, and the flexible 
proctosigmoidoscopy score is 0) (stool frequency = 0, rectal bleeding = 0, patients functional 
assessment = 0, flexible  
proctosigmoidoscopy findings = 0)  
 
1 = Mild disease (mild symptoms and proctoscopic findings that were mildly abnormal) (the 
subscores should be mostly 1’s: stool frequency = 0 or 1; rectal bleeding = 0 or 1; patients 
functional assessment = 0 or 1; sigmoidoscopy findings = 0 or 1)  
 
2 = Moderate disease (more serious abnormalities and proctosigmoidoscopic and symptom 
scores of 1 to 2) (the subscores should be mostly 2’s: stool frequency = 1 or 2; rectal bleeding 
= 1 or 2; patients functional assessment = 1 or 2; sigmoidoscopy findings = 1 or 2)   
 
3 = Severe disease (the proctosigmoidoscopic and symptom scores are 2 to 3 and the patient 
probably requires corticosteroid therapy and possibly hospitalization) (the subscores should 
be mostly 3’s: stool frequency = 2 or 3; rectal bleeding = 2 or 3; patients functional 
assessment = 2 or 3; sigmoidoscopy findings = 2 or 3)  
 


Methotrexate response in treatment of UC 
 
55
** The physician’s global assessment acknowledged the three other criteria, the patient’s 
daily record of abdominal discomfort and general sense of well-being, and other observations, 
such as physical findings and the patient’s performance status.  
Patient’s functional assessment (this variable is not included in the 12 point index calculation 
but is considered as a measure of general sense of well-being when determining the 
physician’s global assessment score)  
0 =  Generally well  
1 =  Fair  
2 =  Poor  
3 =  Terrible  
  
                                                            
1  
 Schroeder KW, Tremaine WJ, Ilstrup DM. Coated Oral 5-aminosalicycli acid therapy  
for mildly to moderate active ulcerative colitis. N Engl J Med 1987; 317: 1625-1629.  


 


Methotrexate response in treatment of UC 
 
57
Clinical Adverse Event Reporting Form 
Randomized, double blind, prospective trial investigating the efficacy of 
Methotrexate in induction and maintenance of steroid free remission in 
ulcerative colitis (MEthotrexate Response In Treatment of UC - Merit-UC) 
Event ID: ________ Study Center: _________ Study ID: ____________ 
DOB: __ __/ __ __/ __ __ __ __  
Gender: ______ 
Race: _______ 
         mm        dd          yyyy 
Name of MD Reporting Event? 
______________________________ 
 
 
Date of Medical Review 
 __ __/ __ __/ __ __ __ __ 
 
     mm   dd        yyyy 
Is this a Serious Adverse Event (SAE)?  
    Yes 
     No 
 
 
Indicate all classifications(s) applicable 
    Death 
    Life threatening 
     Disability 
     Congenital anomaly/birth defect 
     Hospitalization - Initial 
     Hospitalization - Prolongation 
     Other 
 
 
Specify Other 
 
 
 
Relationship/Causality 
(likelihood 
that 
study agent caused the event) 
     Not related 
     Probably not related 
     Possibly related 
     Related 


Methotrexate response in treatment of UC 
 
58
 
 
 
MD Narrative: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Measures at the onset of adverse event 
    None 
  Reduction of the dose of study  
 
medication 
     Withdrawal of the study medication 
     Other 
 
Specify Other: 
 
 
 
Outcome of event (sequelae are defined as 
conditions following and resulting from 
event)  
     Recovered without sequelae 
     Recovered with sequelae 
     Not yet recovered 
     Died 
 
Signature of Study Center MD 
 
 
 
 
__________________________________ 
Date Form Completed 
__ __/ __ __/ __ __ __ __ 
 
  
 
 
 
 
    mm   dd          yyyy  


Methotrexate response in treatment of UC 
 
59
Summary of Abnormal Lab Values and Required Actions 
 
 
Abnormal 
value 
Action after first lab 
Action after FU lab 
Action afte
 
WBC 
 
< 3 x 10 9th L 
 
1. Call patient and advise to 
stop MTX. 
2.  Arrange for FU lab in 14 
days (+/-3 days) 
3.  Send AE report 
4.  Continue study visits 
 
