1. Results from excluded studies.

Study ID	Description	Results
Baron 1993	Open label clinical trial enrolling patients with steroid dependent or steroid refractory IBD (Crohn's n = 10, UC n = 8) Patients received oral methotrexate 15 mg/wk and prednisone The primary outcomes were complete or partial withdrawal from steroids and mean steroid use	UC patients: mean prednisone dose dropped from 26.3 +/‐ 3.2 mg/day to 12.7 +/‐ 2.0 mg/day (P < 0.001) Three patients had a partial response Adverse events were mild
Cummings 2005	Retrospective chart review at two hospitals Steroid dependent or steroid refractory UC patients (n=50) were treated with oral methotrexate (mean dose 19.9 mg/wk for a median of 30 weeks) The primary outcome was remission defined as lack of treatment with steroids for 3 months or more Secondary outcomes: response defined as good, partial or nil, and proportionate reduction of steroids	Remission occurred in 42% of patients The response was good in 54% and partial in 18% Adverse events occurred in 23%; 10% stopped treatment due to adverse events
Dejica 1998	Unrandomized, open label, preliminary trial enrolled twenty‐two patients with chronic active ulcerative colitis, refractory to steroids or sulfasalazine or both for at least 3 months The patients were treated with 25 mg weekly intramuscular injection for 20 weeks The primary outcome was clinical remission with Mayo Clinic Socre ≤ 3, including endoscopy	Clinical remission were obtained in 50% of patients (n=11) Fifteen of 22 patients (68%) had significant clinical improvement in Mayo Clinic score Five patients developed side effects, but the drug‐related adverse effects were not severe enough to warrant discontinuation of therapy
Egan 1999a	Randomized, single‐blind trial comparing two doses of subcutaneous methotrexate (15 mg/wk, n=18, versus 25 mg/wk, n=14) in patients with steroid dependent or refractory IBD The primary outcome was remission at 16 weeks defined as the presence of quiescent disease (IBDQ score > or = 170) and discontinuation of prednisone The secondary outcome was partial response defined as ability to discontinue prednisone without a decrease in IBDQ or a clinically significant improvement in disease activity.	After 16 weeks 17% (3/18) of patients in the 15 mg group achieved remission compared to 17% (2/12) of patients in the 25 mg group (P = N.S.) Improvement occurred in 39% (7/18) of the 15 mg group compared to 33% (4/12) of the 25 mg group (P = N.S.) Adverse events occurred in 11% (2/18) of patients in the 15 mg group compared to 17% (2/12) of patients in the 25 mg group (P = N.S.)
Egan 1999b	Adenosine was thought to play a major role in anti‐inflammatory mechanism of action of methotrexate in animal models The non‐randomized, open‐label pharmacokinetic study investigating the effects of methotrexate on adenosine concentrations in plasma and at the site of the disease in patients with inflammatory bowel disease In 10 patients with Crohn's disease or ulcerative colitis, rectal adenosine and plasma adenosine concentrations were measured before and immediately after a subcutaneous injection of methotrexate at 15 or 25 mg	There were no significant differences between pre‐injection and post‐injection values in both plasma and rectal adenosine concentrations The mean pre‐dose and post‐dose mean rectal adenosine concentrations were 2.4 µmol/L and 2.1 µmol/L, respectively (P = 0.17) The mean pre‐dose and post ‐dose plasma adenosine concentrations were 3.4 µmol/L and 3.4 µmol/L, respectively (P = 0.95) Therefore, the evidence does not support adenosine as the anti‐inflammatory mediator of methotrexate
Egan 2000	Case series Three patients with steroid refractory UC and 2 patients with steroid refractory Crohn's disease who failed monotherapy with subcutaneous methotrexate 25 mg/week for 16 weeks were treated with the combination of methotrexate and low‐dose oral cyclosporine (3 mg/kg/day) for an additional 16 weeks The primary outcome was remission at 16 weeks defined as the presence of quiescent disease (IBDQ score > or = 170) and discontinuation of prednisone The secondary outcome was partial response defined as ability to discontinue prednisone without a decrease in IBDQ or a clinically significant improvement in disease activity	The three patients with UC experienced clinical improvement with a mean increase in IBDQ score from 164 to 190 points (P = 0.01) One patient developed hypertension
