Aliment Pharmacol Ther 2003; 18: 57–63.
`
`doi: 10.1046/j.0269-2813.2003.01614.x
`
`Bioavailability of oral vs. subcutaneous low-dose methotrexate
`in patients with Crohn’s disease
`
`D. KUR NIK*à, R. L OEBST EIN*à, E. FISH BEI N*à, S . ALMOG *à, H . H ALKI N*à, S. BAR- MEI R à &
`Y. CH OWERS à
`*Division of Clinical Pharmacology and Toxicology, and  Department of Gastroenterology, Chaim Sheba Medical Center, Tel
`Hashomer, Israel; àSackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
`
`Accepted for publication 22 April 2003
`
`SUMMARY
`
`Background: Oral methotrexate and folic acid are partly
`absorbed by a common intestinal transporter.
`Aim: To determine the relative bioavailability of oral
`low-dose methotrexate administered with and without
`concomitant folic acid vs. subcutaneous administration
`in patients with stable Crohn’s disease.
`Methods: Ten patients were randomized to receive their
`regular maintenance dose of methotrexate (15–25 mg)
`for three consecutive weeks: orally, orally with 5 mg
`folic acid or subcutaneously. Blood samples were drawn
`at specified intervals during 24 h, and methotrexate
`levels were determined by fluorescence immunoassay.
`Areas under the curve extrapolated to infinity (AUC¥)
`were compared between the three routes.
`Results: The geometric mean AUC¥ values (95% confid-
`ence intervals) were 360 nmol.h/L (301–430 nmol.h/L),
`
`261 nmol.h/L (214–318 nmol.h/L) and 281 nmol.h/L
`(209–377 nmol.h/L) per milligram of methotrexate
`administered for subcutaneous, oral and oral with folic
`acid
`administration,
`respectively
`(P < 0.05 and
`P < 0.01 for oral with folic acid and oral vs. subcuta-
`neous administration, respectively). The geometric mean
`relative bioavailabilities (95% confidence intervals) were
`0.73 (0.62–0.86) and 0.77 (0.60–0.99) for oral and oral
`with folic acid administration, respectively (difference not
`significant).
`Conclusions: In patients with stable Crohn’s disease, the
`oral bioavailability of methotrexate is highly variable
`and averages 73% of that of subcutaneous administra-
`tion. Concomitant folic acid has no significant effect on
`the bioavailability. Dose adjustments based on individ-
`ual pharmacokinetic assessment should be considered
`when switching patients
`from parenteral
`to oral
`therapy.
`
`INTRODUCTION
`
`Since the first report of its use in inflammatory bowel
`disease,1 a number of controlled2, 3 and uncontrolled4–6
`studies (recently summarized in a literature review7)
`have
`shown
`parenteral
`low-dose methotrexate
`(15–25 mg once weekly) to be effective in inducing
`and maintaining remission in patients suffering from
`
`Correspondence to: Dr Y. Chowers, Department of Gastroenterology, Chaim
`Sheba Medical Center, Tel Hashomer 52621, Israel.
`E-mail: ychowers@post.tau.ac.il
`
`steroid-dependent or steroid-refractory Crohn’s disease.
`However, there is conflicting evidence on the efficacy of
`the convenient oral administration route,8, 9 precluding
`a consensus on whether, how or when to recommend the
`oral administration of methotrexate in clinical practice.
`The discrepancy in efficacy between oral and parenteral
`administration may reflect the incomplete and variable
`oral bioavailability of methotrexate. In patient popula-
`tions without intestinal disease, low-dose methotrexate
`has a mean oral bioavailability of 67–106%.10–12
`To date, no methotrexate bioavailability study has been
`performed in patients with Crohn’s disease.
`
`Ó 2003 Blackwell Publishing Ltd
`
`57
`
`Medac Exhibit 2001
`Koios v. Medac
`IPR2016-01370
`Page 00001
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`

`58
`
`D. KURNIK et al.