 
>4 x 10 9th L:   
1.  Call patient and advise to resume MTX at 
15mg/wk. 
2.  Continue study visits 
 
 
1. If value eve
advise patient
2.  Send AE re
3.  Continue s
 
<3 x 10 9th L:   
1. Call patient and advise to discontinue 
MTX  
2.  Continue study visits 
2.  Send AE report 
 
ALT 
AST 
 
> 2X upper limit 
 
1. Call patient and arrange for 
FU lab in 2 weeks 
2.  Continue with current dose 
of MTX 
3.  Send AE report 
4.  Continue study visits 
 
 
 
ALT normalized:   
1.  Continue with current dose of MTX 
2.  Continue study visits 
 
ALT > 2x upper limit 
1.  Call patient and advise to reduce MTX  to 
15mg/wk. 
2.  Recheck ALT in 4 weeks 
3.  Send AE report 
4.  Continue study visits 
ALT > 2x up
1. Call patie
MTX  
2.  Send AE re
3.  Continue s
 
Creatinine 
 
>1.5 mg/dL  
 
 
1. Call patient and advise to 
stop MTX. 
2.  Arrange for FU lab in 14 
days (+/-3 days) 
3.  Send AE report 
4.  Continue study visits 
Creatinine in normal range 
1. Call patient and advise to resume 
 
MTX at 15mg/wk. 
2.  Continue study visits 
 
Creatinine >1.5 mg/dl 
1. Call patient and advise to discontinue 
MTX  
2.  Send AE report 
3.  Continue study visits 
 
 
 

SAP_001.pdf: 
 
1
Randomized, double blind, prospective trial investigating the 
efficacy of Methotrexate in induction and maintenance of steroid 
free remission in ulcerative colitis (MEthotrexate Response In 
Treatment of UC - Merit-UC) 
 
 
DATA ANALYSIS PLAN 
 
Version 1.4 5/30/2015 
 
Anastasia Ivanova, PhD 
Gillings School of Global Public Health 
University of North Carolina at Chapel Hill 
 
Christopher F. Martin, MSPH 
Center of Gastrointestinal Biology and Disease 
Biostatistics Core 
University of North Carolina  
 
Principal Investigator:  
Hans H. Herfarth, MD, PhD 
Division of Gastroenterology and Hepatology 
University of North Carolina 
 
Co-Investigators: 
Kim Isaacs, MD, PhD 
Division of Gastroenterology and Hepatology 
University of North Carolina 
 
James Lewis, MD, MSCE 
Center for Clinical Epidemiology and Biostatistics,  
University of Pennsylvania  
 
Mark Osterman, MD 
Center for Clinical Epidemiology and Biostatistics,  
University of Pennsylvania 
 
Bruce Sands, MD, MS 
Dr. Henry Janowitz Division of Gastroenterology 
The Mount Sinai Medical Center, New York 
 
 


 
2
 
1 
STATISTICAL ANALYSIS 
3 
1.1 
ANALYSIS OF INDUCTION PHASE REMISSION RATES 
3 
1.2 
PRIMARY ANALYSES 
4 
1.3 
DEFINITION OF PRIMARY AND SECONDARY OUTCOMES 
4 
1.4 
PATIENT CHARACTERISTICS OF INTEREST 
5 
2 
ANALYSIS PLAN 
5 
2.1 
DESCRIPTIVE STATISTICS 
5 
2.2 
ANALYSIS OF PRIMARY AND SECONDARY OUTCOMES 
6 
2.3 
EXPLORATORY ANALYSES OF FACTORS ASSOCIATED WITH REMISSION 
6 
2.4 
EXPLORATORY ANALYSES OF FACTORS ASSOCIATED WITH SUCCESSFUL INDUCTION THERAPY 
7 
3 
DSMB INTERIM ANALYSES AND EARLY STOPPING RULES FOR INTERIM ANALYSES 
7 
4 
REFERENCES 
8 
 


 
3
 
The statistical analyses of the primary and secondary endpoints will be performed with the statistical 
support of Joseph Galanko, PhD, who is the biostatistician at the Biostatistics Core of the Center for 
Gastrointestinal Biology and Disease at the University of North Carolina and Christopher Martin, MSPH, 
who is the director of the Biostatistics Core of the Center for Gastrointestinal Biology and Disease at the 
University of North Carolina. SAS software (SAS Institute, Cary NC) will be used to perform all 
analyses. 
 
