Larazotide Acetate for Persistent Symptoms of Celiac Disease Despite a Gluten-Free Diet: a Randomized Controlled Trial Daniel A

Gastroenterology 2015;148:1311–1319 CLINICAL—ALIMENTARY TRACT Larazotide Acetate for Persistent Symptoms of Celiac Disease Despite a Gluten-Free Diet: A Randomized Controlled Trial Daniel A. Lefﬂer,1 Ciaran P. Kelly,1 Peter H. R. Green,2 Richard N. Fedorak,3 Anthony DiMarino,4 Wendy Perrow,5 Henrik Rasmussen,5 Chao Wang,5 Premysl Bercik,6 Natalie M. Bachir,7 and Joseph A. Murray8

1The Celiac Center at Beth Israel Deaconess Medical Center, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts; 2Celiac Disease Center at Columbia University, New York, New York; 3Center of Excellence for Gastrointestinal Immunity and Inﬂammation Research, University of Alberta, Edmonton, Alberta, Canada; 4Thomas Jefferson CLINICAL AT University, Philadelphia, Pennsylvania; 5Alba Therapeutics Corporation, Baltimore, Maryland; 6McMaster University, Hamilton, Ontario, Canada; 7Essentia Health Duluth Clinic, Duluth, Minnesota; 8Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota

BACKGROUND & AIMS: Celiac disease (CeD) is a prevalent symptoms may have a variety of causes, one potential autoimmune condition. Recurrent signs and symptoms are source is sporadic gluten exposure,7 which may contribute common despite treatment with a gluten-free diet (GFD), yet no to persistent enteropathy, continued symptoms, and approved or proven nondietary treatment is available. reduced quality of life. METHODS: In this multicenter, randomized, double-blind, In CeD, paracellular permeability is increased by an in- placebo-controlled study, we assessed larazotide acetate 0.5, flammatory response to gluten entry into the intestinal mu- 1, or 2 mg 3 times daily to relieve ongoing symptoms in 342 cosa.8 Increased permeability promotes gluten peptide adults with CeD who had been on a GFD for 12 months or transport to gut-associated lymphoid tissue, initiating inflam- – longer and maintained their current GFD during the study. The matory cytokine release and T-cell recruitment.8 10 An intes- study included a 4-week placebo run-in, 12 weeks of treatment, tinal permeability–inflammation loop is established, leading to and a 4-week placebo run-out phase. The primary end point a multitude of gastrointestinal and systemic manifestations.11 was the difference in average on-treatment Celiac Disease Larazotide acetate is a novel, locally acting, nonsystemic, Gastrointestinal Symptom Rating Scale score. RESULTS: The – primary end point was met with the 0.5-mg dose of larazotide synthetic, 8 amino acid oral peptide, discovered during – acetate, with fewer symptoms compared with placebo by functional screening of synthetic Vibrio cholera related fi modified intention to treat (n ¼ 340) (analysis of covariance, peptides. Larazotide acetate is a rst-in-class tight junction P ¼ .022; mixed model for repeated measures, P ¼ .005). The (TJ) regulator under development as an adjunct to a GFD. 0.5-mg dose showed an effect on exploratory end points Larazotide acetate appears to prevent opening of intestinal including a 26% decrease in celiac disease patient-reported TJs by promoting TJ assembly and actin filament rear- outcome symptomatic days (P ¼ .017), a 31% increase in rangement, which prevents gluten from reaching the intes- improved symptom days (P ¼ .034), a 50% or more reduction tinal submucosa and triggering an inflammatory response from baseline of the weekly average abdominal pain score for 6 (Supplementary Figure 2).8,12 or more of 12 weeks of treatment (P ¼ .022), and a decrease in Nonclinical studies with larazotide acetate have shown the nongastrointestinal symptoms of headache and tiredness proof-of-concept of TJ regulation including the inhibition of (P ¼ .010). The 1- and 2-mg doses were no different than gliadin-induced TJ alteration, macrophage recruitment, and placebo for any end point. Safety was comparable with placebo. increases in intestinal permeability.8,12,13 In 4 prior clinical CONCLUSIONS: Larazotide acetate 0.5 mg reduced signs and trials, larazotide acetate showed a safety profile comparable symptoms in CeD patients on a GFD better than a GFD alone. with placebo.14–16 In initial clinical trials using gluten Although results were mixed, this study was a successful trial of challenge, larazotide acetate prevented gluten-induced a novel therapeutic agent targeting tight junction regulation in symptoms and blunted increases in anti-tissue trans- patients with CeD who are symptomatic despite a GFD. glutaminase (tTG) antibodies, interferon-g, and intestinal Clinicaltrials.gov: NCT01396213. permeability.14–16 These results provided the foundation for the current phase IIb study. Keywords: Celiac Disease; Gluten; Therapeutic; Tight Junction.