Fraser 2002	Retrospective chart review at two hospitals Seventy patients were reviewed (Crohn's n = 48, UC n = 22) Patients were treated with oral methotrexate (n = 62) or intramuscular methotrexate (n=8) at a mean dose of 20 mg/week for a mean duration of 17.1 months Remission was defined as the lack of a need for oral steroids (either prednisolone or budesonide) for at least 3 months Patients who were well on low doses of prednisolone or budesonide steroids were recorded as ‘remission not achieved’ The continued use of oral 5‐aminosalicylic acid compounds and steroids or 5‐aminosalicylic acid enemas was allowed within the definition of remission Relapse was defined as the need for re‐introduction of steroids, the need for a surgical procedure or the use of infliximab	Remission was achieved in 34 of 55 (62%) of patients who completed more than 3 months of treatment Life‐table analysis showed that the chances of remaining in remission at 12, 24 and 36 months, if treatment was continued, were 90%, 73% and 51% respectively The chances of remaining in remission after stopping treatment at 6, 12 and 18 months were 42%, 21% and 16% respectively
Fraser 2003	Open label clinical trial Eight patients with chronically active moderate to severe UC refractory to corticosteroids and azathioprine or 6‐mercaptopurine were treated with intramuscular methotrexate 25 mg/week (and folic acid) for 16 weeks Efficacy was assessed with the Mayo clinic score	Six of eight patients completed 16 weeks of treatment One patient withdrew due to severe exacerbation and one withdrew due to failure to improve Two patients developed anemia and one patient developed hypertransaminasemia The median Mayo clinic score at 16 weeks was 8 (range 6 to 11) Two patients were referred for colectomy at the end of the study
Gibson 2006	Retrospective chart review at a single IBD clinic including 65 patients (Crohn's n=45, UC n=20) The initial weekly dose was 25 mg in 29 patients, 20 mg in 16 patients, 15 mg in 7 patients or 10 mg in 3 Eighty‐four percent received methotrexate by subcutaneous injection All patients received folate supplementation Response was defined as improvement in bowel symptoms or ability to reduce the dose of steroids Remission was defined as improvement in symptoms with no requirement for steroids for 3 months, or ability to wean off steroids	Remission was achieved by 12 of 19 (63%) patients with UC ‐ an additional patient with UC had a response to treatment The median duration of treatment was 11 months (range 3 to 36) in responders and 6 months (range 1.5 to 10) in non‐responders Fifteen per cent of patients experienced adverse events
González‐Lama 2012	Retrospective chart review of IBD patients treated with methotrexate in eight hospitals in Madrid, Spain Seventy‐seven patients were included (Crohn's disease n = 62, ulcerative colitis n = 15) Methotrexate was initiated at a mean dose of 21 mg/week (range: 15‐30), using parenteral administration in 67% of cases and oral route in 33% of patients Partial response was defined as a decrease in the Harvey‐Bradshaw index of more than three points Remission was defined as a Harvey‐Bradshaw index without steroid treatment below or equal to four	Fourteen out of 15 UC patients received parenteral methotrexate Two patients achieved clinical remission with induction therapy, and 12 (71%) patients gained some response and started maintenance treatment Among the twelve patients, five required dose modification during the follow‐up, three showed loss of response after a mean of 28 weeks, and three more patients achieved clinical remission Adverse events led to methotrexate withdrawal in 5% (4/77) of patients
Hayes 2014	Retrospective chart review of UC patients treated with infliximab (IFX) at a regional referral center Eighty‐five patients with UC were included in the analysis Duration of efficacious IFX therapy, and serum IFX and antibody‐to‐IFX (ATI) levels were compared between patients, who received IFX as monotherapy (n = 39) and in combination with an immunosuppressant (n = 46) Immunosuppressants included azathioprine (65.2% of combination group), mercaptopurine (28.3%), and methotrexate (6.5%)	Concomitant immunosuppressant use was associated with increased duration of IFX therapy (90% in combination group vs. 61% of patients in monotherapy group at 1 year, P = 0.016); greater IFX levels (20.4 mg/L vs. 10.5 mg/L, P = 0.025); and less frequent ATI formation (4.5% vs. 33.3%, P = 0.031)
Herrlinger 2005	Case control study of pharmacogenetics of Mtx therapy in IBD Allele frequencies were assessed in 102 IBD patients treated with methotrexate, 202 patients with Crohn's disease, 205 patients with UC and 189 healthy volunteers All subjects were genotyped for four polymorphisms	No significant difference in allele frequencies were detected between Crohn's disease, UC and healthy volunteers Twenty‐one per cent of methotrexate treated patients experienced adverse events
Houben 1994	Case series of 15 IBD patients (Crohn's disease n=13, UC n=2) treated with intramuscular methotrexate 25 mg/week for 12 weeks, followed by a tapering oral dose One patient was treated twice Disease activity was determined after 1, 2 and 3 months of treatment	The mean defecation frequency went down from 7 to 2 times daily after 12 weeks and prednisone dose could be lowered from 22 mg to 15 mg after 3 months Subjective and objective improvement was noted in 12/15 patients No serious adverse events were reported
