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nature publishing group

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Newly Developed Antibiotic Combination Therapy for Ulcerative Colitis: A Double-Blind Placebo-Controlled Multicenter Trial
Toshifumi Ohkusa, MD, PhD1, Kimitoshi Kato, MD, PhD2, Shuichi Terao, MD3, Toshimi Chiba, MD, PhD4, Katsuhiro Mabe, MD, PhD5, Kazunari Murakami, MD, PhD6, Yuji Mizokami, MD, PhD7, Toshiro Sugiyama, MD, PhD8, Akinori Yanaka, MD, PhD9, Yoshiaki Takeuchi, MD, PhD10, Shigeru Yamato, MD, PhD11, Tetsuji Yokoyama, MD, PhD12, Isao Okayasu, MD, PhD13, Sumio Watanabe, MD, PhD14, Hisao Tajiri, MD, PhD1 and Nobuhiro Sato, MD, PhD14, From the Japan UC Antibiotic Therapy Study Group OBJECTIVES:

Fusobacterium varium may contribute to ulcerative colitis (UC). We conducted a double-blind placebo-controlled multicenter trial to determine whether antibiotic combination therapy induces and/or maintains remission of active UC. Patients with chronic mild-to-severe relapsing UC were randomly assigned to oral amoxicillin 1500 mg/day, tetracycline 1500 mg/day, and metronidazole 750 mg/day, vs. placebo, for 2 weeks, and then followed up. The primary study end point was clinical response (Mayo score at 3 months after treatment completion) and secondary end points were clinical and endoscopic score improvements at 12 months. Anti-F. varium antibodies were measured by enzyme-linked immunosorbent assay. Treatment and placebo groups each had 105 subjects. At the primary end point, response rates were signicantly greater with antibiotics than with placebo (44.8 vs. 22.8%, P = 0.0011). Endoscopic scores signicantly improved at 3 months (P = 0.002 vs. placebo). Remission rates were 19.0% (antibiotics) vs. 15.8% (placebo) at 3 months (P = 0.59). At the secondary end point, response rates were signicantly greater with antibiotics than with placebo (49.5 vs. 21.8%, respectively, P < 0.0001). Endoscopic scores were signicantly improved at 12 months after antibiotic treatment (P = 0.002 vs. placebo). Remission rates had improved to 26.7% with antibiotics vs. 14.9% for placebo, at 12 months (P = 0.041). F. varium antibody titers decreased in responders but not in nonresponders, and more in the antibiotic than in the placebo group. More pretreatment steroid-dependent UC patients discontinued corticosteroids after treatment completion (6 months: 28.6 vs. 11.8%, respectively, P = 0.046; 9 months: 34.7 vs. 13.7%, respectively, P = 0.019; and 12 months: 34.7 vs. 13.7%, respectively, P = 0.019). These effects were greater in the subanalysis of the active group (Mayo scores of 612) than in that of total cases (012). No serious drug-related toxicities occurred. active UC more effectively than a placebo.

METHODS:

RESULTS:

CONCLUSIONS: The 2-week triple antibiotic therapy produced improvement, remission, and steroid withdrawal in
Am J Gastroenterol 2010; 105:18201829; doi:10.1038/ajg.2010.84; published online 9 March 2010

1 Department of Gastroenterology and Hepatology, Jikei University, Tokyo, Japan; 2Department of Gastroenterology and Hepatology, Nihon University, Tokyo, Japan; 3Kyoto Min-Iren Chuo Hospital, Kyoto, Japan; 4First Department of Internal Medicine, Iwate Medical University, Iwate, Japan; 5Division of Internal Medicine, Yamagata Prefectural Central Hospital, Yamagata, Japan; 6Department of Gastroenterology, Oita University, Oita, Japan; 7Department of Internal Medicine 5, Tokyo Medical University, Ibaraki, Japan; 8Third Department of Internal Medicine, Toyama University, Toyama, Japan; 9Division of Gastroenterology, Tsukuba University, Ibaragi, Japan; 10Second Department of Internal Medicine, Showa University, Tokyo, Japan; 11Division of Gastroenterology, International Medical Center of Japan, Tokyo, Japan; 12Department of Technology Assessment and Biostatistics, National Institute of Public Health, Saitama, Japan; 13 Department of Pathology, Kitasato University, Kanagawa, Japan; 14Department of Gastroenterology, Juntendo University, Tokyo, Japan. Correspondence: Toshifumi Ohkusa, MD, PhD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kashiwa Hospital, Jikei University School of Medicine, 163-1 Kashiwashita, Kashiwa, Chiba 277-8567, Japan. E-mail: ohkusa@jikei.ac.jp Received 26 September 2009; accepted 20 January 2010