Abbreviations used in this paper: ANCOVA, analysis of covariance; BMI, eliac disease (CeD), a genetic autoimmune condition, body mass index; CeD, celiac disease; CeD-GSRS, Celiac Disease C affects approximately 1% of the Western popula- Gastrointestinal Symptom Rating Scale; CeD PRO, Celiac Disease 1,2 Patient-Reported Outcome; DGP, deamidated gliadin peptide; GFD, tion. CeD is triggered by ingestion of gluten-containing gluten-free diet; GI, gastrointestinal; MMRM, mixed model for repeated foods and managed by a gluten-free diet (GFD).3,4 Recur- measures; TJ, tight junction; tTG, tissue transglutaminase. rent CeD signs and symptoms resulting from inadvertent or deliberate gluten exposure have been reported in approxi- © 2015 by the AGA Institute 5,6 0016-5085/$36.00 mately 70% of CeD patients on a GFD. Although persistent http://dx.doi.org/10.1053/j.gastro.2015.02.008 1312 Lefﬂer et al Gastroenterology Vol. 148, No. 7

Despite being a common condition that often responds performed centrally using the INOVA (San Diego, CA) Quanta- incompletely to GFD, there is currently no approved non- Flash assay at Mayo Clinical Laboratories. The cut-off value for dietary treatment for CeD.17,18 This multicenter, randomized, levels that were positive for tTG IgA and IgG were 4.0 and 6.0, placebo-controlled trial was a large clinical trial conducted in respectively. The cut-off value for levels that were for positive CeD patients and was designed to assess the efﬁcacy and deamidated gliadin peptide (DGP) IgA and IgG were 20. To safety of larazotide acetate as an adjunct to a GFD in adult evaluate study-related changes in serologic titers, patients with patients with persistent symptoms despite maintenance of a undetectable anti-tTG and anti-DGP antibody levels were long-term GFD. A secondary objective was to validate the excluded. Patients experienced at least 1 gluten-related symp- Celiac Disease Patient-Reported Outcome (CeD PRO) instru- tom (diarrhea, abdominal pain, bloating, nausea) in the month ment as a daily measure of therapeutic effects. before screening, and at screening were required to have a qualifying score of 2 or higher, reﬂecting “mild discomfort” on

LNCLAT CLINICAL the CeD domains of the Gastrointestinal Symptom Rating Scale19 Materials and Methods (CeD-GSRS). The GSRS and CeD-GSRS, which contains 10 items The protocol was approved by relevant institutional review from the GSRS, diarrhea, abdominal pain, and indigestion domains (Supplementary Appendix A), have been used in multiple boards. Patients provided written informed consent, the study – trials of CeD14,20 22 and other gastrointestinal (GI) disor- was conducted according to Good Clinical Practice, and was 23,24 registered on Clinicaltrials.gov (NCT01396213). ders. All survey data were collected daily from patients using an electronic clinical outcome assessment data collection device (Bracket Global, Wayne, PA). Patients Exclusion criteria included refractory CeD, severe CeD com- Entry criteria were as follows: age 18–75 years, body mass plications (eg, enteropathy-associated T-cell lymphoma), other index (BMI) of 16–45 kg/m2, CeD confirmed by intestinal biopsy chronic inflammatory GI disease (eg,inflammatory bowel disease), or capsule endoscopy (capsule endoscopy was the entry criteria diabetes, or autoimmune, psychiatric, or neurologic disease that for 7 of 342 patients randomized) plus positive serology 12 could interfere with assessments. Smoking, pregnancy or breast- months before study entry, maintenance of a GFD for 12 or more feeding, previous exposure to larazotide acetate, concomitant use consecutive months before screening, and adherence to current of systemic or intestinal immune suppressants, continuous anti- GFD on study. Underweight patients (BMI, 16–18.5 kg/m2) were biotics, nonsteroidal anti-inflammatory drugs, and medications included because these patients were believed by the in- that alter gastric pH or intestinal permeability were prohibited. vestigators to reflect patients who were underweight as a result of active celiac disease and thus would be most likely to benefit from therapy underweight due to active celiac disease and thus Study Design and Procedures would be likely to benefit from therapy while not at increased This 20-week study was conducted in 3 phases: a 4-week risk of complications of severe malabsorption owing to severe single-blind placebo run-in phase, a 12-week double-blind malabsorption or other conditions. All celiac serologies were treatment phase, and a 4-week placebo run-out phase

Figure 1. Primary end point: average on-treatment scores on the CeD-GSRS. The 0.5-mg larazotide acetate dose met the primary end point. June 2015 Larazotide Acetate in Celiac Disease 1313

