Safety and Efficacy of AJM300, an Oral Antagonist of α4 Integrin, in Induction Therapy for Patients With Active Ulcerative Colitis

Accepted Manuscript Safety and Efficacy of AJM300, an Oral Antagonist of α4 Integrin, in Induction Therapy for Patients with Active Ulcerative Colitis Naoki Yoshimura, MD, Mamoru Watanabe, MD, Satoshi Motoya, MD, Keiichi Tominaga, MD, Katsuyoshi Matsuoka, MD, Ryuichi Iwakiri, MD, Kenji Watanabe, MD, Toshifumi Hibi, MD, For the AJM300 Study Group PII: DOI: Reference:

S0016-5085(15)01241-X 10.1053/j.gastro.2015.08.044 YGAST 60004

To appear in: Gastroenterology Accepted Date: 21 August 2015 Please cite this article as: Yoshimura N, Watanabe M, Motoya S, Tominaga K, Matsuoka K, Iwakiri R, Watanabe K, Hibi T, For the AJM300 Study Group, Safety and Efficacy of AJM300, an Oral Antagonist of α4 Integrin, in Induction Therapy for Patients with Active Ulcerative Colitis, Gastroenterology (2015), doi: 10.1053/j.gastro.2015.08.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

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ACCEPTED MANUSCRIPT Title:

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Safety and Efficacy of AJM300, an Oral Antagonist of α4 Integrin, in Induction Therapy for

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Patients with Active Ulcerative Colitis

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Short title:

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Oral α4 integrin antagonist for UC

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Authors:

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Naoki Yoshimura MD1$, Mamoru Watanabe MD2$*, Satoshi Motoya MD3, Keiichi Tominaga MD4, Katsuyoshi

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Matsuoka MD2, Ryuichi Iwakiri MD5, Kenji Watanabe MD6, Toshifumi Hibi MD7, For the AJM300 Study Group

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1. Yamate Medical Center, 3-22-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan

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2. Tokyo Medical and Dental University, 1-5-24 Yushima, Bunkyo-ku, Tokyo 113-8573, Japan

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3. Sapporo-Kosei General Hospital, Kita 3-jo Higashi 8-chome 5, Chuo-ku, Sapporo 060-0033, Japan

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4. Dokkyo Medical University, 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan

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5. Saga University, 5-1-1 Nabeshima, Saga, Saga 849-8501, Japan

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6. Osaka City General Hospital, 2-13-22 Miyakojima-hondori, Miyakojima-ku, Osaka 534-0021, Japan

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7. Kitasato University Kitasato Institute Hospital, 5-9-1 Shirogane, Minato-ku, Tokyo 108-8642, Japan

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Author share co-first authorship.

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ACCEPTED MANUSCRIPT Funding:

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The study was sponsored and funded by Ajinomoto Pharmaceuticals Co., Ltd. The trial was designed, managed, and

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data housed and analyzed by Ajinomoto Pharmaceuticals Co., Ltd. in collaboration with investigators. The authors had

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full access to all the data.

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Abbreviations:

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5-ASA, 5-aminosalitylate; CI, confidence interval; IBD, inflammatory bowel disease; JCV, John Cunningham virus;

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MAdCAM-1, mucosal addressin cell adhesion molecule-1; OR, odds ratio; PML, progressive multifocal

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leukoencephalopathy; TNF, tumor necrosis factor; UC, ulcerative colitis; VCAM-1, vascular cell adhesion molecule-1.

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Correspondence:

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*Mamoru Watanabe

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Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 5-45 Yushima 1-chome,

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Bunkyo-ku, Tokyo 113-8510, Japan

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Phone: +81-3-5803-5877

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Fax: +81-3-5803-0268

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e-mail: [email protected]

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Disclosures:

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ACCEPTED MANUSCRIPT The authors disclose the following: Naoki Yoshimura reports grants from Ajinomoto Pharmaceuticals; speaker fees

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from AbbVie, Eisai, Kyowa Hakko Kirin and Mitsubishi Tanabe Pharma. Mamoru Watanabe reports research grants,

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advisory fees and speaker fees from Ajinomoto Pharmaceuticals, AbbVie, Eisai, Kyorin Pharmaceutical, Takeda

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Pharmaceutical, Kyowa Hakko Kirin and Zeria Pharmaceutical; research grants and speaker fees from Mitsubishi

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Tanabe Pharma, Otsuka Pharma, UCB Japan, Toray Industries and JIMRO; research grants from Astellas Pharma,

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Asahi Kasei Medical, MSD, Chugai Pharmaceutical, Gene Care Research Institute, Daiichi-Sankyo, Ono

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Pharmaceutical, Dainippon Sumitomo Pharma and Bristol-Myers; advisory fees from Pfizer Japan, outside the

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submitted work. Satoshi Motoya reports grants from Ajinomoto Pharmaceuticals. Keiichi Tominaga reports grants

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from Ajinomoto Pharmaceuticals.

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Katsuyoshi Matsuoka reports research grants and speaker fees from Mitsubishi Tanabe Pharma and Eisai; research

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grants from Tsumura & Co; speaker fees from AbbVie and Zeria Pharmaceutical; advisory fees and speaker fees from

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Asahi Kasei Medical, outside the submitted work. Ryuichi Iwakiri reports grants from Ajinomoto Pharmaceuticals.

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Kenji Watanabe reports grants from Ajinomoto Pharmaceuticals; research grants, advisory fees and speaker fees from

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AbbVie, Mitsubishi Tanabe Pharma Corporation, Eisai, JIMRO, Asahi Kasei Medical, Ajinomoto Pharmaceuticals,

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Kyowa Hakko Kirin and Kyorin Pharmaceutical; research grants and speaker fees from Astellas Pharma and UCB

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Japan; research grants from Takeda Pharmaceutical, Dainippon Sumitomo Pharma, Horii Pharmaceutical, Janssen

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Pharmaceutical and Pfizer Japan, outside the submitted work. Toshifumi Hibi reports grants and advisory fee from

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ACCEPTED MANUSCRIPT Ajinomoto Pharmaceuticals; research grants and consulting fees from AbbVie and Otsuka Pharma, consulting fees

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from Astra Zeneca Pharmaceutical, research grants and advisory fee from Eizai, research grants and speaker fees from

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JIMRO, Mitsubishi Tanabe Pharma and Zeria Pharmaceutical, speaker fees from Kyorin Pharmaceutical, consulting

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fees from Pfizer Japan and Janssen Pharmaceutical, outside the submitted work.