Khan 2013	Retrospective cohort study using the nationwide Veterans Affairs database to describe the efficacy of methotrexate in achieving steroid‐free remission Ninety‐one patients with UC were included and they were followed for 15 months after methotrexate initiation by tracking prednisone, methotrexate, thiopurines, and infliximab dispensing records Endpoints were: 1) successful remission (cessation of prednisone filling activity while continuing methotrexate); 2) failure with continuance, failure to be weaned off steroids while continuing methotrexate; 3) failure with discontinuance, cessation of methotrexate while continuing steroids	The average weekly prescription dose for oral and parenteral methotrexate was 14 mg/week (range: 2.3‐31.25) and 25 mg/week (range: 5.8‐70), respectively The mean daily prescription dose for oral prednisone within the oral methotrexate group was 12 mg/day (range 0.7‐68 mg/day) and 25 mg/day (range: 5‐113 mg/day) in the parenteral methotrexate group At the twelfth month, 37% of patients on oral methotrexate and 30% of patients on parenteral methotrexate were able to discontinue steroids
Kozarek 1989	Open label clinical trial including 21 patients with refractory IBD (Crohn's n=14, UC n=7) Patients received intramuscular methotrexate 25 mg/week for 12 weeks After 12 weeks, patients were switched to a tapering oral dose if clinical and objective improvement was noted	Five of 7 UC patients had an objective response as measured by the Ulcerative Colitis Activity Index (13.3 to 6.3, P=0.007) Prednisone dosage decreased from 38.6 mg +/‐ 6.35 (SEM) to 12.9 mg +/‐ 3.4, P=0.01 Five of 7 had histological improvement. None of the UC patients had normal flexible sigmoidoscopy results. Adverse events included mild rises in transaminase levels in 2 patients, transient leukopenia in 1, self‐limited diarrhea and nausea in 2 patients, brittle nails (1 case) and atypical pneumonitis (1 case).
Kozarek 1992	Retrospective chart review, over a 4 year period (1987 to 1991) 86 patients with refractory IBD (Crohn's n=37, UC n=30) were started on 25 mg/week parenteral methotrexate Those patients who responded clinically at 12 weeks were offered weekly oral methotrexate therapy (7.5 to 15 mg) Outcomes included the DAI (scored 0 to 15), prednisone dose, and Mtx toxicity	Seventy per cent of UC patients had a symptomatic and objective response At a mean follow‐up of 59 weeks, only 40% of UC patients continued to respond to Mtx (DAI 5.0 +/‐ 0.9; prednisone 12 +/‐ 3.9 mg), 15 of 30 UC patients required colectomy and one patient stopped methotrexate due to hypersensitivity pneumonitis
Mañosa 2011	Retrospective chart review to evaluate the efficacy and safety of methotrexate in UC patients Patients were included in the study if they received methotrexate for steroid dependency or steroid refractoriness and for maintenance of remission Forty patients were identified from databases of 8 Spanish IBD referral hospitals and followed for at least 6 months Therapeutic success was defined as the absence of UC‐related symptoms, complete steroid withdrawal and no‐requirement of rescue therapies within the first 6 months after starting methotrexate	At 6 months, 45% (18/40) achieved therapeutic success Treatment failure were mainly due to inefficacy (11/22, 50%) or intolerance (8/22, 36%) After a median follow‐up of 28 months. 38% (7) of patients with initial therapeutic success required new steroid courses, 22% (4) started biological therapy and 1 of them required colectomy The cumulative probability of maintaining steroid‐free clinical remission was 60%, 48%, and 35% at 6, 12, 24 months after starting methotrexate, respectively In all, 11 out of 40 patients (27.5%) experienced adverse effects, leading to methotrexate discontinuation in 8 patients
Paoluzi 2002	Open label clinical trial including 42 patients with steroid dependent or steroid resistant active UC Patients were treated with a daily dose of azathioprine (2 mg/kg) and, if intolerant or not responding, with intramuscular methotrexate (12.5 mg/week) Efficacy was assessed by clinical, endoscopic and histological examinations at 6 months Patients achieving clinical remission continued with treatment and were followed up Ten patients received methotrexate The achievement of complete remission with the ability to discontinue oral steroids was defined as the primary outcome Response to treatment was defined as follows: complete remission equals achievement of clinical, endoscopic and histological remission; improvement equals disappearance of symptoms (clinical remission) with endoscopic and histological improvement of inflammatory changes; failure equals worsening, no benefit or clinical improvement with the persistence of unmodified inflammatory changes of the mucosa	Methotrexate induced complete remission in six patients (60%) and improvement in four (40%) During follow‐up, a larger number of patients on azathioprine relapsed in comparison with patients on methotrexate [16/28 (57%) vs. 2/10 (20%), respectively; P < 0.05]