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INTRODUCTION
Evidence supporting a pathophysiological role for luminal bacteria in mucosal inflammation has been obtained from models such as non-germ-free, cytokine (interleukin -2 or -10)-knockout and other related gene-knockout mice that developed colitis (18). Recently, it was reported that a novel mouse line with defects in both transforming growth factor- type II receptor and interleukin-10 receptor 2 signaling, as well as the T-bet//RAG2/ mouse model, rapidly and reproducibly developed a disease resembling severe fulminant human ulcerative colitis (UC) (9,10). These disease processes were completely inhibited by a combination of broad-spectrum antibiotics. Microbial agents have long been implicated in the initiation and/or exacerbation of UC in humans, suggesting a possible rationale for antibiotic treatment of UC. Although antibiotic therapy is supported by both clinical and experimental evidence, antibacterial therapy trials have produced conflicting results (1120). In 2002, Fusobacterium varium was reported to be present in the colonic mucosa of a high proportion (84%) of UC patients (21). Butyric acid, a product of F. varium culture supernatants, was also shown to cause UC-like lesions in mice (22). On the basis of these observations, we used an antibiotic combination regimen to which F. varium was susceptible (amoxicillin, tetracycline, and metronidazole (ATM)) and showed in a randomized controlled pilot study that this regimen had significant efficacy in active UC (23,24). We now present the results of a double-blind placebocontrolled multicenter trial of this combination anti-F. varium therapy designed to assess whether ATM induces and maintains remission of active UC.

Study design

METHODS
Study subjects

A multicenter, randomized, double-blind, placebo-controlled study was conducted between January 2004 and July 2006 at 11 hospitals in Japan. This trial was registered at http://www. umin.ac.jp/ctr/index-j.htm:UMIN000000078. The institutional review board or ethics committee at each facility approved the protocol. All subjects gave written informed consent. All eligible patients had an established diagnosis of UC. Study subjects were selected from among patients with chronic relapsing UC (at least one relapse per year) with more than 1 year of follow-up and who regularly visited outpatient clinics. Eligibility criteria for study entry were mild-to-severe disease (11) with a colonoscopy score of at least 1 (erythema, decreased vascular pattern, and mild friability) based on a scale of 0 (normal or inactive) to 3 (spontaneous bleeding and ulceration) (25), and/or watery diarrhea at least 5 times/day with visible blood in stools. Patients with toxic megacolon or penicillin allergy, who were pregnant, or who had serious liver or renal disease or any psychological illness were excluded. Patients who had taken antibiotics within 4 weeks before study entry or had Clostridium difficile or other stool pathogens (Salmonellae, pathogenic Escherichia coli, Campylobacter jejuni, Shigellae, etc.) at entry were also excluded.
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Patients were randomly assigned to either the ATM or the control group. The study used central randomization with a treatment allocation produced by a computer-generated randomization list. Randomization was carried out using numbered containers. The ATM group received a 2-week combination therapy consisting of amoxicillin 500 mg t.i.d, tetracycline 500 mg t.i.d, and metronidazole 250 mg t.i.d. These antibiotics were selected on the basis of F. varium susceptibility test results (23). The controls received three placebo preparations consisting of identical-appearing capsules and tablets containing sugar. Patients received identical drugs in identical bottles and remained blinded throughout the study. All clinical evaluations and endoscopies were carried out by clinicians who were blinded to the patients therapy. Compliance and adverse events were checked after 2 weeks of combination therapy. Doses of any concomitant oral or rectal medication for UC remained constant, except for corticosteroids, which were tapered after week 8 by 5 mg/week until it reached a dose of 20 mg/day. Thereafter, the dose was reduced by 2.5 mg/week until discontinuation. Corticosteroid discontinuation for more than 3 months in steroid-dependent UC was defined as steroid withdrawal. Steroid withdrawal rates at 6, 9, and 12 months after treatment completion were calculated. Patients came to the hospital weekly or monthly, depending on their personal schedules and severity of their symptoms, and were assessed by clinical examination and total colonoscopy at trial start, and again at 3 and 12 months after treatment completion. Stool specimens were cultured for ordinary pathogens (C. difficile, Salmonellae, pathogenic E. coli, C. jejuni, Shigellae, etc.) at the start of the trial, 2 weeks after starting ATM or placebo, and 3 months after treatment completion. The study was originally approved for 12 months and patients continued to be followed up for up to 12 months after completion of treatment. The blinding was not broken until the entire study was completed. Patients who stopped participating in the study included those with relapse, lack of efficacy, loss of responsiveness to ATM, and those without relapse who elected to discontinue the study. For patients who did not complete the 12-month follow-up, the data at 12 months were imputed by the last-observation-carried-forward method.
Clinical scoring, endoscopic scoring, and denitions of response, remission, and relapse