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Transcript Profiling:

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Writing Assistance:

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Author Contributions:

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Naoki Yoshimura contributed to data collection and review of the manuscript. Mamoru Watanabe contributed to study

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design, data interpretation, and writing, review and final approval of the manuscript. Satoshi Motoya contributed to

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data collection, and review of the manuscript. Keiichi Tominaga contributed to data collection and review of the

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manuscript. Katsuyoshi Matsuoka contributed to data collection and writing the manuscript. Ryuichi Iwakiri

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contributed to data collection and review of the manuscript. Kenji Watanabe contributed to data collection and review

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of the manuscript. Toshifumi Hibi contributed to study design, data collection, data interpretation, and review of the

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manuscript.

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BACKGROUND & AIMS: AJM300 is an orally active small molecule antagonist of the α4

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integrin subunit. We performed a randomized trial to investigate the efficacy and safety of

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AJM300 in patients with active ulcerative colitis (UC).

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METHODS: In a double-blind, placebo-controlled, phase 2a study, 102 patients with moderately

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active UC (the Mayo Clinic scores of 6–10, endoscopic subscores ≥2, and rectal bleeding

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subscores ≥1) who had inadequate response or intolerance to 5-aminosalicylic acid or

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corticosteroids were randomly assigned to receive AJM300 (960 mg) or placebo 3 times daily for

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8 weeks. The primary endpoint was a clinical response at week 8, defined as a decrease in the

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Mayo Clinic score of at least 3 points and a decrease of at least 30% from baseline, with a

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decrease in the rectal bleeding subscore of at least 1 point or an absolute rectal bleeding subscore

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of 0 or 1.

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RESULTS: Clinical response rates were 62.7% and 25.5% at week 8 in the AJM300 group and

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placebo group, respectively (odds ratio [OR], 5.35; 95% confidence interval [CI], 2.23–12.82;

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P=.0002). Rates of clinical remission (Mayo Clinic score ≤2 and no subscore >1) were 23.5% and

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3.9% in the AJM300 group and placebo groups, respectively (OR, 7.81; 95% CI, 1.64–37.24;

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P=.0099), and rates of mucosal healing (endoscopic subscores of 0 or 1) were 58.8% and 29.4%

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(OR, 4.65; 95% CI, 1.81–11.90; P=.0014). No serious adverse event, including progressive

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multifocal leukoencephalopathy, was observed although more investigations are needed to

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confirm the safety profile of this drug.

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CONCLUSIONS: AJM300 was safe, well tolerated, and more effective than placebo in inducing

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clinical response, clinical remission, and mucosal healing in patients with moderately active UC.

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ClinicalTrials.jp no: JapicCTI-132293.

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KEYWORDS: integrin; IBD; randomized clinical trial; lymphocyte trafficking

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ACCEPTED MANUSCRIPT Introduction

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Inflammatory bowel disease (IBD) - the main forms of which being Crohn's disease and ulcerative colitis (UC), is a

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chronic inflammatory disease of the gastrointestinal tract. Although the cause of the disease remains unknown, the

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infiltration of lymphocytes into the lamina propria at the sites of inflammation is a well-documented pathogenic

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mechanism of IBD.1, 2 Infiltration of lymphocytes is accelerated by increased expression of adhesion molecules such as

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vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on the

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surface of endothelial cells at the site of inflammatory lesions.3, 4 The binding of lymphocytes to VCAM-1 or

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MAdCAM-1 requires the expression of α4β1 or α4β7 integrin on lymphocytes, respectively. VCAM-1 is expressed on

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the luminal surface of endothelium in numerous types of tissues while MAdCAM-1 is expressed predominantly in the

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intestinal lamina propria.4, 5

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Several clinical studies have demonstrated that α4 integrin blockade is clinically effective as a therapeutic

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approach for IBD, e.g. anti-α4 integrin antibody, natalizumab6, 7 or anti-α4β7 integrin antibody, vedolizumab.8, 9

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Furthermore, anti-β7 integrin antibody, etrolizumab10 and anti-MAdCAM-1 antibodies11 are being developed for the

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treatment of IBD. However, all the agents are biologics and raise some concerns regarding the long-term

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administration. Adverse effects specifically related to biologics have been observed in the treatment with anti-tumor

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necrosis factor α (TNFα) antibodies,12 such as immunogenicity, infusion reaction, and the loss of efficacy related to the

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production of antidrug antibodies. Therefore, the development of orally active chemical medicine is a reasonable and

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preferable approach. Although several small molecule α4 integrin antagonists have been studied in clinical trials for the

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treatment of asthma and multiple sclerosis,13-15 there are no reports on the efficacy of oral α4 integrin antagonist in

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patients with IBD.

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AJM300 is a novel orally active small molecule α4 integrin antagonist which is classified as a

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phenylalanine derivative16 and currently being developed for IBD.17, 18 In animal models, an oral treatment with

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AJM300 inhibited the lymphocyte homing to the gut, and prevented the development of experimental colitis in mice.19

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Orally administrated AJM300 can be absorbed through the intestine, distributed to various tissues through systemic

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blood circulation and mainly excreted into the feces. Pharmacodynamics of α4 integrin antagonist is well known to

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increase the peripheral lymphocyte counts probably due to inhibition of lymphocyte attachment to the vascular

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endothelium. For AJM300, the increase in peripheral lymphocyte counts was maintained throughout the day at 960 mg

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dose, but not at 480 mg dose, three times daily in the healthy volunteers.