(Figure 1). A qualifying score of 2 or higher on the CeD-GSRS laboratory parameters (hematology, chemistry, urinalysis) (Supplementary Appendix A) was required for randomization. (Supplementary Table 1), electrocardiograms, and vital signs. Randomization and masking. Patients were stratified into 4 groups according to sex (85%:15%, female:male) and Statistical Analyses baseline CeD-GSRS scores (<3or3). Randomization was Estimates for treatment effect and variability of changes performed using permuted-block randomization and was kept from baseline in CeD-GSRS scores in the CLIN1001-006 study14 confidential until the study was unblinded. Randomization was were used to determine sample size. Based on a SD of 1:1:1:1 to larazotide acetate 0.5-mg, 1-mg, or 2-mg capsules, or s ¼ 0.548 and a type 1 error rate of a ¼ .05, and assuming a placebo in identical capsules. 14% drop-out rate, 80 subjects per treatment group (320 total) After the 4-week placebo run-in, study drugs were self- would provide 80% power to detect a 0.3-point change from administered 3 times daily, 15 minutes before meals. Patients baseline difference in the CeD-GSRS score between larazotide returned unused capsules for drug compliance assessment. acetate doses and placebo. Blood was collected at visits 2, 4, 6, and 7, or early termi- Efficacy analyses included all patients receiving 1 or more nation for chemistry, hematology, anti-tTG, and anti-DGP doses of study drug during double-blind treatment and had 1 or antibodies. CLINICAL AT more postbaseline assessment (modified intent-to-treat popu- Patients rated CeD symptoms daily on the CeD PRO and lation). Efficacy results for larazotide acetate doses were weekly with the CeD-GSRS. The CeD PRO developed by the compared independently with placebo. An analysis of covari- sponsor in accordance with the US Food and Drug Adminis- ance (ANCOVA) was used for the primary, secondary, and tration’s Study Endpoints and Labeling Development guide- exploratory end points related to GSRS, CeD-GSRS, and CeD lines25 initially was validated in this trial (Supplementary PRO scores, with treatment, sex, baseline CeD-GSRS randomi- Appendix B). Patients completed the CeD PRO using an elec- zation stratum, and randomization cohort as fixed effects, and tronic clinical outcome assessment data collection device. baseline score as a covariate. Sensitivity analyses were con- Responses were scored on an 11-point Likert scale (range, ducted using a mixed model for repeated measures (MMRM). 0–10), with higher scores indicating greater symptom MMRM analyses included treatment and study week as main severity. Results represent averages for each item in a domain effects, sex, baseline CeD-GSRS randomization stratum, or subdomain, aggregated over a 7-day period. randomization cohort, and baseline score as covariates, and weekly CeD PRO scores as repeated measures. Baseline score End Point Measures was the last nonmissing observation before the first dose of The primary end point was the difference in average investigational drug. The proportion of patients with a 50% or weekly on-treatment CeD-GSRS score for each dose vs pla- greater reduction from the baseline CeD PRO abdominal cebo, over the 12-week active treatment period. The CeD-GSRS domain scores for 6 or more of 12 weeks was assessed using captures treatment effect over time in this disease charac- the Cochrane–Mantel–Haenszel test for between-treatment terized by chronic and variable flares with episodic symp- comparison, stratified by sex, baseline CeD-GSRS, randomiza- toms.14,20,26 Symptom improvement was chosen as a primary tion stratum, and randomization cohort. end point in accordance with regulatory guidance.25 Second- Safety assessments were performed for all patients who ary end points were a change from baseline in CeD-GSRS received 1 or more doses of study drug. score, average weekly on-treatment score, and a change from baseline in both the CeD PRO GI and abdominal domain Role of the Funding Source scores. The principal investigator and leading co-investigators Exploratory end points included average weekly on- designed the study in collaboration with Alba Therapeutics treatment differences in total and individual GSRS domain Corporation and Cephalon/Teva, conducted the study, and scores; number of patients experiencing a 50% or more provided oversight for data collection. Statistical analysis reduction from the baseline weekly average CeD PRO abdom- including sensitivity analysis was designed jointly a priori by inal domain scores for 6 or more of 12 weeks; average on- the authors and Alba Therapeutics. Data analysis was per- treatment weekly number of bowel movements and stool formed by Chao Wang, PhD, and John Han, PhD, of PharmaData consistency measured using the Bristol Stool Form Scale; Associates, funded by Alba and Cephalon/Teva. All authors average on-treatment weekly number of CeD PRO GI symp- contributed to data interpretation and writing and editing the fi tomatic days, de ned as the mean CeD PRO abdominal domain manuscript. Drs. Leffler and Murray had full access to all study scores of 3 or higher, or diarrhea and loose stool domain data, contributed equally to manuscript preparation, and had score of 3 or higher; average on-treatment weekly number of final responsibility for the publication. improved symptom days, defined as the mean CeD PRO abdominal domain scores of 1.5 or less, and diarrhea and loose stool domain score of 1.5 or less; CeD PRO non-GI domain Results (headache and tiredness) scores; and changes in anti-tTG and anti-DGP antibody levels over the treatment phase. Study Population GFD compliance was assessed using the Gluten-Free Diet This study was conducted at 74 North American sites. A Compliance Questionnaire (Supplementary Appendix C), total of 783 patients were screened and 454 participants administered at week 16 and assessed voluntary and/or entered the placebo run-in phase (Supplementary Figure 1). inadvertently ingested gluten on-study. At the end of the placebo run-in phase, 342 patients Safety assessments included the frequency and severity of were randomized to receive placebo (n ¼ 84), or larazotide treatment-emergent adverse events (AEs), serious AEs, clinical acetate 0.5 mg (n ¼ 86), 1 mg (n ¼ 85), or 2 mg (n ¼ 87) 1314 Leffler et al Gastroenterology Vol. 148, No. 7

3 times per day. Two randomized patients were lost to phase and then increased at week 0 when patients not follow-up evaluation before receiving the drug, leaving meeting the mean weekly CeD-GSRS score eligibility 340 patients for modified intent-to-treat analysis. The threshold were discontinued (Figure 1). Symptomatic discontinuation rate during double-blind treatment was improvement with larazotide acetate 0.5 mg was evident by consistent across treatment groups (11 patients per arm), treatment week 2 and was sustained over the 12-week most frequently owing to AEs (n ¼ 18) (Supplementary treatment period. Higher larazotide acetate doses were not Table 2) or unwillingness to participate (n ¼ 14). The significantly different from placebo. Per-protocol results mean treatment duration for all patients was 80 days (SD, were similar for the primary end points with an improved 15.5 days). Patient characteristics were similar across average on-treatment CeD-GSRS score vs placebo at the treatment groups (Table 1). 0.5-mg larazotide acetate dose (ANCOVA, P ¼ .007; MMRM, Patients reported multiple CeD symptoms during the P ¼ .001). Similarly, results remained significant after LNCLAT CLINICAL placebo run-in phase: 97% reported gas, 92% reported adjustment for both age (ANCOVA, 0.020; MMRM, 0.005) bloating, 79% reported abdominal cramping, 80% reported and BMI (ANCOVA, 0.017; MMRM, 0.004). pain, and 67% reported loose stools. Constipation, nausea, Secondary end points. Numeric differences in favor diarrhea, and vomiting were reported by 57%, 50%, 44%, of larazotide acetate 0.5 mg were observed in average on- and 4% of patients, respectively. Non-GI symptoms, head- treatment scores, although not all achieved statistical sig- ache and tiredness, each were reported by 70% of patients. nificance (Table 2). Improvement from baseline in the mean CeD-GSRS score was greater in the larazotide acetate 0.5-mg group (MMRM, P ¼ .041). Average on-treatment scores and Efficacy changes from baseline scores in the CeD PRO abdominal and Primary end point. The primary end point, improved GI domains favored the 0.5-mg treatment arm but were not average on-treatment CeD-GSRS score vs placebo, was met statistically significant. No significant improvements were at the 0.5-mg larazotide acetate dose (ANCOVA, P ¼ .022; noted for any secondary end point at higher larazotide ac- MMRM, P ¼ .005), but not for the 1-mg or 2-mg doses etate doses. (Table 2). Consistent with prior studies,14 the mean CeD- Exploratory end points. Exploratory end points also GSRS scores trended down during the placebo run-in supported the effectiveness of 0.5-mg larazotide acetate 3