Richter 2012	Retrospective study using a large U.S. health insurance database to document treatment of new‐onset ulcerative colitis (UC) and ulcerative proctitis (UP) in routine clinical practice One thousand five hundred and sixteen UC patients and 636 UP patients were included in the analysis New‐onset UC or UP were identified based on: 1) initial receipt of an oral 5‐ASA, mesalazine suppository, 5‐ASA enema, steroid, antimetabolite, budesonide or TNF inhibitor; 2) sigmoidoscopy/colonoscopy in prior 30 days resulting in a new diagnosis of UC or UP and 3) no prior encounters for Crohn's disease	In UC, initial therapies most frequently used were oral 5‐ASAs (53%), oral 5‐ASAs and systemic steroids (12%), systemic steroids (8%) and mesalazine suppositories (6%); in UP, mesalazine suppositories (42%) and oral 5‐ASAs (19%), combination therapy (14%), mesalazine enema (11%) and rectal steroids (10%) were the mostly frequently used therapies Few patients received maintenance therapy, and there was a limited use of antimetabolites (0.3% in UC, and lower in UP ‐ no specific figures were provided) and biological agents (0.1% in UC)
Saibeni 2012	Retrospective, observational study using 5420 case histories from 8 referral centres in Italy, to evaluate frequency, indications, efficacy and safety of methotrexate in IBD patients One hundred and twelve patients received methotrexate (2.1%, 89 Crohn's disease, 23 ulcerative colitis)	Indications: first‐line immunosuppressant in 32 (28.6%), alternative (second‐line) to thiopurines in 80 (71.4%) Efficacy: optimal in 39/112 (34.8%), partial in 29/112 (25.9%), absent in 22/112 (19.6%), not assessable in 22/122 (19.6%) Side effects happened in 49/112 patients (43.7%, 39 Crohn's disease, 10 ulcerative colitis), leading to drug discontinuation in 38 patients (33.9%) Folic acid use was related to the lower side effects (35/93, 37.6% in those who received folic acid vs. 14/19, 73.7% in those who did not)
Siveke 2003	Case series of 3 patients with steroid dependent or steroid resistant UC Patients were treated with intramuscular methotrexate 25 mg/week These patients received 10 mg of folate orally on the day after injection An additional patient received 15 mg of methotrexate, with the dose being adjusted to 25 mg following increased activity of colitis	Three of 4 patients achieved remission One patient had to discontinue methotrexate due to an increase in aspartate aminotransferase and alanine aminotransferase levels despite dose reduction and prophylactic supplementation of folate
Soon 2004	Retrospective chart review including 72 patients (Crohn's n=66, UC n=6) Patients were treated with mean dose of 18.2 mg/week of methotrexate for six months Methotrexate was given orally in 64 patients and intramuscularly in eight patients Clinical response was defined as sustained withdrawal of oral steroids within 3 months of starting treatment and sustained for a further 3 months or fistula improvement New episodes of steroid therapy, infliximab or surgery during the first 6 months were considered as failure to achieve clinical response	Fifty‐four patients completed six months of treatment Clinical response was achieved in 22 (40.7%) patients [19 of 48 (39.6%) with CD and three of six (50%) with UC]
Te 2000	Retrospective chart review looking at hepatotoxicity among IBD patients who had received a minimum cumulative dose of 1500 mg of methotrexate	In 20 patients who had liver biopsies, the mean cumulative methotrexate dose was 2633 mg (range, 1500–5410 mg), given for a mean of 131.7 wk (range, 66–281 weeks) Nineteen of 20 patients (95%) had mild histological abnormalities (Roenigk’s grade I and II), and one patient had hepatic fibrosis (Roenigk’s grade IIIB)
Wahed 2009	Retrospective chart review to examine the efficacy and safety profile of methotrexate in patients with CD or UC who were either intolerant or non‐responsive to azathioprine/mercaptopurine (AZA/MP) One hundred and thirty‐one patients with IBD treated with MTX were included (99 CD, 32 UC) Clinical response was assessed at 6 months and it was defined as steroid withdrawal, normalization of previously raised CRP or physician's clinical assessment of improvements	In CD, clinical response occurred in 18/29 patients (62%) refractory to AZA/MP and 42/70 patients (60%) intolerant to AZA/MP (P = 1.0) In UC, clinical response occurred in 7/9 patients (78%) refractory to AZA/MP and 15/23 (65%) intolerant to AZA/MP Side effects were seen in 23 (17.4%) patients and led to discontinuation in 11 (8.3%) patients