Patients

We enrolled patients (18 to 75 years of age) who had moderate-to-severe Crohn’s disease (CDAI score of 220 to 400; scores can range from 0 to 600, with higher scores indicating greater disease activity) 1 week or more before enrollment,4 with inflammatory lesions in the terminal ileum, right colon, or both, as documented with the use of ileocolonoscopy and contrast ultrasonography of the small intestine, magnetic resonance enterography, or computed tomographic enterography within 1 year before enrollment, and had steroid-dependent or glucocorticoid-resistant disease, as defined by guidelines of the European Crohn’s and Colitis Organization.13

Because the active compound of mongersen is released in the terminal ileum and right (proximal) colon, we excluded patients with known lesions in the stomach, proximal small intestine, transverse colon, or left colon. Patients were also excluded if they had strictures, fistulae, perianal disease, extraintestinal symptoms, active or recent infections, or a history of cancer.

Patients could continue to receive stable doses of oral prednisolone (≤40 mg per day), budesonide (≤9 mg per day), or mesalamine during the 2-week treatment period; they could also receive a stable dose of immunomodulators (e.g., azathioprine, mercaptopurine, or methotrexate) if therapy had been initiated 6 or more months before initiation of the study treatment. Antibiotic agents, glucocorticoids, immunosuppressive drugs, and biologics could not be initiated before study entry or during the 2-week treatment period.

Patients received no treatment with anti–TNF-α antibodies or other biologic agents within 90 days or antibiotics within 3 weeks before the date of their enrollment in the trial. Female participants used two forms of contraception throughout the study. We excluded women who were pregnant or breast-feeding, as well as persons with previous proctocolectomy or intestinal resection resulting in the short-bowel syndrome and persons with a clinically significant abnormality on electrocardiography or laboratory testing. Patients who had worsening of disease (increase of ≥70 points in the CDAI score) could receive rescue therapy with biologic agents, immunosuppressive drugs, or both after the 2-week treatment period, and for those who were in clinical remission after the 2-week treatment period (CDAI score <150 at both day 15 and day 28), glucocorticoids could be tapered.

The study protocol was approved by the institutional review board or ethics committee at each of the 17 study centers in Italy and Germany. Written informed consent was obtained from patients before they underwent screening for eligibility (Table S1 in the Supplementary Appendix). Eligible patients underwent randomization between September 2011 and June 2013.

Mongersen

Mongersen is a 21-base oligonucleotide with the sequence 5′-GTC GCC CCT TCT CCC CGC AGC-3′. The phosphorothioate chemistry consists of replacement of a nonbonding oxygen with a sulfur atom in each of the internucleotide linkages. The cytosine residues at nucleotide positions 3 and 16 are modified by 5-methylation.

Study Design

In this multicenter, randomized, placebo-controlled, double-blind, phase 2 clinical trial, patients were randomly assigned to receive one of three doses of mongersen (10, 40, or 160 mg per day) or placebo in a 1:1:1:1 ratio by means of a computer-generated randomization schedule without stratification or block allocation. The placebo and active drug were identical in appearance and taste. The three doses of mongersen and the treatment duration were selected on the basis of preclinical, toxicologic, and phase 1 studies.9,11 Patients received treatment daily for 2 weeks and were evaluated at days 15, 28, and 84.

The study was sponsored by Giuliani, acting under contract to Nogra Pharma; employees of Giuliani collected and had access to the data, participated in its analysis, and participated in discussions about its interpretation. The study was designed by the first author, who also wrote the first draft of the manuscript; all authors contributed equally to the gathering and analysis of data, and each author had access to the full data set. Editorial assistance with the preparation of the manuscript for resubmission was received from an editor (from Precise Publications) and from Peloton Advantage; both were supported by Celgene, which had no additional role in the study. The authors vouch that the study was conducted in accordance with the protocol and statistical analysis plan, both of which are available at NEJM.org. Each author vouches for the accuracy and completeness of the reported data, and each author agreed with the decision to submit the final version for publication.

Efficacy and Safety Assessment

The primary end point of the study was the percentage of patients who were in remission at day 15 (defined as a CDAI score of <150) and who remained in remission for at least 2 weeks.14 Evaluation of the safety of mongersen treatment was another objective. Clinical, biochemical, and hematologic variables were assessed on days −7, 1, 15, 28, and 84. An enzyme-linked immunosorbent assay (ELISA) was used to monitor the patients for complement activation (a side effect of systemic antisense exposure15). The severity of adverse events and their cause (study drug or procedure) were determined.

Secondary end points included the rates of clinical response, defined as a decrease in the CDAI score of 100 points or more or a decrease of 70 points or more, at days 15 and 28, as well as the percentages of patients with a CDAI score of less than 150 at days 15, 28, and 84. We also assessed the CDAI score before treatment and during the week preceding days 15, 28, and 84, as well as changes in median CDAI scores from baseline to each time point. Percentages of patients who had normalization of C-reactive protein after treatment, had elevated C-reactive protein levels at baseline and reached clinical remission, and were in glucocorticoid-free remission at day 84 were also evaluated. Additional end points included changes in plasma levels of proinflammatory cytokines (e.g., interleukin-8 and TNF-α), measured with the use of commercial ELISA kits (R&D Systems).

Statistical Analysis

Sample size was determined with the use of a one-sided testing framework with an alpha error of 0.1 and a beta error of 0.1, because the prospective primary hypothesis was that 14 days of treatment with the highest mongersen doses (40 and 160 mg per day) would result in a higher proportion of patients in clinical remission than would 14 days of placebo treatment and that the lowest dose of mongersen (10 mg per day) would not be effective. Rates of remission were assumed to be 50% with the highest mongersen doses and 20% with placebo. It was estimated that 40 patients per group would be needed for the study to have at least 90% power to detect significant differences in remission rates between one of the two groups treated with the highest mongersen doses and the placebo group. All efficacy analyses were conducted according to the intention-to-treat principle with all patients who underwent randomization and who received at least one dose of study medication. Patients with missing primary end-point data at day 15 or week 4 were classified as not having a response. Patients who received rescue therapy because of worsening of disease after day 28 were classified as not having a response for the secondary end-point analyses. Missing data for continuous end points and secondary variables were imputed with the use of the last-observation-carried-forward method.

The primary efficacy and adverse-event analyses included all 166 randomly assigned and treated patients. Remission and response rates were compared with the use of Pearson’s chi-square test or, when not applicable, with Fisher’s exact test. Although sample size was determined with the use of a one-sided testing framework, we present results in the conventional two-sided framework. We compared each of the three treatment groups with the placebo group and each group treated with either 160 or 40 mg per day of the drug with the 10-mg dose group.

Demographic characteristics, rates of adverse events, and proportions of patients who discontinued glucocorticoid treatment were compared with the use of descriptive methods. Proportions of patients with changes from baseline in the C-reactive protein level were also documented. Changes in median CDAI scores and cytokine levels from baseline to each time point were analyzed with the Mann–Whitney test and Student’s t-test, respectively.