`
`In patients with rheumatoid arthritis, daily folate
`supplements reduce the toxicity of weekly low-dose
`methotrexate therapy13 and, accordingly, have been
`recommended in methotrexate-treated patients with
`inflammatory bowel disease.14 Folic acid and methot-
`rexate are structurally similar, and the absorption of
`both substances is at least partly mediated by a common
`membrane transporter
`in the proximal
`intestinal
`mucosa.15 Thus, folic acid could theoretically compro-
`mise methotrexate absorption by competitive inhibition
`of these intestinal carriers.
`We therefore designed a randomized, open-label, cross-
`over study to determine the oral bioavailability of
`low-dose methotrexate (12.5–25 mg) relative to subcu-
`taneous administration in patients with Crohn’s disease,
`and the influence of simultaneous folic acid adminis-
`tration on the absorption of methotrexate.
`
`methotrexate was administered by subcutaneous injec-
`tion (Amp. Abitrexate 50 mg/2 mL, Pharmachemie,
`Teva Group, Netanya, Israel). In schedule B, methot-
`rexate was administered as oral tablets (Tab. Methot-
`rexate 2.5 mg, Lederle, Wolfratshausen, Germany) after
`discontinuing oral
`folic acid for 24 h.
`In schedule
`C, methotrexate was administered as oral
`tablets
`simultaneously with a tablet of folic acid (5 mg, Rekah,
`Azor, Israel). On each treatment day, methotrexate
`was administered in the morning (08.00–09.00 h)
`after an overnight fast, and food was allowed only 2 h
`after drug administration. Other chronic concomitant
`medication
`(steroids,
`infliximab) was
`continued
`unchanged throughout the study. All patients contin-
`ued to receive chronic daily folic acid supplements
`(5 mg/day), except on the day of administration of
`schedule B.
`
`METHODS
`
`Patients
`
`Patients were recruited from the referral-based gastro-
`enterology out-patient clinic of a university-affiliated
`teaching hospital (Chaim Sheba Medical Center, Tel
`Hashomer, Israel) during the period October 2001 to
`June 2002. Patients were eligible if they suffered from
`Crohn’s disease (based on clinical and endoscopic,
`histological or radiographic findings) and had been
`treated with chronic low-dose methotrexate (at con-
`stant doses of 10–25 mg once weekly, orally or
`subcutaneously,
`for at least 3 months) and attained
`significant improvement or remission. Patients were
`excluded if the disease activity was unstable during the
`month preceding the study, as assessed by changes in
`the Crohn’s disease activity index of more than 70
`points,16 or if they had taken antibiotics, non-steroidal
`anti-inflammatory drugs or any other intermittent
`medication during the 2 weeks preceding the study or
`during the study period itself. The study protocol was
`approved by the local institutional review board, and all
`subjects gave written informed consent.
`
`Study design
`
`Patients received their regular once-weekly doses of
`methotrexate according to three administration sched-
`ules over three consecutive weeks in a randomized,
`three-way, cross-over block design.
`In schedule A,
`
`Data collection
`
`the
`for
`On each treatment day, blood samples
`determination of methotrexate serum levels were
`drawn through an indwelling intravenous catheter
`immediately before and at pre-defined time intervals
`(15, 30, 45, 60, 90, 120, 150 min and 4, 6, 8
`and 24 h) after the administration of methotrexate.
`Demographic data and details on the type, duration,
`extent (endoscopic/radiological) and activity of disease
`(as assessed by the Crohn’s disease activity index)
`were collected from patient files and a structured
`interview on the days of treatment. Body weight was
`measured, and routine blood tests
`(blood count,
`biochemistry and serum folate levels) were performed
`on each treatment day.
`
`Measurements
`
`Blood samples were centrifuged and the serum was
`stored at ) 20 °C until assay, which was performed in
`batches within 4 weeks of collection. Methotrexate
`concentrations were measured using a fluorescence
`polarization immunoassay technique
`(Abbott TDx
`Methotrexate Reagent Kit, Abbott Diagnostics, North
`IL, USA).17 The lowest
`Chicago,
`limit of detection
`reported by the manufacturer was 0.01 lmol/L, and
`the inter- and intra-day coefficients of variation were
`4% and 5%, respectively. For each patient, samples
`from the three treatment schedules were analysed in
`the same batch.