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Meanwhile, progressive multifocal leukoencephalopathy (PML) has been known as the side effect of anti-α4

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integrin therapy. PML is an opportunistic brain infection that is caused by the John Cunningham virus (JCV), and

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usually leads to death or severe disability. Natalizumab is currently available through a special restricted distribution

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program to mitigate the risk of PML in clinical practice. The clinical development of oral α4 integrin antagonists also

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needs to minimize the potential risk of PML carefully. Therefore, we designed the 8-week, placebo-controlled trial to

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investigate the efficacy and safety of AJM300 as remission induction therapy in patients with moderately active UC,

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under the study protocol to mitigate the potential risk of PML.

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ACCEPTED MANUSCRIPT Methods

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Ethics Statement

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The study was approved by the Institutional Review Board at each center and conducted in accordance with the Good

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Clinical Practice, other relevant rules and regulations, and Helsinki declaration. All patients provided written informed

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consent. All authors had access to the study data and reviewed and approved the final manuscript.

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Study Design and Patients

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This was a randomized, double-blind, placebo-controlled, phase 2a study, conducted at 42 medical centers in Japan

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between May 2012 and July 2013 [ClinicalTrials.jp number: JapicCTI-132293]. This study was conducted to obtain

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the information on the efficacy and safety of AJM300 as induction therapy in patients with moderately active UC. The

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target number of subjects was decided to be 60 per group, 120 in total.

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Eligible patients were 20 to 65 years of age with a diagnosis of moderately active UC, with the Mayo Clinic

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score20 of 6-10, a rectal bleeding subscore of 1 or higher, and an endoscopic subscore of 2 or higher. Additional

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inclusion criteria were documentation of inadequate response or intolerance to 5-aminosalitylate (5-ASA) (4.0g or

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higher of salazosulfapyridine, 4.0g of Pentasa® or 3.6g of Asacol® per day) and/or corticosteroids (30-40 mg of oral

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prednisolone or the equivalent per day). Patients continued to take 5-ASA at a constant dose for at least 4 weeks or

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prednisolone for at least 2 weeks before enrollment. Although concomitant oral 5-ASA was administrated at a constant

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dose throughout the study, concomitant corticosteroid dose was allowed to taper within 5 mg per 2 weeks according to

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a predefined regimen for patients showing any clinical response to the study drug. Patients were ineligible if they had

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received azathioprine or 6-mercaptopurine within 8 weeks before enrollment, or TNF antagonists, cyclosporine,

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tacrolimus, or methotrexate within 12 weeks before enrollment. Rectal therapy with 5-ASA or corticosteroids as well

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as leukocytapheresis was discontinued 4 weeks before enrollment. Other criteria for exclusion included proctitis,

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corticosteroid-dependence, severe colonic stricture, infectious enteritis, a history of bowel surgery, major organ

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dysfunction, malignant neoplasm, drug hypersensitivity or dependence, alcoholism, psychiatric symptom, pregnancy

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or lactation, and a white blood-cell count of 3.0 x 103/µL or less, concomitant use of immunosuppressants e.g.,

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azathioprine, 6-mercaptoopurine, cyclosporine, methotrexate, or tacrolimus, and the presence of neurological symptom

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e.g., motor paralysis, impaired vision, alogia, symptoms of dementia, facial nerve palsy for an increased potential risk

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of PML.

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Randomization and Blinding

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Patients were randomized to the active treatment group or placebo group in a 1:1 ratio by dynamic balancing allocation

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with minimization method based on the strata of the inadequate response or intolerance to 5-ASA and/or

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corticosteroids and the baseline Mayo Clinic score (6-7 or 8-10). Randomization was performed centrally. Patients,

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assessing physicians, and the funder were blinded to the assignment of treatment throughout the study.

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Study Procedures

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Patients were assigned to receive either AJM300 960 mg or placebo three times daily for 8 weeks. The dose regimen of

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AJM300 was determined according to the following data from the phase 1 multiple oral dose study (data not shown).

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For pharmacokinetics, although the drug exposure (Cmax and AUC) was mostly saturated at 480 mg dose or above

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three times daily, the trough drug concentration in plasma was higher at 960 mg dose than that at 480 mg dose, three

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times daily. In addition, the pharmacological activity of AJM300 (surrogated by the increase in peripheral lymphocyte

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count) was maintained throughout the day at 960 mg dose, but not at 480 mg dose, three times daily.

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The patients visited at weeks 0, 2, 4 and 8. At each visit, a partial Mayo Clinic score (excluding endoscopic

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subscore) was calculated, adverse events were noted and neurological symptom questionnaires were administrated.

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Blood samples for hematologic testing were obtained at each visit. The peripheral lymphocyte counts at weeks 0, 2, 4

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and 8 was measured as the pharmacodynamics parameter, whereas neutrophil counts was assessed to investigate the

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effect on leukocytes lacking α4 integrin. Only at week 8, the administration of AJM300 or placebo was terminated in

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the morning on the day before the last visit, for the purpose of confirmation of the recovery of pharmacodynamics.

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Pharmacokinetics measurement was not performed in this study.

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Colonoscopy or sigmoidscopy was performed at baseline and week 8 with biopsy. Endoscopic subscores

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were primarily assessed by the on-site investigators. The central evaluation committee for colonoscopy was established

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to assure the reliability of the assessment by the on-site investigators.

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Endpoints

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The primary endpoint was a clinical response, defined as a decrease in the Mayo Clinic score of at least 3 points and a

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decrease of at least 30% from the baseline score, with a decrease of at least 1 point on the rectal bleeding subscore or

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an absolute rectal bleeding subscore of 0 or 1. The main secondary endpoints were clinical remission, defined as a

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Mayo Clinic score of 2 or lower and no subscore higher than 1, mucosal healing, defined as an endoscopic subscore of

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0 or 1, and partial Mayo Clinic score. The endoscopic subscores assessed by the on-site investigators (investigator or

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subinvestigator) were applied to the Mayo Clinic score. The on-site investigators evaluated and determined the most

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severely affected site up to the sigmoid colon from the anus at week 0, and evaluated the same site at week 8. In

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addition, the endoscopic subscores were also evaluated by the central evaluation committee for endoscopic evaluation

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to assure the reliability of the evaluation by the on-site investigators. The on-site investigators collected two biopsy

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specimens of colonic mucosal tissue in the marginal region of the most severely affected site during colonoscopy at

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week 0 and in the same region at week 8. The histological activity was evaluated using the Riley score21 by the central

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evaluation committee for histopathological evaluation.