At each assessment, patients completed a symptom questionnaire and underwent clinical examinations to determine Mayo scores (25). Mayo scores provide an assessment of disease activity on the basis of a combination of symptoms, signs, and sigmoidoscopic findings, with scores ranging from 0 to 12. The initial endoscopy was performed no more than 2 weeks before starting therapy, with follow-up, including total colonoscopy, and biopsy at 3 and 12 months after treatment completion. After total colonoscopy, the endoscopic findings of the sigmoid colon and rectum were evaluated according to the Mayo system, with scores of 03. Zero represents normal findings or an appearance consistent with inactive UC. In all three scoring systems, a higher score indicates more severe disease.
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For the Mayo system, clinical response was defined as a decrease from baseline in the total score of at least 3 points (26) if the baseline score was > 3 points, or a decrease of 2 points if the baseline disease activity index was 3, or a decrease of 1 point if the baseline disease activity index was 1 or 2. Clinical remission was defined as a total score of 2 points or lower with no individual subscore exceeding 1 point (27). Clinical relapse was defined as reappearance of visible blood in stools for two consecutive days and/or recurrence of frequent diarrhea (five or more bowel movements/day), nocturnal diarrhea, or abdominal cramps. If a patient relapsed or showed exacerbation with severe or fulminant UC symptoms, study participation was stopped and the patient was treated appropriately.
Bacterial assessment

relapsed, and/or were lost to follow-up. The proportions showing a clinical response at 3 months (primary end point) in the ATM and placebo groups were calculated, and the difference was statistically tested using Fishers exact test. For analyses of secondary end points, changes in clinical endoscopic scores at 12 months (x12) from the baseline score (xb) were calculated as d12 = x12xb. The data for d12 are summarized as mean and percentile values, and the d12 difference between the ATM and placebo groups was tested using the MannWhitney U-test. Differences with two-sided P < 0.05 were considered statistically significant. SAS version 9.1 and STAT VIEW software version J 5.1 (SAS Institute, Cary, NC) were used for all analyses.

RESULTS
Baseline characteristics

At study entry, and at 3 and 12 months, serum titers of immunoglobulins (IgG, IgM, and IgA) to F. varium were measured by enzyme-linked immunosorbent assay, performed blinded according to previously described methods (21,23). The primary antibodies (Protein Purify, Maebashi, Japan) for the standard solution were from rabbits immunized with a whole-cell preparation of F. varium. Dilutions of 6.25, 12.5, 25, 50, and 100 were performed to calculate the standard curve, and the optical density of dilution was defined as 1 unit/ml. Thus, 5 units/ml was equivalent to the optical density cutoff value of 0.25 (21), such that 5 units/ml was defined as positive for F. varium infection.
Study end points

The distributions of age, sex, disease duration, disease severity, extent of disease, concomitant medication, rates of corticosteroid-refractory and -dependent disease, and Mayo scores were well balanced between the two groups (Table 1). Out of 101 controls, 10 (9.9%) and out of 105 ATM patients, 12 (11.4%) were receiving long-term sulfasalazine or steroid therapy by enema at the time of recruitment.
Primary end point

The primary study end point was a clinical response documented by the Mayo score at 3 months after treatment completion; secondary end points were clinical and endoscopic score improvements at 12 months.
Subset analysis

Subset analyses with respect to the severity of disease at entry were carried out at the primary and secondary end points.
Statistical analysis

In our randomized controlled pilot trial (23), clinical responses were observed in 50% of the ATM and 30% of the placebo group. These results were used for sample size calculations. To detect a significant difference in these proportions at P < 0.05 with 80% power, 93 patients would be required in each treatment group. Assuming a maximal dropout rate of 10%, the sample size in each group was determined to be 103. We recruited 210 patients, 105 in each group (Figure 1). After random allocation, four patients in the placebo group did not meet the inclusion criteria (inactive UC, no mucosal inflammatory findings, and no symptoms) and were excluded because further improvement in inactive UC would be unlikely, (i.e., inclusion could have biased the placebo group in the no-effect direction). Therefore, 206 patients were included in the full analysis set for an intention-to-treat analysis. For full analysis set analysis, the last-observation-carried-forward method was used to impute incomplete data, including those of patients who dropped out,
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Mayo score improvements were significantly greater in the ATM ( mean; 2.10) than in the placebo group ( 0.52), at 3 months after treatment (Table 2 and Figure 2, P < 0.0001). Using the Mayo system, the subscores for stool frequency, rectal bleeding, physicians global assessment, and endoscopic score (Table 2) in the ATM group were 0.50, 0.35, 0.67, and 0.58, respectively, and those in the placebo group were 0.07, 0.16, 0.14, and 0.16, respectively, at 3 months. The means of all scores except rectal bleeding differed significantly between the ATM and placebo groups. The clinical response rate was significantly higher in the ATM (47 of 105, 44.8%; 95% confidence interval (CI) 35.154.8) than in the placebo (23 of 101, 22.8%; 95% CI 15.032.2%) group at 3 months after treatment completion (Figure 3a, P = 0.0011). Remission was seen in 20 of 105 ATM patients (19.0%, 95% CI 12.027.9%) and in 16 of 101 controls (15.8%, 95% CI 9.324.9%) at 3 months (Figure 3b, P = 0.59).
Secondary end points

At 12 months after treatment completion, Mayo scores remained significantly lower in the ATM than in the placebo group (Table 2 and Figure 2; P < 0.0001). The subscores for stool frequency, physicians global assessment, and endoscopic score (Table 2) showed significant improvement at 12 months in the ATM group than in the placebo group (stool frequency, P < 0.0001; global assessment, P < 0.0001; and endoscopic score, P = 0.0002). The clinical response rate was significantly higher in the ATM (49.5%, 95% CI 39.659.5%) than in the placebo group (21.8%, 95% CI 14.231.1%) at 12 months after treatment completion
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210 Patients randomly allocated