`
`Ó 2003 Blackwell Publishing Ltd, Aliment Pharmacol Ther 18, 57–63
`
`Page 00002
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`

`

`BIOAVAILABILITY OF METHOTREXATE IN CROHN’S DISEASE
`
`59
`
`Data analysis
`
`Methotrexate concentration–time profiles were analysed
`using the WinNonlin software package (Pharsight,
`Mountain View, CA, USA). Non-compartmental analy-
`sis was used to estimate the area under the concentra-
`tion–time curve extrapolated to infinity (AUC¥) (based
`on the trapezoidal rule and the terminal phase elimin-
`ation constant kz).18 The relative bioavailabilities Fo
`(oral vs.
`subcutaneous administration) and Fo + f
`(oral + folic acid vs. subcutaneous administration) were
`defined as the ratio of the respective AUC¥ value to the
`subcutaneous AUC¥ value. For subcutaneous adminis-
`tration, the relative clearance (Cl/F) was obtained by
`dividing the dose by the AUC¥ value, and the relative
`terminal phase volume of distribution (Vz/F) was
`obtained by dividing the dose by the AUC¥ value and
`kz.18 The maximal concentration cmax and time to the
`maximal concentration tmax were determined directly
`from the observed data. Creatinine clearance was
`calculated according to the Cockcroft–Gault formula.19
`
`Statistical analysis
`
`Data are summarized and presented as the geometric
`mean (for cmax and AUC) or arithmetic mean (for all
`
`other data) and 95% confidence intervals (95% CI).
`Pharmacokinetic data (logarithmically transformed
`and dose normalized for AUC and cmax) were
`compared between the three treatment schedules by
`one-way repeated measures analysis of variance
`(anova),
`followed by a post hoc Fisher’s least sig-
`nificant difference (LSD) analysis of multiple compar-
`isons. The bioavailabilities for the oral and oral +
`folic acid schedules were compared by the paired
`t-test. All
`tests were two-tailed, and P < 0.05 was
`considered to be significant. All calculations were
`performed using the GB-STAT statistical
`software
`package (Dynamic Microsystems Inc., Silver Spring,
`MD, USA).
`
`Sample size calculation
`
`Defining a difference of 25% between oral and subcu-
`taneous bioavailability as clinically significant
`(i.e.
`requiring the addition of at least one methotrexate
`tablet of 2.5 mg when changing from parenteral to oral
`administration at the usual maintenance doses), and
`assuming a standard deviation of 20% for bioavailabil-
`ity, a sample size of 10 patients was calculated to
`provide a power of 80% (1 ) b) at the usual level of
`statistical significance (a ¼ 0.05).
`
`Table 1. Demographic data and details of the history, treatment and activity of Crohn’s disease
`
`Weight
`(kg)
`
`Methotrexate
`dose (mg)
`
`Year of
`diagnosis
`
`Extent of
`disease
`
`Previous intestinal
`resection
`
`Concomitant
`medication
`
`Patient
`
`Age
`(years)
`
`1
`
`2
`
`3
`4
`5
`
`6
`
`7
`
`8
`9
`
`10
`
`49
`
`32
`
`62
`47
`49
`
`54
`
`46
`
`33
`27
`
`27
`
`Gender
`
`Female
`
`Female
`
`Male
`Female
`Male
`
`Female
`
`52
`
`89
`
`75
`52
`94
`
`63
`
`12.5
`
`25
`
`17.5
`15
`12.5
`
`12.5
`
`Male
`
`52
`
`12.5
`
`Male
`Male
`
`87
`100
`
`Female
`
`64
`
`25
`25
`
`20
`
`1976
`
`Ileum
`
`1991
`
`Ileum
`
`Terminal ileum
`(70 cm)
`—
`
`1982
`1998
`1989
`
`1967
`
`1981
`
`1990
`1999
`
`1992
`
`Colon
`Colon
`Colon
`
`Terminal
`ileum
`
`Terminal
`ileum
`Colon
`Terminal
`ileum
`Ileum,
`jejunum
`and colon
`
`—
`—
`Partial colectomy
`(50 cm)
`Terminal ileum
`(60 cm)
`
`—
`
`—
`—
`
`Terminal ileum
`(40 cm)
`
`—
`
`Budesonide
`(9 mg/day)
`Prednisone
`(20 mg/day),
`infliximab
`—
`—
`Infliximab
`
`Prednisone
`(15 mg/day),
`infliximab
`—
`
`Infliximab
`—
`
`Mean
`CDAI
`
`207
`
`27
`
`157
`129
`147
`
`150
`
`320
`
`35
`63
`
`53
`
`Mean CDAI, average Crohn’s disease activity index over the study period.