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Safety evaluations, which included all reports of adverse events and clinical laboratory tests, were

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conducted throughout the study. The investigators and the sponsor were not informed of the patients' differential counts

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of white blood cells, so that the increase in circulating lymphocyte counts observed in the active treatment group would

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not lead to unblinding of the treatment assignments.

For all patients, new occurrence of neurological signs and symptoms potentially consistent with PML were

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assessed cautiously throughout the study using the standardized questionnaires. If PML-related signs or symptoms

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were suspected, first of all, the administration of the study drug was to be forcedly terminated. Then neurological

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examination using MRI scans and PCR analyses was available as necessary. Independent safety assessment committee

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consisting of a panel of PML experts was organized to secure patient’s safety for the onset of PML and to provide a

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further guidance for the on-site investigators and the sponsor.

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Statistical Analysis

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The full analysis set, which consisted of all patients who were randomized, received at least 1 dose of study drug, and

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had at least 1 available efficacy data, was used for the analysis of efficacy. The safety analysis set, which consisted of

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all patients who were randomized and received at least 1 dose of study drug, was used for the analysis of safety. The

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last observation carried forward method was used for statistical analyses on the efficacy variables to handle of the

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missing data, especially for withdrawal. On the other hand, for the safety variables, the imputation methods were not

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used. The primary endpoint was analyzed by the logistic regression model, after adjustment for the inadequate

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response or intolerance to 5-ASA and/or corticosteroids, and the baseline Mayo Clinic score (6-7 or 8-10) to compare

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the clinical response between the treatment groups. Additionally, the proportion of patients who had good clinical

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response in each treatment group and its difference between the treatment groups were calculated. The secondary

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endpoint, the clinical remission was analyzed by the logistic regression model, after adjustment for the baseline Mayo

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Clinic score (6-7 or 8-10). The mucosal healing was analyzed by the logistic regression model, after adjustment for the

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inadequate response or intolerance to 5-ASA and/or corticosteroids, and the baseline Mayo Clinic score (6-7 or 8-10).

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The proportions of patients in clinical remission and patients with mucosal healing were calculated by treatment group.

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Summary statistics of the change from baseline in the partial Mayo Clinic score and the peripheral lymphocyte counts

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at each evaluation time were calculated by treatment group. A t-test was used to compare the change in the partial

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Mayo Clinic scores, and the peripheral lymphocyte counts between the two treatment groups. For evaluating the safety,

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the adverse events were tabulated by treatment group. All statistical tests were conducted at the 0.05 level of

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significance (two-sided). No adjustment was made for multiple comparisons.

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The determination of sample size was based on the following calculations. The clinical response rate was

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estimated to be 54.8% to 66.0% for the active treatment group whereas 33.0% to 35.6% for the placebo group. The

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number of subjects required to provide power of 80% with two-sided significance level of 5% was computed.

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ACCEPTED MANUSCRIPT Results

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Randomization and Baseline Characteristics

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Of 127 patients that gave informed consent, 3 patients withdrew the study before randomization. Excluding 22 patients

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who did not meet inclusion criteria or met exclusion criteria, 102 patients underwent randomization. Those patients

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were randomly assigned to receive the active treatment (AJM300) or placebo (51 patients for each) (Figure 1). Study

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treatment was started in all the 102 patients. The full analysis set was equivalent to the safety analysis set.

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Demographics and baseline characteristics were similar between the placebo and active treatment groups (Table 1). In

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the 102 randomized patients, the therapeutic outcomes of the current relapse were an inadequate response to 5-ASA

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(n=92, 90.2%), intolerance to 5-ASA (n=3, 2.9%), an inadequate response to corticosteroid (n=3, 2.9%), and

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intolerance to corticosteroids (n=4, 3.9%).

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Efficacy

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A total of 32 of the 51 patients receiving the active treatment (62.7%) and 13 of the 51 patients receiving placebo

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(25.5%) had a clinical response at week 8 (OR, 5.35; 95% CI, 2.23 to 12.82; logistic regression, P = .0002) (Figure 2).

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A total of 12 patients receiving the active treatment (23.5%) and 2 receiving placebo (3.9%) had clinical remission (OR,

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7.81; 95% CI, 1.64 to 37.24; logistic regression, P = .0099). Proportions of patients with mucosal healing were 58.8%

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(30 of 51 patients) with the active treatment and 29.4% (15 of 51 patients) with placebo (OR, 4.65; 95% CI, 1.81 to

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11.90; logistic regression, P = .0014) (Figure 2). The efficacy of the active treatment was generally consistent across

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subgroups (Supplementary Figure 1).

The proportion of patients achieving endoscopic subscore of 0 at week 8 was also higher in the active

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treatment group than that in the placebo group, although the difference was not statistically significant (15.7% versus

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5.9% in the active treatment group and placebo group, respectively; difference, 9.8; 95% CI, -2.7 to 22.7). The

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significant efficacies of the active treatment (clinical response, clinical remission and mucosal healing) were also

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confirmed by the assessment of the central evaluation committee for endoscopic evaluation as well (data not shown).

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Consistency of the endoscopic subscores between the on-site investigators and the central evaluation committee was

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64% at baseline and 59% for outcome measure.

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Patients receiving the active treatment had greater improvement in the partial Mayo Clinical score

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compared to patients receiving placebo (Figure 3). Each subscore (stool frequency, rectal bleeding, endoscopic findings,

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or physician’s global assessment) of the Mayo Clinic score improved significantly in the active treatment group

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compared with that in the placebo group at week 8 (Supplementary Table 1). In addition, the endoscopic improvement

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was accompanied by a significant histological improvement in the Riley score (Supplementary Table 2).