105 Patients randomized to placebo 100 received placebo for 2 weeks 1 withdrawal of consent 4 excluded (criteria not met)

22 Discontinued follow-up 2 adverse events 12 relapse & exacerbation 7 lack of efficacy or responsiveness 0 withdrawal of consent 1 other reasons 1 lost to follow-up due to residence change

31 Discontinued follow-up 0 adverse events 22 relapse & exacerbation 6 lack of efficacy or responsiveness 3 withdrawal of consent 0 other reasons 0 lost to follow-up

81 Completed follow-up at 3 months

3 mo

69 Completed follow-up at 3 months

105 Analyzed for primary end point

101 Analyzed for primary end point 4 excluded (criteria not met)

22 Discontinued follow-up 0 adverse events 15 relapse & exacerbation 6 lack of efficacy or responsiveness 1 withdrawal of consent 0 other reasons 4 lost to follow-up due to residence change

27 Discontinued follow-up 0 adverse events 15 relapse & exacerbation 12 lack of efficacy or responsiveness 0 withdrawal of consent 0 other reasons 3 lost to follow-up due to residence change

55 Completed follow-up at 12 months

12 mo

39 Completed follow-up at 12 months

Figure 1. Enrollment, randomization, and follow-up. One patient discontinued treatment and another dropped out (1%) in both the placebo and the ATM (amoxicillin, tetracycline, and metronidazole) group. At the primary end point of 3 months after treatment completion, the study was discontinued prematurely by 31 patients in the placebo (30.7%) and 22 in the ATM (21.0%) group, all of whom experienced adverse events, had symptom relapse and/or exacerbation, showed lack of efcacy or responsiveness, withdrew consent, or dropped out for other reasons. At the secondary end point of 12 months after treatment completion, the study was discontinued prematurely by 58 patients in the placebo (57.4%) and 44 in the ATM (41.9%) group. Three patients in the placebo and ve in the ATM group dropped out because they changed residence and lived too far away to return for follow-up.

(Table 2 and Figure 3a; P < 0.0001). Remission was achieved in 28 of 105 ATM patients (26.7%, 95% CI 18.536.2%) but in only 15 of 101 controls (14.9%, 95% CI 8.623.3%) at 12 months (Table 2 and Figure 3b; P = 0.041). Pretreatment steroid-dependent UC was present in 51 placebo and 49 ATM group patients. The proportions experiencing remission and corticosteroid discontinuation were higher in the ATM than in the placebo group at 6, 9, and 12 months after treatment completion (Table 2 and Figure 4; 28.6 vs. 11.8%; 34.7 vs. 13.7%; and 34.7 vs. 13.7% at 6, 9, and 12 months, respectively). A total of 55 (28 at 3 months, 27 at 12 months; 54.5% of total) subjects in the placebo group and 40 (19 at 3 months, 21 at 12 months; 38.1% of total) in the ATM group showed relapse, exacerbation, lack of efficacy, or loss of responsiveness, a statistically significant difference (P = 0.0251 by Fishers exact test). Therefore,
2010 by the American College of Gastroenterology

at the secondary end point, 50 ATM and 61 placebo patients were lost to follow-up. At 6, 9, and 12 months, more patients in the ATM group, which had a higher rate of clinical remission, were available for follow-up. The rate of long-term follow-up was thus higher in the ATM than in the placebo group (Table 2).
Subset analysis

According to disease severity at entry, patients were divided into active and inactive groups with Mayo scores of 612 and < 6, respectively. At the primary end point in those with active disease (51 placebo and 65 ATM patients), the clinical response rate was significantly higher in the ATM than in the placebo group (Figure 5a, 34 of 65 vs. 11 of 51, 52.3% (95% CI 39.564.9) vs. 21.6% (CI 11.335.3), P = 0.0010). When mucosal healing was defined as an absolute subscore for endoscopy of 0 or 1, the
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105 Patients randomized to ATM 105 received AMPC 500mg, TC 500mg and metronidazole 250mg all t.i.d, for 2 weeks 1 withdrawal of consent

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Table 1. Baseline characteristics of patients*

Characteristic Age at entry, years (mean s.d.) Males, no. (%) Duration of disease, years (mean s.d.) Severity
a

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Placebo group (n =101) 39.3 13.1 70 (69.3) 7.3 6.9

ATM group (n =105) 38.5 14.1 69 (65.7) 6.6 5.3

medication, rates of corticosteroid-refractory and -dependent disease, and Mayo scores were well balanced between the ATM and placebo groups.
Antibodies against F. varium

Mild, no. (%) Moderate, no. (%) Severe, no. (%) Colonic involvement Extensive, no. (%) Left side, no. (%) Proctitis, no. (%) Concomitant medication, no. (%) Corticosteroid Sulfasalazine 5-Aminosalicylic acid Immunosuppressant Corticosteroid-refractory disease, no. (%) Corticosteroid-dependent disease, no. (%) Corticosteroid-non-refractory/nondependent disease, no. (%) Mayo score, median (range) b