Table 1.Patient Demographics and Disease Characteristics

Larazotide acetate (n ¼ 258)

Placebo 0.5 mg TID 1.0 mg TID 2.0 mg TID Total (n ¼ 84) (n ¼ 86) (n ¼ 85) (n ¼ 87) (N ¼ 342)

General characteristics Mean age [SD], y 45.5 [14.6] 44.2 [14.4] 46.2 [15.0] 44.7 [16.0] 45.2 [15.0] Female, n (%) 69 (82.1) 73 (84.9) 71 (83.5) 72 (82.8) 285 (83.3) White, n (%) 83 (98.8) 84 (97.7) 85 (100) 86 (98.9) 338 (98.8) Weight, mean [SD], kg 74.3 [16.8] 75.5 [16.0] 72.2 [15.4] 73.4 [15.3] 73.9 [15.8] Height, mean [SD], cm 167.3 [9.6] 165.4 [8.9] 166.2 [8.6] 167.4 [8.5] 166.5 [8.9] Body mass index, mean [SD], kg/m2 26.6 [5.4] 27.6 [5.3] 26.0 [4.6] 26.1 [4.6] 26.6 [5.0] Clinical characteristics Time since CeD symptom onset, mean [SD], mo 145.5 [141.1] 149.2 [144.8]a 162.9 [150.6]a 174.6 [174.7] 158.1 [158.3]a Time since CeD diagnosis,b mean [SD], mo 60.3 [51.3] 58.0 [50.1]c 66.7 [68.2]c 71.5 [65.07]c 64.1 [59.2]c Time on most recent GFD,b mean [SD], mo 62.1 [58.7] 60.3 [60.1] 71.4 [86.2] 70.9 [63.7] 66.2 [68.0] Symptoms at diagnosis, n (%) Diarrhea 67 (79.8) 68 (79.1) 69 (81.2) 64 (73.6) 268 (78.4) Bloating 75 (89.3) 77 (89.5) 78 (91.8) 78 (89.7) 308 (90.1) Abdominal distention/ stomach swelling 64 (76.2) 62 (72.1) 68 (80.0) 68 (78.2) 262 (76.6) Recurrent abdominal pain 72 (85.7) 78 (90.7) 76 (89.4) 75 (86.2) 301 (88.0) Scores at baseline, mean [SD] CeD-GSRS 3.26 [0.92] 3.05 [0.84] 3.21 [0.77] 3.25 [0.96] Not calculated Total GSRS 3.03 [0.82] 2.89 [0.80] 3.03 [0.75] 3.09 [0.89] CeD PRO abdominal domain 2.56 [1.71] 2.19 [1.34] 2.63 [1.59] 2.90 [1.58] CeD PRO GI domain 2.27 [1.46] 1.86 [1.10] 2.23 [1.29] 2.35 [1.29] Average weekly number of bowel movementsd 9.00 [7.11] 7.59 [4.62] 7.25 [4.78] 7.95 [5.45]

NOTE. There were no signiﬁcant differences between cohorts in any measure. TID, 3 times daily. aData missing for 1 patient each in the 0.5-mg and 1.0-mg TID group. bAssessed at screening visit. cData missing for 1 patient in each of the 3 larazotide acetate treatment groups. dAs recorded in the Bristol Stool Form Scale daily diary. June 2015 Larazotide Acetate in Celiac Disease 1315

Table 2.Efﬁcacy End Points in the Modiﬁed Intent-to-Treat Population

Placebo (n ¼ 84) 0.5 mg TID (n ¼ 86) 1.0 mg TID (n ¼ 85) 2.0 mg TID (n ¼ 87)