`
`Ó 2003 Blackwell Publishing Ltd, Aliment Pharmacol Ther 18, 57–63
`
`Page 00003
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`

`

`60
`
`D. KURNIK et al.
`
`RESULTS
`
`Ten subjects were enrolled. One patient did not
`complete the study for personal reasons, and the data
`on treatment schedule C are therefore incomplete.
`Demographic data and information on the extent and
`activity of Crohn’s disease are presented in Table 1. The
`mean methotrexate dose was 17.8 mg (95% CI,
`13.8–21.7 mg;
`range, 12.5–25 mg). The mean
`creatinine
`clearance was 90.3 mL/min (95% CI,
`69.8–110.8 mL/min;
`range, 65–142 mL/min) and
`the mean folate level was 11.9 ng/dL (95% CI,
`8.3–15.5 ng/dL; range, 3.1–17.6 ng/dL). One patient
`with low baseline folate levels at the beginning of the
`study admitted low compliance to folate supplements,
`but his folate levels normalized during the study with
`regular folic acid administration. The disease activity, as
`assessed by the Crohn’s disease activity index, did not
`change significantly (> 70) during the study period in
`any of
`the patients. Concomitant medication was
`continued unchanged throughout the study. None of
`the four patients treated with infliximab (administered
`at a dose of 5 mg/kg every 2 months) received the drug
`during the 3-week study period. No adverse effects of
`methotrexate were recorded.
`The results of
`the pharmacokinetic analyses are
`presented in Table 2. Oral administration, both with
`and without folic acid, resulted in significantly lower
`AUC0–¥ values when compared with the subcutaneous
`
`route (P < 0.05 and P < 0.01, respectively). The mean
`relative bioavailability was similar for the oral admin-
`istration schedules, both without and with folic acid,
`with a non-significant trend towards a larger between-
`patient variability for the latter (coefficients of variation
`for Fo and Fo + f of 0.23 and 0.33, respectively) (Table 2
`and Figure 1). There was a non-significant
`trend
`towards lower bioavailability in the five patients taking
`a higher dose (20–25 mg) when compared with the
`four patients taking a lower dose (12.5–15 mg) [0.62
`(95% CI, 0.38–0.97) vs. 0.80 (95% CI, 0.65–1.0),
`respectively].
`tmax was significantly longer in the oral regimens and
`longest when methotrexate was administered with folic
`acid [mean tmax (95% CI): 1.53 h (1.08–1.97 h) and
`1.71 h (1.19–2.24 h) for oral administration without
`and with folic acid, respectively, vs. 0.91 h (0.72–
`1.09 h) for subcutaneous administration; P < 0.05 and
`P < 0.01, respectively], although the difference be-
`tween the two oral regimens was not significant. The
`mean dose-corrected cmax values were similar in all the
`administration schedules [geometric mean (95% CI) of
`50 nmol/L
`(41–61 nmol/L),
`cmax:
`dose-corrected
`48 nmol/L
`(33–71 nmol/L)
`and
`51 nmol/L
`(34–75 nmol/L) per milligram dose for subcutaneous,
`oral and oral + folic acid administration, respectively].