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Pharmacodynamics

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Anti-α4 integrin therapeutic agents are known to increase peripheral-blood total lymphocyte counts. The significant

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increase in peripheral lymphocyte counts (as the pharmacodynamic action of AJM300) was observed at week 2 (mean,

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2.7 x 103/µL versus 1.8 x 103/µL in the active treatment group and placebo group, respectively; difference, 0.8 x

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103/µL; 95% CI, 0.5 to 1.2 x 103) as well as week 4 (mean, 2.7 x 103/µL versus 1.8 x 103/µL; difference, 1.0 x 103/µL;

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95% CI, 0.6 to 1.3 x 103) (Figure 4). At week 8, the day after last administration of the active treatment, the

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pharmacodynamic action was disappeared (mean, 2.0 x 103/µL versus 1.8 x 103/µL; difference, 0.2 x 103/µL; 95% CI,

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-0.1 to 0.5 x 103). As for the effect on leucocytes lacking α4 integrin, no change in neutrophil counts was observed

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throughout the study (data not shown).

Safety

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Data for all randomized patients were used for the safety analysis. No important differences were observed between the

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treatment groups in the incidence of adverse events, 49.0% (25 of 51 patients) in the active treatment group and 56.9%

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(29 of 51 patients) in the placebo group (Table 2). The grades of all the adverse events were mild or moderate in

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severity, and no serious adverse events were observed in this study. The most common adverse events were

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nasopharyngitis and UC. The incidence of nasopharyngitis was similar between the treatment groups, 9.8% (5 of 51

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patients) in the active treatment group and 7.8% (4 of 51 patients) in the placebo group. The adverse events related to

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UC were higher in the placebo group (17.6%; 9 of 51 patients) than that in the active treatment group (3.9%; 2 of 51

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patients). Adverse events leading to discontinuation of the study drug included the exacerbation of UC (1 patient in the

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active treatment group and 8 in the placebo group) and abnormality of liver function (1 patient in the placebo group).

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The incidence of drug-related adverse events were 21.6% (11 of 51 patients) in the active treatment group and 7.8% (4

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of 51 patients) in the placebo group, all of which were mild. There was no frequent drug-related adverse events

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(>5.0%). Although higher incidence of drug-related adverse events, particularly abnormalities in the laboratory test

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values, were observed in the active treatment group than in the placebo group, these changes were mild and recovered

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without any treatment. Overall, neither infection, neurological symptom nor the onset of PML was observed in this

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study.

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ACCEPTED MANUSCRIPT Discussion

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This is the first study to demonstrate the clinical benefit of an orally active small molecule α4 integrin antagonist for

3

UC. In this phase 2a study, AJM300 960 mg three times daily significantly improved clinical response and clinical

4

remission at week 8 compared with placebo in patients with moderately active UC. Subgroup analysis showed the

5

therapeutic benefit of the active treatment regardless of disease duration, extent of disease, and baseline Mayo Clinic

6

scores. A higher proportion of patients achieved mucosal healing in the active treatment group compared with the

7

placebo group. Recent evidence has emphasized the importance of mucosal healing as a treatment goal for UC.22 It has

8

been reported that the most favorable subsequent clinical outcomes were observed in patients with early achievement

9

of endoscopic subscore of 0.23 There was a trend towards an increase in the number of patients with endoscopic

10

subscore of 0 in the active treatment group compared to the placebo group. Furthermore, a significantly better

11

histological improvement at week 8 was obtained in patients treated with the active treatment compared to those with

12

placebo.

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Rationales for the development of AJM300 were the achievements of the ‘oral’ anti-α4 integrin therapy.

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Oral drug formulations often provide good drug adherence and may resolve some issues in biological therapy. Adverse

15

effects specifically related to biologics, such as immunogenicity, infusion reaction, and the loss of efficacy related to

16

the production of antidrug antibodies, could be observed in the clinical practice.12 Antibodies to infliximab develop in

17

8-60% of patients with IBD.12 The increase in the number of patients with loss of response or intolerance may be also

20

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observed during the treatment with the anti-integrin antibodies in the future.9, 24 Orally active small molecule medicine

2

with a similar pharmacological activity to biologics could be beneficial for patients with IBD by overcoming these

3

problems of biologics.

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As for safety of AJM300, there was no serious adverse event in this study. The incidence of adverse events

5

in the active treatment group was similar to that in the placebo group. Only 1 patient (2.0%) receiving the active

6

treatment discontinued the study due to adverse event, whereas 9 patients (17.6%) receiving placebo discontinued the

7

study mainly due to aggravation of UC. All the drug-related adverse events were mild and self-limiting. Furthermore,

8

neither infection nor neurological symptom was observed.

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The most concerned adverse event of α4 integrin blockade is the development of PML which is a

10

demyelinating disorder of the central nervous system caused by JCV infection and reactivation. PML occurs in

11

severely immunocompromised patients such as acquired immune deficiency syndrome as well as the use of

12

immunosuppressants or some biologics, and usually leads to death or severe disability. As of February 2012, 212

13

confirmed PML cases among 99,751 patients treated with natalizumab were reported (2.1 cases per 1,000 patients).25

14

Although no cases of PML had been observed among approximately 500 IBD patients and healthy volunteers treated

15

with AJM300 for maximum of 8 weeks in the clinical studies yet, such a short term safety data does not preclude the

16

possibility of PML risk with this agent. Clinical development of AJM300 should be carefully conducted under the risk

17

management program to mitigate the potential risk of PML.