Primary end point Average on-treatment CeD-GSRS score, 2.88 [0.72] 2.59 [0.7] 2.84 [0.8] 2.92 [0.8] mean [SD] P value vs placebo–ANCOVA, MMRM - P ¼ .022, P ¼ .005 NS, NS NS, NS Secondary end points CeD-GSRS score change from baseline to -0.50 [1.0] -0.54 [0.9] -0.40 [1.0] -0.37 [1.0] end of treatment, mean [SD] P value vs placebo - ANCOVA, MMRM NS, P ¼ .041 NS, NS NS, NS Average on-treatment score - CeD PRO 2.27 [1.3] 2.04 [1.3] 2.59 [1.6] 2.77 [1.5] abdominal domain, mean [SD] P value vs placebo–ANCOVA, MMRM NS, NS NS, P ¼ .036 NS, NS Average on-treatment score– CeD PRO GI 2.05 [1.19] 1.69 [1.01] 2.21 [1.33] 2.31 [1.26] domain, mean [SD] CLINICAL AT P value vs placebo–ANCOVA, MMRM NS, NS NS, NS NS, NS Change from baseline to end treatment - -0.43 [1.73] -0.26 [1.47] -0.18 [1.27] -0.16 [1.32] CeD PRO Abdominal domain, mean [SD] P value vs placebo–ANCOVA, MMRM NS, NS NS, NS .023, NS Change from baseline to end treatment - -0.38 [1.45] -0.28 [1.24] -0.13 [1.00] 0.00 [1.23] CeD PRO GI Domain, mean [SD] P value vs placebo–ANCOVA, MMRM NS, NS NS, NS .013, .028 Exploratory end points Average on-treatment total GSRS score, 2.70 [0.7] 2.47 [0.7] 2.70 [0.8] 2.76 [0.8] mean [SD] P value vs placebo–ANCOVA, MMRM .017, .004 NS, NS NS, NS Average on-treatment scores of individual GSRS domains, mean [SD] ANCOVA P value vs placebo Diarrhea 2.77 [1.2] 2.40 [1.1], NS 2.61 [1.1], NS 2.74 [1.21], NS Indigestion 3.18 [0.8] 2.87 [0.8] .029 3.18 [1.0], NS 3.23 [0.88], NS Constipation 2.58 [1.1] 2.45 [1.0], P ¼ .050 2.64 [1.1], NS 2.61 [1.14], NS Abdominal pain 2.58 [0.9] 2.42 [0.8], P ¼ .024 2.62 [0.8], NS 2.68 [0.94], NS Reﬂux 2.00 [1.1] 1.87 [0.9], NS 2.08 [1.1], NS 2.19 [1.07], NS - 50% reduction from baseline in weekly average score for 6 weeks, n (%) CMH P value vs placebo CeD-GSRS 1 (1.2) 6 (7.2), NS 1 (1.2), NS 1 (1.2), NS CeD PRO abdominal domain 12 (14.3) 24 (28.6), P ¼ .022 12 (14.3), NS 17 (19.5), NS CeD PRO GI domain 14 (16.7) 29 (34.5) P ¼ .002 15 (17.9), NS 15 (17.2), NS Average weekly number of bowel 8.96 [7.0] 6.90 [4.2] 7.20 [4.2] 8.72 [5.9] movements (BSFS), mean [SD] ANCOVA P value vs placebo NS NS NS Average on-treatment weekly number of 2.38 [2.01] 1.73 [1.7] 2.48 [2.0] 2.63 [1.8] CeD PRO symptomatic days ANCOVA P value vs placebo P ¼ .017 NS NS Average on-treatment weekly number of 1.99 [2.0] 2.51 [1.9] 1.80 [1.9] 1.46 [1.6] CeD PRO improved symptom days ANCOVA P value vs placebo P ¼ .034 NS NS Average on-treatment CeD PRO non-GI 2.75 [1.55] 2.44 [1.4] 2.73 [1.4] 3.11 [1.8] domain score, mean [SD] ANCOVA, MMRM P value vs placebo - NS, P ¼ .010 NS, NS NS, NS

NOTE. All statistical analyses include baseline score, sex, baseline GSRS stratum, and randomization cohort as covariates. BSFS, Bristol Stool Form Scale; CMH, Cochrane–Mantel–Haenszel. times per day (Table 2). The average on-treatment total larazotide acetate group (ANCOVA, NS; MMRM, P ¼ .010) GSRS score decreased in the 0.5-mg larazotide acetate group (Supplementary Figure 1). vs placebo (ANCOVA, P ¼ .017; MMRM, P ¼ .004). The Patients receiving 0.5 mg larazotide acetate had 26% average on-treatment CeD PRO non-GI domain scores for fewer CeD PRO GI symptomatic days (deﬁned a priori as a headache and tiredness also were lower in the 0.5-mg day with a mean score of 3 on either the abdominal 1316 Lefﬂer et al Gastroenterology Vol. 148, No. 7

symptom or diarrhea/loose stool domains) than patients Safety and Tolerability receiving placebo. The average on-treatment weekly number Larazotide acetate safety and tolerability were compa- of CeD PRO GI symptomatic days was 1.73 for 0.5 mg lar- rable with placebo at all dose levels (Supplementary azotide acetate vs 2.38 for placebo (ANCOVA, P ¼ .017) Tables 1 and 3). No significant changes were noted in vital (Figure 2), indicating reductions of 0.56 days/wk, and an signs, laboratory measures, or electrocardiograms at any overall reduction of 6.72 symptomatic days over the treat- larazotide acetate dose. The most frequent treatment- ment period vs placebo. This finding was accentuated in a emergent AEs by system organ class were GI disorders subanalysis of patients with 3 or more symptomatic days per and were equally frequent in all treatment groups. No sig- week at baseline (n ¼ 60). In this cohort, there was a nificant change in iron status was noted with treatment in reduction of 1.89 days per week in the 0.5-mg arm compared any of the treatment groups. There were no drug-related with 0.58 days/wk in the placebo arm, a net decrease of 15.72 LNCLAT CLINICAL serious AEs. fewer symptomatic days during the 12 weeks of treatment. Similarly, in the 0.5-mg larazotide arm there was a 31% increase in average on-treatment weekly number of CeD Discussion PRO improved symptom days (2.51 vs 1.99 with placebo; Larazotide acetate 0.5 mg 3 times per day improved ANCOVA, P ¼ .034), an increase of 0.49 days/wk or 5.88 signs and symptoms of CeD among patients with persistent days over the treatment period. The weekly average CeD symptoms despite a GFD. Conversely, higher treatment PRO abdominal domain scores were reduced 50% or more doses showed no effect. This was a large randomized from baseline for 6 or more weeks in the larazotide acetate controlled trial in CeD patients of a novel TJ therapeutic 0.5-mg arm (ANCOVA, P ¼ .022). agent for CeD that met its primary end point. Larazotide In the subset of patients reporting the highest number of acetate reduced GI and non-GI symptoms of CeD, decreased GI symptomatic days scores (5–7/wk), there was a median the weekly number of CeD PRO GI symptomatic days, reduction of 2.21 GI symptomatic days per week with increased the number of improved symptom days, and 0.5 mg larazotide acetate, vs a median increase of 0.08 reduced abdominal domain symptom severity scores by days/wk with placebo. 50% or more for at least half of the active treatment period. There was no change or worsening from baseline anti- In the 0.5-mg larazotide acetate group, the subset of tTG or anti-DGP antibody titers in any treatment group patients reporting the highest number of GI symptomatic (Supplementary Figure 3). On the Gluten-Free Diet days scores (5–7/wk) at baseline experienced approxi- Compliance Questionnaire, 35% and 59% of patients in the mately 30 fewer GI symptomatic days in contrast to an in- larazotide acetate 0.5-mg group reported having voluntary crease of approximately 10 GI symptomatic days in the or accidental gluten exposure, respectively, compared with placebo group over the 12-week treatment period. This 27% and 52% in the placebo group. level of improvement is similar to what is regarded as