`Other pharmacokinetic parameters [for subcutaneous
`schedule: Vz/FÆkg, 0.49 L/kg (95% CI, 0.38–0.63 L/kg);
`Cl/FÆkg, 1.45 mL/min.kg (95% CI, 1.12–1.86 mL/min.kg);
`
`Table 2. Comparison of pharmacokinetic parameters by administration route in individual patients
`
`Subcutaneous
`
`Oral
`
`AUC0–¥
`per mg Mtx
`(nmol.h/L)
`
`AUC0–¥
`per mg Mtx
`(nmol.h/L)
`
`405
`245
`376
`394
`417
`368
`309
`604
`280
`308
`360 (301–430)
`
`297
`232
`281
`255
`390
`366
`232
`293
`153
`200
`261  (214–318)
`
`Fo
`
`0.73
`0.94
`0.75
`0.65
`0.94
`0.99
`0.75
`0.49
`0.55
`0.65
`0.73  (0.62–0.86)
`
`Oral + folic acid
`
`AUC0–¥
`per mg Mtx
`(nmol.h/L)
`
`279
`148
`414
`260
`540
`312
`287
`268
`186
`NA
`281* (209–377)
`
`Fo + f
`
`0.69
`0.60
`1.10
`0.66
`1.30
`0.85
`0.93
`0.45
`0.67
`NA
`0.77* (0.60–0.99)
`
`Patient
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`Geometric mean (95% CI)
`
`AUC0–¥, area under the curve from zero time to infinity; CI, confidence interval; F, relative bioavailability; Mtx, methotrexate; NA, not assessed.
`* P < 0.05,   P < 0.01 for comparison with subcutaneous administration using repeated measures analysis of variance (anova) with Fisher’s least
`significant difference for multiple comparisons (performed on logarithmically transformed data for AUCs). One patient (10) did not complete the study.
`
`Ó 2003 Blackwell Publishing Ltd, Aliment Pharmacol Ther 18, 57–63
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`BIOAVAILABILITY OF METHOTREXATE IN CROHN’S DISEASE
`
`61
`
`to 10 mg12, 23, 24 and 0.67–0.92 at doses up to
`25 mg.10–12, 25, 26 However, differences in the study
`protocols (e.g. methotrexate assay, dose, parenteral
`reference route) render direct comparisons of these
`studies problematic. Our study was not designed to
`compare methotrexate
`bioavailability
`in different
`patient populations and, in view of the wide between-
`patient variability, a larger comparative study is
`required to rule out meaningful differences. Only one
`small Israeli study has directly compared AUC values
`up to 2 h (AUC0)2) after the oral administration of
`low-dose methotrexate (12.5 mg) in five patients with
`Crohn’s disease,
`four patients with ulcerative colitis
`and six patients with rheumatoid arthritis.27 As there
`was no parenteral reference, the bioavailability could
`not be assessed, but
`the peak concentrations and
`AUC0)2 values did not differ significantly between the
`patient groups. However,
`this
`study was under-
`powered to detect differences in AUC values as large
`as 30%, and the short observation period of 2 h (just
`beyond the mean time to the peak concentration)
`may not have adequately reflected systemic drug
`exposure.
`An inverse relation between dose and oral bioavaila-
`bility, as described previously for intermediate and high
`doses,28 also seems to exist within the low-dose range
`(7.5–25 mg).10–12, 23–26 We noted an overall non-
`significant
`trend towards
`lower bioavailability in
`patients taking higher (20–25 mg) rather than lower
`(12.5–15 mg) doses, but this study was not designed or
`powered to address this question.
`The range (0.49–0.99) and between-patient variability
`(coefficient of variation, 0.23) of
`the relative oral
`bioavailability in our study were similar to those derived
`in other studies. In individual patients, the relative oral
`bioavailability of
`low-dose methotrexate ranges from
`0.28 to 1.5, with coefficients of variation between
`0.15% and 0.31%.10–12, 23, 25 Differences in age, fasting
`state, dose, drug interactions (non-steroidal anti-inflam-
`matory drugs, ciprofloxacin) and, possibly, the expression
`of cellular drug efflux transporters (multi-drug resistance
`proteins MRP1 and MRP3) may account for such inter-
`individual differences.20, 29 This
`two-
`to five-fold
`between-patient variability precludes precise forecasts
`for the individual patient. In contrast, within-patient
`variability is comparatively low (< 20%).30, 31 Thus, the
`routine assessment of individual bioavailability has been
`suggested for the individualization of oral methotrexate
`therapy. For this purpose, AUC values can be estimated
`
`1.4
`
`1.2
`
`1.0
`
`.8
`
`.6
`
`.4
`
`.2
`
`0.0
`
`Relative bioavailability
`
`Oral
`
`Oral +
`folic acid
`
`Figure 1. Relative oral methotrexate bioavailability after oral
`administration with and without folic acid. Data are shown for
`nine patients who completed both administration schedules. The
`bold horizontal lines represent the geometric means.