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Based on the large post-marketing surveillance data derived from more than 100,000 natalizumab-treated

2

patients, especially two important findings have been obtained as for risk factors for PML. First, no cases of PML have

3

been observed with the therapy for 8 months or less.26 Second, the risk of PML was much lower among the patients

4

who were negative for anti–JCV antibodies than those who were positive (the incidence of PML; 0/1,000 (95% CI, 0 to

5

0.32) and 3.87/1,000 (95% CI, 2.91 to 5.05), respectively).25, 26 A prospective study further confirmed that no cases of

6

PML were observed in natalizumab-treated patients who were negative for anti-JCV antibodies.27 These data suggest

7

that the potential PML risk of AJM300 may be minimized by limiting a treatment duration to less than 8 months. In

8

addition, the measurement of the anti-JCV antibodies may provide the information to make a decision to continue the

9

treatment with this agent. Taken together, the clinical development of AJM300 should be focused on the induction

10

therapy in active UC patients, with PML risk stratification by measurement of anti-JCV antibodies, at the moment. The

11

long-term safety study should be conducted to preclude a possible increased risk of PML with this agent in the future.

12

The potential risk of PML should be mitigated by a special restricted distribution program under the risk management

13

action plan in clinical practice as well as in clinical trials. The patients who were in immunocompromised conditions or

14

treated with concomitant immunosuppressants or anti-TNFα antibodies should be precluded. Furthermore, the patients

15

with any neurological symptom should be precluded to make a quick detection of new neurological symptoms

16

suggesting PML development. In the event of suspected PML, early removal of drug from the body might lead to more

17

acceptable outcomes of the complication.26, 28 For instance, a timely discontinuation of treatment with natalizumab

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followed by the plasma exchange and immunoadsorption has been recommended.26,

2

advantage in the outcome of PML, because of extremely shorter duration of pharmacodynamic action (within a day

3

after the last dose in this study) than natalizumab (approximately 4 months).29 However, careful monitoring and risk

4

minimization strategies for PML will still be required to use this drug.

AJM300 may have an

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We concluded that the dose of 960 mg three times daily was optimal, because this dose was well tolerated

6

and demonstrated the expected clinical efficacy. Furthermore, the increase in peripheral lymphocyte counts, which is

7

one of the pharmacological activities of α4 integrin blockade, was maintained throughout the day at this dose regimen.

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This study has the following limitations: small sample size (a total of 102 patients, 51 patients per group), a

9

short period of study drug medication (8 weeks) and extremely low enrollment of an inadequate response or

10

intolerance to corticosteroids. There was no information of medical history regarding an inadequate response or

11

intolerance to corticosteroids, immunosuppressants and anti-TNFα antibodies. Larger or longer-term clinical trials are

12

required to further confirm the efficacy and safety of AJM300.

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corticosteroids have been, for decades, the first and almost exclusive therapeutic option for such patients. Given the

15

diverse adverse effects of corticosteroids, reasonable alternatives to them have been desired. AJM300 can be

16

administered on outpatient basis without any infusion equipment or instruction. Thus, AJM300 could be suitable for

17

induction therapy in patient with moderately active UC refractory to 5-ASA.

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In conclusion, oral treatment with the small molecule α4 integrin antagonist AJM300 was safe, well

2

tolerated and more efficacious than placebo in inducing clinical response, clinical remission and mucosal healing in

3

patients with moderately active UC in this study. AJM300 may become a novel oral therapeutic option especially for

4

patients who had an inadequate response to 5-ASA with moderately active UC, and phase 3 study for induction therapy

5

is currently ongoing in Japan.

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ACCEPTED MANUSCRIPT Acknowledgements

2

The authors would like to honor the memory of Prof. Takayuki Matsumoto who contributed to study design.

3

The authors would like to thank the members of the central evaluation committee following below.

4

The central evaluation committee for endoscopic evaluation: Fumiaki Ueno (Gokeikai Ofuna Chuo Hospital),

5

Masahiro Igarashi (The Cancer Institute Hospital of JFCR) and Haruhiko Ogata (Keio University Hospital). The

6

central evaluation committee for histopathological evaluation: Yoichi Ajioka (Niigata University Graduate School of

7

Medical and Dental Sciences), Masahiro Ikegami (Tokyo Jikei Medical Science University Hospital) and Ryoji

8

Kushima (National Cancer Center Hospital). The independent safety assessment committee: Hidehiro Mizusawa

9

(Tokyo Medical and Dental University) and Shuji Kishida (Hatsuishi Hospital).

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The authors would also like to thank the following investigators who participated in this study:

11

Hideyuki Hiraishi (Dokkyo Medical University Hospital), Hirokazu Yamagami (Osaka City University Hospital),

12

Fuminao Takeshima (Nagasaki University Hospital), Tadashi Yokoyama (Yokoyama Gastrointestinal Division

13

Hospital), Yasuo Suzuki (Toho University Sakura Medical Center), Yoshiaki Ukai (Sendai Medical Center), Atsuo

14

Kitano (Wakakusa-Daiichi Hospital), Katsuya Endo (Tohoku University Hospital), Kiyonori Kobayashi (Kitasato

15

University East Hospital), Masaaki Sumioka (Hiroshima Prefectural Hospital), Sakiko Hiraoka (Okayama University

16

Hospital), Takafumi Ando (Nagoya University Hospital), Tomoki Inaba (Kagawa Prefectural Central Hospital),

17

Yuichiro Kojima (Yamanashi Prefectural Central Hospital), Kazuhiko Kawakami (Matsuda Hospital), Koichiro

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Matsuda (Toyama Prefectural Central Hospital), Mikihiro Fujiya (Asahikawa Medical College Hospital), Shinji

2

Tanaka (Hiroshima University Hospital), Takanori Kanai (Keio University Hospital), Takayuki Yamamoto (Yokkaichi

3

Social Insurance Hospital), Takuya Inoue (Osaka Medical College Hospital), Tatsuya Toyokawa (Fukuyama Medical

4

Center), Toshifumi Ashida (Sapporo Higashi Tokushukai Hospital), Toshiyuki Matsui (Fukuoka University Chikushi

5

Hospital), Hiroshi Yamamoto (Kurashiki Central Hospital), Keiichi Mitsuyama (Kurume University Hospital), Ken

6

Haruma (Kasawaki Medical School Hospital), Masakazu Nagahori (Tokyo Medical and Dental University), Nobuyuki

7

Hida (The Hospital of Hyogo College of Medicine), Toshimi Chiba (Iwate Medical University Hospital), Tomoyuki

8

Tsuzuki (Nagoya Medical Center), Akira Ando (Shiga University of Medical Science Hospital), Hitoshi Nishiyama

9

(Nagasaki Medical Center), Katsuyuki Fukuda (St. Luke’s International Hospital), Kazuhito Sugimura (Niigata City

10

General Hospital), Koichi Oshimoto (Isesaki Municipal Hospital), Kunihiko Aoyagi (Fukuoka University Hospital),

11

Nobuhide Oshitani (Izumiotsu Municipal Hospital) and Terasu Honma (Saiseikai Niigata Daini Hospital).