41 (40.6) 57 (56.4) 3 (3.0)

42 (40.0) 62 (59.0) 1 (1.0)

52 (51.5) 32 (31.7) 17 (16.8)

48 (45.7) 45 (42.9) 12 (11.4)

In each group, 98 patients had serum samples available for assessment of antibodies to F. varium. The clinical characteristics of 10 patients whose sera were not available were similar to those of the other 196. Before treatment, baseline titers of immunoglobulins to F. varium were similar in the two groups (mean s.e., ATM: 9.1 0.7, placebo: 9.4 0.6, P = 0.541). Mean serum titers of immunoglobulins to F. varium in the ATM group were decreased at 3 ( mean: 0.02 U/ml) and 12 months ( mean: 0.11 U/ml) and were significantly lower than those in the placebo group at 12 months (P = 0.0071; mean: + 0.12 at 3 months, + 0.17 at 12 months).
Responses in F. varium antibody-positive patients

52 (51.5) 29 (28.7) 71 (70.3) 2 (2.0) 9 (8.9) 51 (50.5) 41 (40.6) 6 (2 12)

59 (56.2) 27 (25.7) 71 (67.6) 6 (5.7) 13 (12.4) 49 (46.7) 43 (40.9) 6 (2 12)

ATM, amoxicillin, tetracycline, and metronidazole. *There were no signicant differences between the two groups. a Severities are based on Truelove and Witts severity index (11). b Mayo scores range from 0 to 12 according to Schroeder et al. (25), with a higher score indicating more severe disease.

mucosal healing rate was significantly higher in the ATM than in the placebo group at 3 months after treatment completion (Figure 5b, 32 of 65 vs. 11 of 51, 49.2% (CI 36.661.9) vs. 21.6% (CI 11.335.3), P = 0.0034). Remission was seen in 10 of 65 ATM patients (15.4% (CI 7.626.5)) and in 6 of 51 placebo patients (11.8% (CI 4.423.9)) at 3 months (Figure 5c, P = 0.79). At 12 months after treatment completion, the clinical response rate and the mucosal healing rate were significantly higher in the ATM than in the placebo group (Figure 5a, 39 of 65 vs. 11 of 51, 60.0% (CI 47.172.0) vs. 21.6% (CI 11.335.3), P < 0.0001, and Figure 5c, 36 of 65 vs. 12 of 51, 55.4% (CI 42.567.7) vs. 23.5% (CI 12.837.5), P < 0.0001). The remission rate in the ATM group at 12 months was significantly higher than that in the placebo group (Figure 5c, 19 of 65, 29.2% (CI 18.641.8) vs. 6 of 51, 11.8% (CI 4.423.9), P = 0.025). In those with inactive UC who had a Mayo score of < 6, there were no significant differences in response and remission rates at 3 and 12 months between the ATM and placebo groups. In those with active and inactive UC, the distributions of age, sex, disease duration, disease severity, extent of disease, concomitant
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In this study, 142 of the 196 patients (72.4%) were positive for F. varium antibodies ( 5 U/ml; 76 of 98 placebo and 66 of 98 ATM patients). Among 66 ATM patients positive for F. varium antibodies, a clinical response was achieved in 30 patients (45.5%) at 3 months and in 32 patients (48.5%) at 12 months after treatment completion. In responders, serum titers of immunoglobulins to F. varium were decreased at 3 months (mean s.e.; pretreatment, 12.2 1.6 U/ml; 3 months, 11.7 1.5 U/ml; mean 0.55 U/ml, P = 0.080) and significantly decreased at 12 months (mean s.e.; before treatment, 11.8 1.5 U/ml; 12 months, 10.9 1.5 U/ml; mean 1.20 U/ml, P = 0.036). No serum titer decreases were observed in nonresponders at 3 months (pretreatment, 12.7 0.9; 3 months, 12.7 0.9; mean + 0.03, P > 0.2) or at 12 months after treatment completion (pretreatment, 13.2 0.9; 12 months, 12.7 0.9; mean 0.50, P = 0.15). Among the patients positive for F. varium antibodies ( 5 U/ml), the clinical response rate was significantly higher in the ATM (30 of 66, 45.5%) than in the placebo group (16 of 76, 21.1%) at 3 months (P = 0.0023) and at 12 months (32 of 66, 48.5%) vs. the placebo group (14 of 76, 18.4%, respectively; P < 0.0001), after treatment completion. Among patients negative for F. varium antibodies ( < 5 U/ml), the response rates in the ATM group were 37.5% (12 of 32) at 3 months and 46.9% (15 of 32) at 12 months but only 27.2% (6 of 22) at 3 months and 31.8% (7 of 22) at 12 months in the placebo group (P > 0.2).
Compliance and adverse events