Figure 2. Larazotide acetate 0.5 mg 3 times per day reduced the average on-treatment weekly number of CeD PRO symptomatic days. Symptomatic days defined a priori as a day with a mean score of 3 on either the abdominal symptom or diarrhea/loose stool domains. June 2015 Larazotide Acetate in Celiac Disease 1317 clinically meaningful in other conditions with episodic but may involve peptide aggregation at higher doses, symptoms.27–29 reducing activity in vivo. Results of this trial are consistent with previous studies, Second, although prevention of an increase of celiac which showed a reduction in gluten-induced signs and antibody titers despite reported gluten exposure is reas- symptoms during a gluten challenge.14–16 An effective suring, we did not document a reduction in serologic titers adjunct to the GFD has the potential to transform CeD in any treatment arm. This likely is because the majority of treatment and improve the lives of patients. Practicing a participants entered the study with serologic titers in the strict GFD is a continuous burden and often an unsuccessful normal range, and thus were not expected to be responsive struggle for many people with CeD.30–32 Gluten exposure is to change over the course of the real-life study. Larazotide one of the common causes of ongoing or recurrent symp- acetate did prevent an increase of anti-tTG antibody titers toms in patients with celiac disease on a GFD. The preva- during the gluten challenge, suggesting disease modifica- lence of persistent symptoms suggests that there is a tion.14 Whether larazotide acetate may result in a reduction substantial unmet medical need for pharmacologic ap- of serologic titers over a longer time period in individuals proaches that can improve CeD signs and symptoms beyond with persistently high serologic titers will be evaluated in CLINICAL AT what is possible with the GFD alone.30–32 future studies. It also is possible that larazotide acetate may CeD PRO results show that moderate-to-severe CeD nonspecifically alleviate symptoms because celiac disease symptoms are common despite a GFD. In the 4-week may co-exist with other common conditions including irri- placebo-run in phase, GI symptoms were reported by table bowel syndrome. Although efficacy during gluten more than 90% of patients, and more than two thirds of challenge is suggestive of a mechanism of action relevant to patients reported tiredness and headache, suggesting that celiac disease, the specificity of larazotide acetate for celiac extraintestinal symptoms have a significant impact on well- disease and its utility in other conditions remains to be being in patients with CeD. The placebo run-in phase determined. In addition, there were few participants showed substantial day-to-day variability in symptoms. This enrolled older than the age of 65, so efficacy in this age was not well described in the literature but it is not sur- group cannot be inferred from the current study. Finally, prising because symptoms may be linked to gluten expo- histology was not an end point evaluated in this study. sure, which is highly variable and intermittent for patients Although the role of histology for celiac diagnosis is clear, its attempting a GFD. Although the observed reduction of GI utility in monitoring is controversial because there is poor symptoms may reflect nonspecific effects, this is consistent correlation between symptoms, serology, histology, and with the proposed mechanism of action of larazotide acetate quality of life in treated patients, and recent data have of limiting gluten entry into the lamina propria by pre- suggested that ongoing intestinal inflammation is not venting intestinal TJ opening. Although larazotide acetate is associated with significant long-term complications.34,35 a locally acting peptide restricted to the luminal surface of Furthermore, although the kinetics of histologic deteriora- the small intestine,16 the 0.5-mg dose also reduced tiredness tion during gluten challenge are well understood, the de- and headache. This suggests that larazotide acetate might gree, timing, and clinical significance of improvements with reduce extraintestinal symptoms, potentially through adjunctive therapy in treated patients is unknown. Because reduced local inflammation, with subsequent reduction in of these limitations, histology may not be an appropriate cytokine release. primary end point for clinical trials designed to improve Although we believe that this study supports the safety symptoms in patients with CeD on the GFD. and efficacy of TJ modulation as a therapeutic modality in Regulating TJ activity represents a potential novel mo- celiac disease, we do recognize potential limitations. First, dality for treatment of diseases that involve epithelial bar- direct comparison between the present study and the prior riers. Altered intestinal permeability is associated with gluten challenge studies is difficult because of the lack of a many autoimmune disorders including Crohn’s disease, dose response and the different doses used between studies. multiple sclerosis, and type 1 diabetes.36 The potential In the first study published, doses of 0.25 mg, 1 mg, 4 mg, therapeutic activity of larazotide acetate in other disorders and 8 mg 3 times per day were administered and both the associated with TJ dysregulation should be explored further. 0.25-mg and 4-mg doses prevented symptoms recorded by In summary, larazotide acetate 0.5 mg reduced GI and the GSRS; only the 0.25-mg dose prevented symptoms non-GI symptoms, resulting in fewer GI symptomatic days. recorded by the CeD-GSRS.15 In the second gluten challenge Furthermore, in studies now including a total of 828 sub- study, 1 mg, 4 mg, and 8 mg 3 times per day were admin- jects, larazotide acetate has not been associated with safety istered and only the 1-mg group was effective in amelio- concerns. This study represents a therapeutic trial in CeD rating symptoms induced by gluten challenge measured by patient to meet a primary end point of reducing symptoms the CeD-GSRS.14,15 Although the results of this study overall in patients attempting to maintain a GFD. Larazotide acetate, are consistent with prior studies, we acknowledge that the the first of a novel class of agents targeting TJ regulation, dose range chosen was based on earlier gluten challenge thus may represent an important therapeutic option for CeD data, and, for this reason, the optimal dose to study during a patients with persistent symptoms, although the overall real-life study may not be fully understood. This inverse efficacy and risk/benefit ratio of this therapy remain to be dose effect is not unique to larazotide acetate and has been assessed fully. Our results contribute to a growing body of seen for other minimally or nonabsorbed oral peptides.33 evidence of the safety and efficacy of larazotide acetate and Why higher doses appear to be less effective is unclear, support further investigation. 1318 Leffler et al Gastroenterology Vol. 148, No. 7