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ACCEPTED MANUSCRIPT Figure Legends

2

Figure 1. Enrollment and treatment

3

In the study, a total of 102 patients with ulcerative colitis were randomly assigned to receive placebo (n=51) or

4

AJM300 (n=51).

5

Figure 2. Proportion of patients with a clinical response, in clinical remission, and with mucosal healing at week

6

8

7

A clinical response was defined as a reduction in the Mayo Clinic score of at least 3 points and a decrease of at least

8

30% from the baseline score, with a decrease of at least 1 point on the rectal bleeding subscore or an absolute rectal

9

bleeding subscore of 0 or 1. Clinical remission was defined as a Mayo Clinic score of 2 or lower and no subscore

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higher than 1. Mucosal healing was defined as an absolute endoscopic subscore of 0 or 1.

11

The logistic regression model adjusted for the inadequate response or intolerance with 5-ASA and/or corticosteroids,

12

and the baseline Mayo Clinic score (6-7 or 8-10) was used for the statistical analysis of clinical response and mucosal

13

healing. For the clinical remission, the logistic regression model adjusted for the baseline Mayo Clinic score (6-7 or

14

8-10) was used.

15

Figure 3. Change from baseline in the partial Mayo Clinic score

16

The partial Mayo Clinic score consists of the Mayo Clinic score minus the endoscopic subscore; ranging 0 to 9, with

17

higher scores indicating more severe disease. The change from baseline in the partial Mayo Clinic score at each visit

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(weeks 2, 4 and 8) was indicated by the mean value with SD. Asterisk denotes significant difference between treatment

2

groups at each visit (t-test, *P < .05, **P < .01).

3

Figure 4. Peripheral lymphocyte counts

4

Peripheral lymphocyte counts of patients with active ulcerative colitis before and after receiving placebo or AJM300.

5

Blood samples were obtained at weeks 0, 2, 4 and 8. Study drug treatment was terminated in the morning on the day

6

before the last visit (week 8) to confirm the disappearance of the pharmacological activity of AJM300 at week 8. Bars

7

are ±SD, and asterisk denotes significant difference between treatment groups at each evaluation time (t-test, P < .05).

8

Supplementary Figure 1. Subgroup analysis

9

Plot of risk differences (dots) and 95% confidence intervals (CIs; horizontal bars) for comparing the proportion of

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patients with clinical response between AJM300 and placebo at week 8 by baseline characteristics

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1.

3

2.

5

3.

4.

5.

6.

17

Sandborn WJ, Rutgeerts P, Colombel JF, et al. Natalizumab induction and maintenance therapy for

Crohn’s disease. N Engl J Med 2005;353:1912-1925.

7.

15

16

Briskin M, Winsor-Hines D, Shyjan A, et al. Human mucosal addressin cell adhesion molecule-1 is

preferentially expressed in intestinal tract and associated lymphoid tissue. Am J Pathol 1997;151:97-110.

13

14

González-Amaro R, Mittelbrunn M, Sánchez-Madrid F. Therapeutic anti-integrin (α4 and αL)

monoclonal antibodies: two-edged swords? Immunology 2005;116:289-296.

11

12

Arihiro S, Ohtani H, Suzuki M, et al. Differential expression of mucosal addressin cell adhesion molucule-1

(MAdCAM-1) in ulcerative colitis and Crohn’s disease. Pathol Int 2002;52:367-374.

9

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Bowel Dis 2008;14:1298-1312.

7

8

Eksteen B, Liaskou E, Adams DH. Lymphocyte homing and its role in the pathogenesis of IBD. Inflamm

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2007;369:1627-1640.

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Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet

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Targan SR, Feagan BG, Fedorak RN, et al. Natalizumab for the treatment of active Crohn’s disease: results

of the ENCORE trial. Gastroenterology 2007;132:1672-1683.

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Feagan BG, Rutgeerts P, Sands BE, et al, for the GEMINI 1 Study Group. Vedolizumab as induction and

maintenance therapy for ulcerative colitis. N Engl J Med 2013;369:699-710.

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and maintenance therapy for Crohn's disease. N Engl J Med 2013;369:711-721.

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controlled, phase 2 trial. Lancet 2014;384:309-318

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Vermeire S, O'Byrne S, Keir M, et al. Etrolizumab as induction therapy for ulcerative colitis: a randomised,

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Sandborn WJ, Feagan BG, Rutgeerts P, et al, for the GEMINI 2 Study Group. Vedolizumab as induction

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Nanda KS, Cheifetz AS, Moss AC. Impact of antibodies to infliximab on clinical outcomes and serum

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infliximab levels in patients with inflammatory bowel disease (IBD): a meta-analysis. Am J Gastroenterol

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inflammatory diseases? Drug Discovery Today 2007;12:569-576.

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Hijazi Y, Welker H, Dorr AE, et al. Pharmacokinetics, safety, and tolerability of R411, a dual α4β1-α4β7

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Davenport RJ, Munday JR. Alpha4-integrin antagonism — an effective approach for the treatment of

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volunteers. J Clin Pharmacol Ther 2004;44:1368-1378.