1 
STATISTICAL ANALYSIS 
 
The primary aim is to test effectiveness of MTX in maintaining clinical remission over 32 weeks of 
therapy compared to placebo. Analyses of this aim will be conducted at three time points: (1) final 
analyses at the conclusion of the trial and (2) one interim analysis, one each at the end of years two and 
three, for review by the DSMB. The interim analysis is planned to allow the DSMB to determine whether 
treatments are so convincingly different that continuation of the trial would be unethical, and also whether 
side effects of treatment are too severe to warrant continued therapy given the potential risk:benefit ratio. 
Details and methods of these final and interim analyses are described below. 
 
In addition, we plan to conduct a single test of open label induction phase remission rates after the first 75 
patients have completed this phase of the trial. Accrual of sufficient numbers of patients into the 
randomized maintenance trial depends on the effectiveness of MTX during the 16-week open label 
induction phase. Therefore, after the first 75 patients have completed the induction phase, we will assess 
whether observed response rates are sufficient to meet goals for accrual to randomization in a timely 
manner, and also to determine whether inclusion/exclusion criteria should be modified to target subgroups 
of patients most likely to benefit from induction treatment. 
 
1.1 
Analysis of induction phase response rates 
Each patient will be categorized as a success or failure with respect to response at 16 weeks. Response at 
week 16 is defined as either clinical remission or clinical response:  (1). Clinical remission is a partial 
Mayo score of no more than 2 points with no individual subscore exceeding 1 point, and no use of 
steroids at week 16; (2) clinical response, defined by a reduction of the partial Mayo score ≥ 2 points and 
at least 25% with an accompanying decrease in the subscore for rectal bleeding of at least 1 point or an 
absolute subscore for rectal bleeding of 0 or 1, and no use of steroids at week 16 and a partial Mayo score 
≤5. 
 
Because only patients who achieve response during the induction phase are randomized into the 
maintenance therapy, successful accrual into the randomized maintenance trial depends on the minimal 
proportion achieving response during induction. Based on expected numbers of patients available for 
enrollment into induction phase, and the number required to meet minimum power in the randomized 
trial, we have determined that induction rates need to a minimum of 30% or more to ensure sufficient 
accrual into the trial during the time allowed. 
 
Patients who discontinue treatment prior to 16 weeks for any reason will be categorized as failures. As 
soon as 75 patients have completed the 16-week induction phase, we will compute the point estimate and 
Clopper-Pearson exact 95% confidence interval of the proportion of successful induction therapy.  
 


 
4
Patients who discontinue treatment prior to 16 weeks for any reason will be categorized as failures. To 
achieve our enrollment goals in the planned time frame, we estimate that the rate of remission/response 
during induction should be at least 30%. Therefore, we will assess the success rate as soon as the first 75 
patients have completed the 16-week induction phase. If the proportion achieving remission does not 
exceed 30% (23 or more patients achieving this outcome), the sponsor and the DSMB will review the data 
and decide if the trial should be stopped or modified and continued.  
 
If the proportion achieving remission/response exceeds 30%, we will continue enrollment as planned. 
However, we will utilize this assessment of remission/response rates to investigate possible revision of 
inclusion criteria to improve accrual. Specifically, we will compare remission/response rates across strata 
of patients defined by pre-enrollment therapy: steroid-dependent only, azathioprine/6-MP failure, 
azathioprine/6-MP intolerance, anti-TNF failure, or anti-TNF loss of response. If one or more of these 
subgroups has significantly higher remission/response rates, we will explore whether revision of inclusion 
criteria to preferentially recruit patients most likely to respond to induction therapy will enhance accrual 
rates into the randomized phase. 
 