Compliance was similar in the placebo (mean 99.6%, range 0100%) and ATM (mean 94.8%, range 30100%) groups. No serious drug-related toxicities were observed during the trial. Significantly more ATM (52.4%) than placebo (14.9%) group patients experienced adverse events (P < 0.0001). The proportions of ATM patients with nausea, fever, and watery diarrhea were 17.1, 12.4, and 7.6%, respectively. These symptoms all resolved promptly after treatment completion. Six ATM patients had reversible urticaria and fever, which resolved rapidly after antibiotic discontinuation. Stool cultures revealed no pathogens in any of our patients at any time during this study.
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Table 2. Mayo score improvements and corticosteroid discontinuation in ATM and placebo groups
Variable Mean duration of follow-up, mean s.d. (weeks) 26.9 20.6 Mayo score Total score Before treatment At 3 months At 12 months Stool frequency Before treatment At 3 months At 12 months Rectal bleeding Before treatment At 3 months At 12 months Physicians global assessment Before treatment At 3 months At 12 months Sigmoidoscopy subscore Before treatment At 3 months At 12 months Clinical response, no. (%) At 3 months At 12 months Clinical remission, no. (%) At 3 months At 12 months 16 (15.8) 15 (14.9) 20 (19.1) 28 (27.6) > 0.2 0.044 23 (22.8) 22 (21.8) 47 (44.8) 52 (49.5) 0.0011 < 0.0001 1.78 1.59 1.56 0.16 0.19 1.91 1.33 1.29 0.58 0.63 0.0002 0.0002 1.74 1.60 1.55 0.14 0.19 1.94 1.28 1.20 0.67 0.74 < 0.0001 < 0.0001 0.98 0.82 0.82 0.16 0.16 1.06 0.70 0.66 0.35 0.40 0.1186 0.0596 1.29 1.22 1.23 0.07 0.05 1.47 0.96 0.89 0.50 0.58 0.0001 < 0.0001 5.79 5.24 5.18 0.52 0.58 6.38 4.28 4.02 2.10 2.36 < 0.0001 < 0.0001 Mean Mean Mean 32.6 20.2 Mean 0.0416 Placebo (n =101) ATM (n =105) P value a

Clinical remission and corticosteroid discontinuation in corticosteroid-dependent patients No./total no. (%) At 6 months At 9 months At 12 months 6/51 (11.8) 7/51 (13.7) 7/51 (13.7) 14/49 (28.6) 17/49 (34.7) 17/49 (34.7) 0.046 0.019 0.019

ATM, amoxicillin, tetracycline, and metronidazole. a P value for difference in mean between ATM and placebo groups by MannWhitney U-test.

DISCUSSION
Bacteria have increasingly been recognized as having important roles in mucosal inflammation (28), and there is a growing body of evidence that colonic luminal bacteria contribute to clinical activity in UC (29,30), suggesting that, in addition to traditional immunosuppression, appropriate antimicrobial therapy may be important in UC management. Origi 2010 by the American College of Gastroenterology

nally, antibacterial treatment for UC focused on sulfasalazine. However, definitive antibiotic activity has never been shown for this agent. Truelove and Jewell (11) treated severe UC with prednisolone and tetracycline for 5 days, and 36 of 49 patients (73%) achieved complete remission. Their patients received both drugs and there were no controls. Dickinson et al. (13) reported that UC
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12

50

*P = 0.0462

*P= 0.019

*P = 0.019

10

Steroid withdrawal rate (%)

40 Placebo ATM

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Mayo score (total)

P<0.0001

P<0.0001

30

0.52 2.10

0.58 2.36

20

10

2 0 6 Months 0 Placebo ATM Before Placebo ATM 3 Months Placebo ATM 12 Months 9 Months 12 Months

Figure 2. Changes in the Mayo score before vs. after treatment. The comparison of ATM (amoxicillin, tetracycline, and metronidazole) and placebo therapy is shown as box plots. The ends of the boxes dene the 25th and 75th percentiles, with the bold line at the mean. The Mayo score decrease in the ATM group, compared with that in the control group, was statistically signicant. P values are for the difference in mean between the ATM and placebo groups.

Figure 4. Steroid withdrawal rates in steroid-dependent cases (n = 100). The rates of patients with clinical remission and discontinuation of steroid therapy at 6, 9, and 12 months after treatment completion were 28.6% (95% condence interval (CI) 16.643.3), 34.7% (95% CI 21.749.6), and 34.7% (95% CI 21.749.6) in the ATM (amoxicillin, tetracycline, and metronidazole) group and 11.8% (95% CI 4.423.9), 13.7% (95% CI 5.726.3), and 13.7% (95% CI 5.726.3) in the placebo group, respectively.

a
Patients with a sustained response (%) 100 80 60 40 20 0

Response rate based on Mayo score

P = 0.0010 P < 0.0001 Placebo ATM

a
100

Response rate based on Mayo score

Pateints with a sustained response (%)

80 60

P = 0.0011

P < 0.0001 Placebo ATM

3 Months

12 Months

b
Patients with a sustained response (%) 100 80 60 40 20 0

Remission rate based on Mayo score

Placebo P = 0.79 P = 0.025 ATM

40

20 0 3 Months 12 Months

3 Months

12 Months

b
100

Remission rate based on Mayo score

Placebo

c
Patients with a sustained response (%) ATM 100 80 60 40 20 0

Mucosal healing rate based on Mayo score

P = 0.0034 P < 0.0001 Placebo ATM

Pateints in sustained remission (%)