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Miller DH, Weber T, Grove R, et al. Firategrast for relapsing remitting multiple sclerosis: a phase 2,

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Villablanca EJ, Cassani B, von Andrian UH, et al. Blocking lymphocyte localization to the gastrointestinal

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Takazoe M, Watanabe M, Kawaguchi T, et al. Oral alpha-4 integrin inhibitor (AJM300) in patients with

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Schreoder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to

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Author names in bold designate shared co-first authorship

ACCEPTED MANUSCRIPT Table 1. Demographics and baseline characteristics* Placebo (N = 51)

AJM300 (N = 51)

Total (N = 102)

Age — yr

42.6±13.4

41.7±11.4

42.2±12.4

Male sex — no. (%)

26 (51.0)

31 (60.8)

57 (55.9)

Body weight — kg

60.7±12.0

62.0±11.3

61.4±11.6

Current smoker — no. (%)

3 (5.9)

4 (7.8)

7 (6.9)

Duration of disease — yr

9.3±8.9

C-reactive protein — mg/dL

0.60±1.16

Disease extent — no. (%) Left sided

31 (60.8) 20 (39.2)

8.6±9.2

9.0±9.0

0.50±0.82

0.55±1.00

29 (56.9)

60 (58.8)

22 (43.1)

42 (41.2)

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Pancolitis or extensive

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†

7.7±1.2

7.8±1.2

7.8±1.2

Score 6-7 — no. (%)

22 (43.1)

20 (39.2)

42 (41.2)

Score 8-10 — no. (%)

29 (56.9)

31 (60.8)

60 (58.8)

2.0±0.8

2.1±0.8

2.1±0.8

1.6±0.6

1.6±0.6

1.6±0.6

2.2±0.4

2.1±0.3

2.1±0.4

1.9±0.2

1.9±0.2

1.9±0.2

11.8±3.3

11.8±2.8

11.8±3.0

46 (90.2)

46 (90.2)

92 (90.2)

1 (2.0)

2 (3.9)

3 (2.9)

Inadequate response to corticosteroids

1 (2.0)

2 (3.9)

3 (2.9)

Intolerance to corticosteroids

3 (5.9)

1 (2.0)

4 (3.9)

Components of Mayo Clinic score† Stool frequency subscore Rectal bleeding subscore Endoscopic subscore Physician’s global assessment subscore ‡

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Riley score

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Response to treatment of current relapse — no. (%) Inadequate response to 5-ASA

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Intolerance to 5-ASA

* Plus–minus values are means ±SD

The Mayo Clinic score ranges from 0 to 12, with higher scores indicating more severe disease. Each subscore ranges

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from 0 to 3. ‡

The Riley score ranges from 0 to 18, with higher scores indicating more severe disease.

ACCEPTED MANUSCRIPT Table 2. Summary of adverse events Overall safety population N = 102, No.(%) Placebo (n = 51) Event

AJM300 (n = 51)

Study drug-related

All

Study drug-related

Any adverse events

29 (56.9)

4 (7.8)

25 (49.0)

11 (21.6)

Discontinued because of adverse event

9 (17.6)

1 (2.0)

1 (2.0)

0

0

0

Nasopharyngitis

4 (7.8)

0

Headache

1 (2.0)

1 (2.0)

Upper respiratory tract inflammation

3 (5.9)

0

Ulcerative colitis

9 (17.6)

Nausea

1 (2.0)

Abdominal bloating

2 (3.9)

Upper abdominal pain

Serious adverse event

0

0

5 (9.8)

0

2 (3.9)

1 (2.0)

2 (3.9)

0

0

2 (3.9)

0

0

2 (3.9)

2 (3.9)

1 (2.0)

1 (2.0)

1 (2.0)

2 (3.9)

0

0

0

0

0

2 (3.9)

2 (3.9)

0

0

2 (3.9)

2 (3.9)

1 (2.0)

0

2 (3.9)

1 (2.0)

0

0

2 (3.9)

1 (2.0)

C-reactive protein increased

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White blood cell count increased

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Blood amylase increased

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Supplementary Table 1. Change from baseline to week 8 in the Mayo Clinic score and each subscore

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Placebo

AJM300

Difference from Placebo

95% CI

P-value‡

LSM* ±SE† (no.) Mayo Clinic score

-1.87±0.61 (44)

-4.05±0.60 (47)

-2.18±0.49

-3.14, -1.21

< .0001

Stool frequency

-0.36±0.22 (51)

-1.06±0.22 (51)

-0.70±0.17

-1.03, -0.36

< .0001

Rectal bleeding

-0.38±0.23 (51)

-0.96±0.23 (51)

-0.58±0.18

-0.93, -0.22

.0016

Endoscopic findings

-0.55±0.18 (44)

-1.01±0.18 (47)

-0.46±0.15

-0.75, -0.17

.0024

Physician’s global assessment

-0.53±0.16 (51)

-0.96±0.16 (51)

-0.43±0.13

-0.67, -0.18

.0009

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* LSM, least squares mean † SE, standard errors ‡ ANCOVA models were adjusted for stratification factors at randomization.

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Supplementary Table 2. Change from baseline to week 8 in Riley score and each subscore

ACCEPTED MANUSCRIPT Placebo

AJM300

Difference from Placebo

95% CI

P-value‡

LSM* ±SE† (no.) Riley score#

-0.94±1.03 (40)

-3.03±1.01 (46)

-2.09±0.86

-3.79, -0.39

.0017

Round cells in the lamina propria

-0.47±0.22 (42)

-0.93±0.21 (46)

-0.46±0.18

-0.81, -0.10

.012

Polymorphonuclear cells in the lamina propria

-0.33±0.26 (42)

-0.89±0.25 (46)

-0.56±0.21

-0.97, -0.14

.0096

Mucin depletion

-0.085±0.251 (42)

-0.50±0.25 (46)

-0.41±0.21

-0.82, -0.0052

.047

Crypt abscesses

-0.31±0.26 (42)

-0.65±0.25 (46)

-0.34±0.21

-0.77, 0.086

.12

Surface epithelial integrity

-0.38±0.30 (42)

-0.16±0.29 (46)

-0.22±0.24

-0.70, 0.26

.36

Crypt architectural irregularities

-0.17±0.19 (42)

-0.23±0.19 (46)

-0.053±0.160

-0.37, 0.27

.74

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