Supplementary Material of single doses of AT-1001 in coeliac disease subjects: a Note: To access the supplementary material accompanying proof of concept study. Aliment Pharmacol Ther 2007; 26:757–766. this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http://dx.doi.org/10.1053/ 17. Crowe SE. Management of celiac disease: beyond the gluten-free diet. Gastroenterology 2014;146:1594–1596. j.gastro.2015.02.008. 18. Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology 2009; – References 137:1912 1933. 19. Svedlund J, Sjodin I, Dotevall G. GSRS–a clinical rating 1. Mooney PD, Hadjivassiliou M, Sanders DS. Coeliac dis- scale for gastrointestinal symptoms in patients with irri- ease. BMJ 2014;348:g1561. table bowel syndrome and peptic ulcer disease. Dig Dis LNCLAT CLINICAL 2. Green PH, Cellier C. Celiac disease. N Engl J Med 2007; Sci 1988;33:129–134. – 357:1731 1743. 20. Mustalahti K, Lohiniemi S, Collin P, et al. Gluten-free diet 3. Ludvigsson JF, Bai JC, Biagi F, et al. Diagnosis and and quality of life in patients with screen-detected celiac management of adult coeliac disease: guidelines from disease. Eff Clin Pract 2002;5:105–113. the British Society of Gastroenterology. Gut 2014; 21. Lohiniemi S, Maki M, Kaukinen K, et al. Gastrointestinal – 63:1210 1228. symptoms rating scale in coeliac disease patients on 4. Ludvigsson JF, Leffler DA, Bai JC, et al. The Oslo defi- wheat starch-based gluten-free diets. Scand J Gastro- nitions for coeliac disease and related terms. Gut 2013; enterol 2000;35:947–949. – 62:43 52. 22. Leffler D, Schuppan D, Pallav K, et al. Kinetics of the 5. Leffler DA, Dennis M, Hyett B, et al. Etiologies and pre- histological, serological and symptomatic responses to dictors of diagnosis in nonresponsive celiac disease. Clin gluten challenge in adults with coeliac disease. Gut 2013; Gastroenterol Hepatol 2007;5:445–450. 62:996–1004. 6. Hall NJ, Rubin GP, Charnock A. Intentional and inad- 23. Kulich KR, Madisch A, Pacini F, et al. Reliability and vertent non-adherence in adult coeliac disease. A cross- validity of the Gastrointestinal Symptom Rating Scale sectional survey. Appetite 2013;68:56–62. (GSRS) and Quality of Life in Reflux and Dyspepsia 7. Catassi C, Fabiani E, Iacono G, et al. A prospective, (QOLRAD) questionnaire in dyspepsia: a six-country double-blind, placebo-controlled trial to establish a safe study. Health Qual Life Outcomes 2008;6:12. gluten threshold for patients with celiac disease. Am J 24. Verma S, Giaffer MH. Helicobacter pylori eradication Clin Nutr 2007;85:160–166. ameliorates symptoms and improves quality of life in pa- 8. Gopalakrishnan S, Durai M, Kitchens K, et al. Larazotide tients on long-term acid suppression. A large prospective acetate regulates epithelial tight junctions in vitro and study in primary care. Dig Dis Sci 2002;47:1567–1574. in vivo. Peptides 2012;35:86–94. 25. McLeod LD, Coon CD, Martin SA, et al. Interpreting 9. Lammers KM, Lu R, Brownley J, et al. Gliadin induces an patient-reported outcome results: US FDA guidance and increase in intestinal permeability and zonulin release by emerging methods. Expert Rev Pharmacoecon Out- binding to the chemokine receptor CXCR3. Gastroen- comes Res 2011;11:163–169. terology 2008;135:194–204 e3. 26. Kurppa K, Paavola A, Collin P, et al. Benefits of a gluten- 10. Turner JR. Molecular basis of epithelial barrier regulation: free diet for asymptomatic patients with serologic from basic mechanisms to clinical application. Am J markers of celiac disease. Gastroenterology 2014;147: Pathol 2006;169:1901–1909. 610–617. 11. Matysiak-Budnik T, Candalh C, Dugave C, et al. Alterations 27. Aurora SK, Winner P, Freeman MC, et al. Onabotuli- of the intestinal transport and processing of gliadin peptides numtoxinA for treatment of chronic migraine: pooled in celiac disease. Gastroenterology 2003;125:696–707. analyses of the 56-week PREEMPT clinical program. 12. Gopalakrishnan S, Tripathi A, Tamiz AP, et al. Larazotide Headache 2011;51:1358–1373. acetate promotes tight junction assembly in epithelial 28. Macdougall JE, Johnston JM, Lavins BJ, et al. An eval- cells. Peptides 2012;35:95–101. uation of the FDA responder endpoint for IBS-C clinical 13. Black KE, Murray JA, David CS. HLA-DQ determines the trials: analysis of data from linaclotide phase 3 clinical response to exogenous wheat proteins: a model of trials. Neurogastroenterol Motil 2013;25:481–486. gluten sensitivity in transgenic knockout mice. J Immunol 29. Lembo AJ, Schneier HA, Shiff SJ, et al. Two randomized 2002;169:5595–5600. trials of linaclotide for chronic constipation. N Engl J Med 14. Kelly CP, Green PH, Murray JA, et al. Larazotide acetate 2011;365:527–536. in patients with coeliac disease undergoing a gluten 30. Shah S, Akbari M, Vanga R, et al. Patient perception of challenge: a randomised placebo-controlled study. treatment burden is high in celiac disease compared with Aliment Pharmacol Ther 2013;37:252–262. other common conditions. Am J Gastroenterol 2014; 15. Leffler DA, Kelly CP, Abdallah HZ, et al. A randomized, 109:1304–1311. double-blind study of larazotide acetate to prevent the 31. Barratt SM, Leeds JS, Sanders DS. Quality of life in activation of celiac disease during gluten challenge. Am J coeliac disease is determined by perceived degree of Gastroenterol 2012;107:1554–1562. difficulty adhering to a gluten-free diet, not the level of 16. Paterson BM, Lammers KM, Arrieta MC, et al. The safety, dietary adherence ultimately achieved. J Gastrointestin tolerance, pharmacokinetic and pharmacodynamic effects Liver Dis 2011;20:241–245. June 2015 Larazotide Acetate in Celiac Disease 1319