80 P > 0.2 P = 0.041

60

40

3 Months

12 Months

20 0 3 Months 12 Months

Figure 3. Response and remission rates at 3 and 12 months after treatment. (a) In the ATM (amoxicillin, tetracycline, and metronidazole) group at 3 and 12 months after treatment completion, 44.8% (95% condence interval (CI) 35.154.8) and 49.5% (95% CI 39.659.5) of patients (47 and 52 of 105) showed a clinical response, compared with 22.8% (95% CI 15.032.2) and 21.8% (95% CI 14.231.1) in the placebo group (23 and 22 of 101, P = 0.0011 and P < 0.0001, respectively). (b) In the ATM group at 12 months after treatment completion, 26.7% (95% CI 18.536.2) of patients (28 of 105) showed clinical remission, compared with 14.9% (95% CI 8.623.3) in the placebo group (15 of 101, P = 0.041).

Figure 5. Subset analyses of patients with active ulcerative colitis (UC; Mayo scores of 612). The rates of (a) response, (b) remission, and (c) mucosal healing at 3 and 12 months after treatment were signicantly higher in the ATM (amoxicillin, tetracycline, and metronidazole) group than in the placebo group, except for the remission rate at 3 months after treatment in patients with active UC.

temporarily stabilized in 16 of 18 (89%) patients who were given oral vancomycin, in contrast to only 8 of 15 (53%) placebo controls, although there was no significant difference in long-term outcomes. Intravenous metronidazole has been used for severe UC as an adjunct to intensive intravenous regimens (14). Although a double-blind, placebo-controlled trial found
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no benefit in the metronidazole group, no studies have focused on the possible efficacy of oral metronidazole. Burke et al. (15) studied 84 patients with acute relapse or a first attack of UC who were randomized to receive either oral tobramycin or placebo for 7 days as an adjunct to steroid therapy. At the 18- to21-day end point, 31 of 42 patients (74%) in the tobramycin and 18 of 42 (43%) in the placebo group showed clinical remission (P = 0.008). However, on the basis of the same study, Lobo and Burke et al. (16) reported that although this difference between the two groups remained significant at 6 months, it had disappeared by 1 year. Furthermore, Mantzaris et al. (17) reported that a combination of intravenous tobramycin and intravenous metronidazole, administered with steroid therapy, provided no benefit for acute, severe UC. Mantzaris et al. (18) also reported that a short course of ciprofloxacin did not increase the proportion of patients with active UC achieving remission. In contrast, in a double-blind, placebo-controlled study, Turunen et al. (19) showed that adding 6 months of ciprofloxacin to conventional therapy with steroids, mesalazine, or sulfasalazine was significantly superior to treatment with placebo during the first 6 months of administration. However, there was no significant difference between the ciprofloxacin and placebo groups in endoscopic, histological, or clinical findings at 12 months. Oral rifaximin was reportedly effective for severe UC attacks refractory to steroid treatment in a small, double-blind, placebo-controlled trial (20). However, the response rate was not significantly higher in the rifaximin compared with the placebo group, and long-term follow-up results have not yet been reported. Therefore, previous antimicrobial therapy for UC has tended to be associated with short-term benefits and none has translated into long-term improvement. In contrast, in this multicenter, randomized, double-blind, placebo-controlled study, the addition of a short course of therapy with three antibiotics to traditional therapy proved effective in patients with any stage of active UC, from mild to severe, despite conventional treatment. We showed ATM administration for only 2 weeks to be effective in UC patients with active disease, not only in the short term, but also for at least 12 months after treatment completion. The numbers needed to treat to obtain one clinical response at 3 and 12 months were estimated to be 4.5 (95% CI 2.910.6) and 3.6 (CI 2.56.6), respectively, and that to obtain one clinical remission at 12 months was 8.5 (CI 4.4114.0). These small numbers needed to treat support the use of ATM as an adjunct to conventional therapies. Interestingly, in this study, clinical response and remission rates in the ATM vs. the placebo group were higher at 12 than at 3 months. Mayo scores, the subscores for stool frequency, physicians global assessment, and the endoscopic score, except for rectal bleeding, all showed more improvement at 12 months in the ATM than in the placebo group. When patients were divided into active and inactive groups with a Mayo score of 612 vs. < 6, ATM treatment was more effective in the active group, as reflected by the clinical response and mucosal healing rates at 3 and 12 months, and the remission rate at 12 months after treatment completion. It is reasonable that there were no significant effects in the inactive group. It is noteworthy
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that approximately half of the patients in these two groups were steroid dependent, and that ATM was not only effective in these patients but actually facilitated tapering and withdrawal of corticosteroids. Because previous reports showed a low eradication rate for Helicobacter pylori using a single drug (31,32), we chose three antibiotics to which F. varium was highly susceptible. The decreases in F. varium antibody titers and mucosal F. varium densities at both short- and long-term follow-up in the ATM group, in both the present and our previous study (23,24), suggest that this therapy can suppress F. varium in the mucosa. Interestingly, F. varium antibody titers of nonresponders in the ATM group were not decreased, suggesting that additional studies are needed to identify a regimen that will routinely yield high eradication rates (e.g., longer treatment duration or the use of other antibiotics to which this microorganism is susceptible). The cumulative remission rate on long-term follow-up was significantly higher in the ATM than in the placebo group, suggesting a possible contributory relationship between F. varium, or an as yet unidentified but susceptible organism, and UC, possibly similar to that between H. pylori and gastroduodenal ulcers. However, as these three antibiotics are lethal to many bacterial species besides F. varium, we cannot rule out the possibility that a nonspecific sterilization effect may explain these UC remissions. Although the proportion of patients with adverse events was significantly higher in the ATM than in the placebo group, the frequency and types of adverse events, such as nausea, watery diarrhea, and fever, were similar to those reported in up to 50% of patients undergoing eradication therapy for H. pylori (31,32). The safety findings in these studies were similar to the data obtained in clinical studies of antibiotic treatments in patients with UC or those receiving H. pylori eradication treatment. It is not possible to avoid adverse events with antibacterial treatment, but the events in this trial were mild to moderate and the completion and compliance with treatment rates were similar in the ATM and control groups. A limitation of this study is that a rather large proportion of patients were unavailable for follow-up at 12 months. Many refractory UC cases were included in both groups (59.4% in the placebo and 59.1% in the ATM). Thus, these patients, especially those in the placebo group, tended to relapse and were unavailable for longterm follow-up. In analyzing those lost to follow-up, there were fewer dropouts in the ATM than in the placebo group and followup duration was much longer in the former. This confirmed the advantage experienced by the ATM group. In conclusion, a 2-week antibiotic combination therapy was effective and safe in patients with active UC in this double-blind placebo-controlled multicenter trial. We propose ATM to be considered alongside conventional therapy in patients with relapsing UC as an alternative to undertaking surgery.
ACKNOWLEDGMENTS