We recognize that there is a degree of uncertainty around the remission/response rate yet our decision to 
stop the trial would be based on a point estimate of response of at least 30%. With 75 evaluable subjects, 
the confidence interval around the 30% response rate would be 20% to 42%.  Thus, with 75 subjects, we 
will have relatively precise estimates of response/remission rates. Even were the response/remission rate 
as high as 42%, the medication would be viewed as relatively modestly effective. We would reserve the 
right to discuss this stopping rule with the DSMB if the proportion of the responders who have achieved 
remission is high, understanding that greater value may be placed on remission than response by patients 
and treating physicians.   
1.2 
Primary analyses 
 
The primary aim of the trial is to test whether primary and secondary outcomes after 32 weeks of 
maintenance therapy differ between patients randomized to MTX versus those randomized to placebo. As 
described previously, one interim analysis of primary outcomes is planned for DSMB review during the 
course of the trial. Both the primary and interim analyses will be based on intent-to-treat therapy. That is, 
patients will be classified according to the treatment arm to which they were randomized, without respect 
to compliance, drop out, or other drugs taken. We also plan secondary per-protocol analyses after 
completion of primary intent-to-treat analyses. 
 
The total period of enrollment is 48 weeks, consisting of 16 weeks open label induction, followed by 32 
weeks of randomized maintenance therapy for patients who achieve remission during the induction phase. 
The follow-up time for planned primary comparisons of treatment-specific treatment survivor functions is 
restricted to the 32 week maintenance phase. The colonoscopy exam at the end of follow-up occurs at 
week 48 of enrollment, which is week 32 of follow-up. Thus, we refer to outcome assessment below as 
occurring at week 32.  
 
1.3 
Definition of primary and secondary outcomes 
 
The primary outcome is relapse-free survival, comprised of two components, both of which must be met 
to be categorized as relapse-free: total week 32 Mayo score not exceeding 2 points, with all individual 
subscores not exceeding 1 point and relapse free survival defined by a numerical stable Mayo score 
throughout 32 weeks of maintenance therapy without increase of 3 or more points in the partial Mayo 
clinic score (excluding sigmoidoscopy) compared to the partial Mayo score of the individual patient at 


 
5
randomization at week 16 and no steroid use or other immunosuppressive medication (anti-TNF agents, 
thiopurines, cyclosporine, tacrolimus) to control disease activity throughout the 32 week maintenance 
period. For most patients, time-to-failure or censoring will occur at the final visit (32 weeks) when the 
Mayo score is determined, however for patients who relapse due to initiation of steroid treatment or the 
need for other prohibited medications to control disease activity before 32 weeks, time-to-failure will be 
the week in which steroid therapy was started. 
 
Six secondary outcomes are also defined:  
(1) mucosal healing, defined as an absolute subscore for endoscopy no more than 1 at week 32,  
(2) relapse of disease, defined as an increase of 3 or more points in the partial Mayo clinic score 
(excluding sigmoidoscopy) with an absolute clinical Mayo score ≥ 4, or re-treatment with steroids during 
maintenance. Any patients for whom outcomes cannot be assessed for any reason will be classified as 
failures (relapses) with the event time assigned as the date that the patient was withdrawn from the study. 
(3) steroid free clinical remission as defined as a Mayo score of ≤ 2 points with no individual subscore 
exceeding 1 point or steroid free clinical response defined as a reduction from baseline in the clinical 
Mayo score of ≥ 2 points and at least 25%, with an accompanying decrease in the rectal bleeding subscore 
of ≥ 1 point or an absolute rectal bleeding subscore of 0-1 point and a clinical Mayo score ≤5 and stool 
calprotectin levels <250 mcg/g stool at week 16 of the induction period in the subgroup of patients with 
calprotectin >250mcg/g stool at screening. 
(4) steroid free clinical remission as a Mayo score of ≤ 2 points with no individual subscore exceeding 1 
point and stool calprotectin levels of ≤ 50mcg at week 16 of the induction period in the subgroup of 
patients with calprotectin >250mcg/g stool at screening. 
(5) steroid free clinical remission as defined as a Mayo score of ≤ 2 points with no individual subscore 
exceeding 1 point or steroid free clinical response defined as a reduction from baseline in the clinical 
Mayo score of ≥ 2 points and at least 25%, with an accompanying decrease in the rectal bleeding subscore 
of ≥ 1 point or an absolute rectal bleeding subscore of 0-1 point and a clinical Mayo score ≤5 and stool 
calprotectin levels <250 mcg/g stool at week 32 of the maintenance period in the subgroup of patients 
with calprotectin >250mcg/g stool at screening. 
(6) steroid free clinical remission as a Mayo score of ≤ 2 points with no individual subscore exceeding 1 
point and stool calprotectin levels of ≤ 50mcg at week 32 of the maintenance period in the subgroup of 
patients with calprotectin >250mcg/g stool at screening. 
 