`
`terminal phase half-life, 3.88 h (95% CI, 3.56–4.23 h)]
`did not differ significantly between the three schedules
`when corrected for F (data not shown), and were all
`within the previously published range.20
`
`DISCUSSION
`
`Our study is the first to assess the oral bioavailability of
`methotrexate in patients with Crohn’s disease. We
`chose to examine the relative oral bioavailability
`compared with subcutaneous administration, the stand-
`ard of care for parenteral low-dose methotrexate, thus
`addressing the need for dose adjustment when switch-
`ing from parenteral to oral treatment. The relative oral
`bioavailability compared with subcutaneous adminis-
`tration for doses between 12.5 and 25 mg was 0.73
`(95% CI, 0.62–0.86). As both subcutaneous and
`intramuscular administration have an absolute sys-
`temic bioavailability of 0.90–1.0 when compared with
`intravenous administration,11, 21, 22
`the
`calculated
`mean absolute oral bioavailability in our patients was
`0.66–0.73.
`Our results in Crohn’s disease patients largely concur
`with those derived from patients without small bowel
`affection, e.g. with rheumatoid arthritis, psoriasis and
`asthma.
`In these patients,
`the mean relative oral
`bioavailability was in the range 0.93–1.06 at doses up
`
`Ó 2003 Blackwell Publishing Ltd, Aliment Pharmacol Ther 18, 57–63
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`

`62
`
`D. KURNIK et al.
`
`from a limited sampling protocol (two to three plasma
`samples).30, 32 Accordingly, a single limited sampling
`pharmacokinetic study may be warranted when trans-
`ferring a patient from parenteral to oral
`long-term
`methotrexate maintenance therapy. If such an assess-
`ment is not feasible, the parenteral route is preferred in
`view of the unpredictable individual oral bioavailability,
`especially at the higher methotrexate doses typically used
`for the induction of remission.
`Folic acid and methotrexate are structurally similar
`and are partially absorbed by common membrane
`transporters;
`therefore, co-administration may affect
`their absorption.15 Patients on chronic low-dose met-
`hotrexate generally receive daily folic acid supplements
`to reduce the adverse effects of methotrexate. Therefore,
`we sought to examine whether the timing of the daily
`folic acid supplement with respect
`to methotrexate
`administration affected the bioavailability of methotrex-
`ate. In our patients, the mean oral bioavailability was
`not
`significantly affected when methotrexate was
`administered simultaneously with folic acid or 24 h
`after the last folic acid administration. However, this
`study was not powered to detect smaller, clinically less
`significant differences in bioavailability, and the results
`cannot be extrapolated to patients not consuming daily
`folic acid supplements.
`Owing to the small number of patients in our study, no
`attempt was made to examine the influence of the
`activity and extent of Crohn’s disease on the oral
`bioavailability of methotrexate. Most of our patients
`were in remission (as defined by a Crohn’s disease
`activity index of < 150),16 and further studies may be
`necessary to investigate the bioavailability of methot-
`rexate in patients with active Crohn’s disease.
`
`CONCLUSIONS
`
`In patients with stable Crohn’s disease on chronic
`weekly low-dose methotrexate therapy, the relative oral
`methotrexate bioavailability (compared to subcuta-
`neous administration) is highly variable and averages
`0.73, well within the range of that in patients without
`bowel affection. Simultaneous folic acid administration
`does not affect the mean systemic bioavailability. Dose
`adjustment
`should be considered when switching
`patients from parenteral to oral therapy if the same
`systemic exposure is to be achieved. In view of the
`considerable inter-individual variability of
`the oral
`bioavailability, an individual pharmacokinetic study
`
`based on a limited sampling strategy would be helpful to
`individualize such dose adjustments.
`
`REFERENCES
`
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`2 Feagan BG, Rochon J, Fedorak RN, et al. Methotrexate for
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`292–7.
`3 Feagan BG, Fedorak RN, Irving EJ, et al. A comparison of
`methotrexate with placebo for the maintenance of remission
`in Crohn’s disease. N Engl J Med 2000; 342: 1627–32.
`4 Chong RY, Hanauer SB, Cohen RD. Efficacy of parenteral
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