We thank Professor David Y. Graham, Baylor College of Medicine, for his critical review of the paper and helpful advice. We thank Dr S. Mizuno, Dr T. Terai, Dr T. Okimoto, Professor M. Imawari,
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Ohkusa et al.

and Professor K. Suzuki for their cooperation in the clinical study, and Dr B. Barfod for assistance in preparing the paper.

CONFLICT OF INTEREST

Guarantor of the article: Toshifumi Ohkusa, MD, PhD. Specific author contributions: Study concept and design: Toshifumi Ohkusa, Nobuhiro Sato, and Tetsuji Yokoyama; acquisition of data: Toshifumi Ohkusa, Kimitoshi Kato, Shuichi Terao, Toshimi Chiba, Katsuhiro Mabe, Kazunari Murakami, Yuji Mizokami, Toshiro Sugiyama, Akinori Yanaka, Yoshiaki Takeuchi, and Shigeru Yamato; analysis and interpretation of data: Tetsuji Yokoyama and Toshifumi Ohkusa; drafting of the article: Toshifumi Ohkusa and Kimitoshi Kato; clinical revision of the article for important intellectual content: Toshifumi Ohkusa, Kimitoshi Kato, Tetsuji Yokoyama, and Isao Okayasu; statistical expertise: Tetsuji Yokoyama; obtainment of funding: Toshifumi Ohkusa, Nobuhiro Sato, Sumio Watanabe, and Hisao Tajiri; administrative, technical, or material support: Nobuhiro Sato, Sumio Watanabe, and Hisao Tajiri; study supervision: Toshifumi Ohkusa, Kimitoshi Kato, and Tetsuji Yokoyama. Financial support: This work was funded by the Department of Gastroenterology, Juntendo University School of Medicine, and the Kanagawa Nanbyo Foundation. Potential competing interests: None.

Study Highlights
WHAT IS CURRENT KNOWLEDGE

3Luminal bacteria in mucosal inammation contribute 3Spontaneous colitis in animal models is completely inhibited by a combination of broad-spectrum antibiotics. 3Microbial agents have long been implicated in the 3Antibacterial therapy trials for UC have produced conicting
results. initiation and/or exacerbation of ulcerative colitis (UC) in humans, suggesting a possible rationale for antibiotic treatment of UC. to spontaneous colitis in models such as cytokine (interleukin (IL)-2 or IL-10)-knockout and other related gene-knockout mice.

WHAT IS NEW HERE

3 A 2-week antibiotic combination therapy consisting of 3Fusobacterium varium antibody titers decreased in responders 3We propose ATM (amoxicillin, tetracycline, and metronidazole)
to be considered alongside conventional therapy in patients with relapsing UC as an alternative to undertaking surgery. but not in nonresponders, and more in the antibiotic than in the placebo group. amoxicillin 500 mg t.i.d., tetracycline 500 mg t.i.d., and metronidazole 250 mg t.i.d. produced improvement, remission, and steroid withdrawal in active UC more effectively than a placebo.

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