 
 
Patient characteristics of interest 
For assessment of randomization success, and for assessment of possible confounding of treatment effects 
with respect to primary and secondary outcomes, we will use the following list of patient factors: age, sex, 
previous medication use (5-ASA, azathioprine/-MP, anti-TNF agents), extent of disease (proctitis, left-
sided disease, or pan colitis), steroid dependent disease at the time of enrollment, entry Mayo disease 
activity score, calprotectin at inclusion and smoking status. 
 
2 
ANALYSIS PLAN 
 
2.1 
Descriptive Statistics 
 
We will compute the treatment-arm-specific proportions and exact 95% confidence intervals of patients 
achieving each primary and secondary outcome of interest. 


 
6
 
We will describe and summarize the bivariate distributions of patient characteristics (age, sex, duration of 
disease, previous therapies, smoking status, disease extent, disease severity, calprotectin) within treatment 
arms. We will compute medians, interquartile ranges, means and standard errors of continuous variables. 
Categorical variables will be summarized using proportions in each level. We will compare distributions 
across treatment arms as follows: (1) for categorical variables, Fisher's exact chi-square tests of 
association in 2-by-X tables, or (2) for continuous variables, either Student's t-tests of differences in 
means (for normally distributed variables), or Wilcoxon rank-sum tests for non-normally distributed 
variables. Because any imbalance in the two randomized groups is by definition a chance occurrence, 
these descriptive analyses will be used to highlight potential areas of substantial unbalance between the 
study arms and to inform adjusted analyses of treatment effect. 
 
2.2 
Analysis of primary and secondary outcomes 
 
All statistical tests for the primary and secondary outcomes will be 2-sided using alpha=0.05, except as 
described below with regard to the pre-specified interim analyses of the primary outcome for the DSMB.  
 
The primary aim is to test whether the relapse-free remission in patients randomized to MTX maintenance 
therapy following MTX-induced remission is superior to that in similar patients randomized to placebo 
maintenance using the intention-to treat (ITT) principle. This primary outcome will be determined for 
each patient, along with the time-to-failure or, for patients who do not relapse, time-to-censoring of 32 
weeks. These data will be used to compute survivor functions within treatment groups. We will then 
compare treatment-group-specific survivor functions using the non-parametric logrank test. This tests the 
null hypothesis  
 
 
H0 : S1(t) = S2(t) for all t ≤32 
 
where Sj (t) is the survival function in group j at time t. For this test, we will use alpha=.05 to account for 
the two planned interim analyses (see below).  
 
We fully expect randomization to result in balanced distribution of potential confounders across treatment 
groups. Further, as randomization will be by permuted blocks within site, we also do not expect 
confounding by study site. We will nonetheless assess possible confounding by any variables found to 
differ across treatment groups, and, also by site if treatment distribution differs across sites.  
 
We will also assess heterogeneity of effect by covariates of interest, and by site, using Mantel-Haenzel 
methods to compute and compare crude and summary incidence rate ratios, with accompanying tests for 
heterogeneity. If any heterogeneity is observed, we will report stratum-specific treatment effects. 
 
We will repeat these analyses for the secondary outcomes. 
2.3 
Exploratory analyses of factors associated with remission at week 32 of Maintenance Period 
 
If a treatment effect on relapse-free survival is observed, we will also conduct exploratory analyses of 
patient characteristics predicting remission versus failure among MTX-treated patients. We will use 
bivariate methods to assess associations of each patient factor of interest (described above) with relapse-
free remission at week 32, followed by multivariable logistic regression modeling having relapse-free 
remission as the dependent variable.  
 


 
7
2.4 
Exploratory analyses of factors associated with successful induction therapy 
 
We will conduct exploratory analyses of patient factors predicting achievement of remission and 
clinical response following 16 week of open label MTX treatment (Induction Period). The 
methods used will parallel those just described used for exploratory analyses of factors 
associated with relapse-free remission at week 32 of the Maintenance Period, except the 
outcomes of interest will be remission and clinical response at 16 weeks induction therapy. 
 
3 
DSMB INTERIM ANALYSES AND EARLY STOPPING RULES FOR INTERIM ANALYSES 
 
Interim analyses of treatment effect will be conducted when 50% of patients expected to enrol in the 
maintenance phase will have completed the study (which will be approx. at the end of year 2). Results 
of the interim analyses will only be provided only to the Data Safety Monitoring Board, not to the 
investigators. This interim analysis will use the methods described above using logrank tests to detect 
treatment group differences in survivor functions with respect to the primary outcome, relapse-free 
maintenance of remission. The absolute and relative difference in survivor functions, if any, will also be 
summarized. Accompanying these results will be summaries of the distributions of covariates of interest 
by treatment group, as explained in Descriptive Analyses. In addition, summaries of adverse events will 
be calculated by treatment group with accompanying chi-square tests to indicate statistically different 
incidence of adverse events by treatment group. 
 
By definition, interim analyses require multiple analyses of the data. To avoid inflating the overall type I 
error rate for the principal analysis of efficacy, we will use the O'Brien-Fleming method of adjusting type 
I error rate for planned interim analyses. This design utilizes very low type I error rates early in the trial, 
to preserve a level at or near the traditional 0.05 for the final analysis. In addition, an inflating type I error 
rate is used for sequential interim analyses, increasing the probability of stopping the trial in later analyses 
when more data are available. As this trial accrues patients for over three years, there will be a total of 
two analyses (one interim analyses and one final analysis). The suggested stopping boundaries for the 
analysis of primary treatment effect are shown in table 1.  
 
Table 1. Proposed stopping boundaries 
Analysis 
Z 
P 
1 
2.800 
.0050 
2 
1.977 
.0480 
 
Since the DSMB might not meet when exactly half of the patients completed the study, it might be 
preferable to use the error spending function f(t) = min{2-2(0.025/t),0.05}, where  is the 
cumulative distribution function of standard normal and t is the information fraction observed in the trial 
at the time of interim analysis.  
 
Any decision to stop the trial early due reasons of efficacy or safety will be made soley by the Data Safety 
Monitoring Board; these a priori stopping boundaries are merely intended to serve as a guideline to those 
decisions. 
 


 
8
 
4 
REFERENCES 
 
 
Armitage P, Berry G. Statistical Methods in Medical Research. 2nd ed Oxford: Blackwell Scientific Publications; 
1987. 
 
Chow, S., Shao, J., and Wang, H. (2003), Sample Size Calculations in Clinical Research, Boca Raton, FL: CRC 
Press. 
 
Clopper, C. J., and Pearson, E. S. (1934), "The Use of Confidence or Fiducial Limits Illustrated in the Case of the 
Binomial," Biometrika 26, 404–413. 
 
Collett, D. 2003a. Modelling Binary Data. 2nd ed. London: Chapman & Hall/CRC. 
 
Freedman, L. S. 1982. Tables of the number of patients required in clinical trials using the logrank test. Statistics in 
Medicine 1: 121–129. 
 
Landis J, Sharp T, Kuritz S, Koch G. Mantel-Haenszel Methods. In: Armitage P, ed. Encyclopedia of Biostatistics. 
London: Wiley & Sons; 1997. 
 
Machin,  D.,  M.  J.  Campbell,  S.  B.  Tan,  and  S.  H.  Tan. 2009. Sample  Size  Tables  for  Clinical  Studies. 3rd  
ed. Chichester, UK: Wiley–Blackwell. 
 
Mickey RM, Greenland S. The impact of confounder selection criteria on effect estimation. American Journal of 
Epidemiology. 1989;129:125-37. 
 
Schoenfeld, D. A. 1981. The asymptotic properties of nonparametric tests for comparing survival distributions. 
Biometrika 68: 316–319.