32. Aziz I, Evans KE, Papageorgiou V, et al. Are patients with by Daniel Leffler, with writing assistance from Beth Llewellyn, BA, and Kate Huber, MBA. coeliac disease seeking alternative therapies to a gluten- The principal investigator and leading co-investigators designed the study in free diet? J Gastrointestin Liver Dis 2011;20:27–31. collaboration with Alba Therapeutics Corporation and Cephalon/Teva, 33. Brenneman DE, Spong CY, Hauser JM, et al. Protective conducted the study, and provided oversight for data collection. Data analysis was performed by Chao Wang, PhD, and John Han, PhD, of peptides that are orally active and mechanistically non- PharmaData Associates, funded by Alba and Cephalon/Teva. All authors chiral. J Pharmacol Exp Ther 2004;309:1190–1197. contributed to data interpretation and writing and editing the manuscript. Drs Leffler and Murray had full access to all study data, contributed equally to 34. Rubio-Tapia A, Rahim MW, See JA, et al. Mucosal re- manuscript preparation, and had final responsibility for the publication. covery and mortality in adults with celiac disease after treatment with a gluten-free diet. Am J Gastroenterol Conflicts of interest These authors disclose the following: Daniel Leffler has served as a consultant 2010;105:1412–1420. and/or provided research support for Alba Therapeutics, Alvine 35. Lebwohl B, Granath F, Ekbom A, et al. Mucosal healing Pharmaceuticals, INOVA Diagnostics, Genzyme, Coronado Biosciences, Sidney Frank Foundation, Shire Pharmaceuticals, Ironwood Pharmaceuticals, and mortality in coeliac disease. Aliment Pharmacol Ther and GI Supply; Ciaran Kelly has served as a consultant and scientific advisor 2013;37:332–339. related to celiac disease for Alba, Alvine, ImmusanT, and Pfizer; Peter Green has served on the scientific advisory boards of Alba Therapeutics, Alvine 36. Arrieta MC, Bistritz L, Meddings JB. Alterations in in- Pharmaceuticals, and ImmusanT; Richard Fedorak has served as a CLINICAL AT testinal permeability. Gut 2006;55:1512–1520. consultant/advisory board member for Abbvie, Ferring, Janssen, Shire, VSL#3, and Celltrion, and has received clinical/basic research grants from Abbvie, Alba Therapeutics, Bristol Myers Squibb, Centocor, GSK, Genentec, Received November 3, 2014. Accepted February 10, 2015. Janssen, Merck, Millennium, Novartis, Pfizer, Proctor & Gamble, Roche, VSL#3, and Celltrion, and is an owner/shareholder in Metabolomic Reprint requests Technologies, Inc; Anthony DiMarino has received grant/research support Address requests for reprints to: Joseph A. Murray, MD, Division of from Alba Therapeutics; Wendy Perrow is the CEO and an employee of Alba Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Therapeutics; Henrik Rasmussen and Chao Wang have served as a Rochester, Minnesota 55905. e-mail: [email protected]; fax: (507) consultant for Alba Therapeutics; Premysl Bercik has received research grant 255-6318. support from Nestle Switzerland, and has served on the advisory board for Janssen Canada and Forest Laboratories Canada; and Joseph Murray has Acknowledgment received research support from Alba Therapeutics (>$50,000), Alvine The authors acknowledge Sheila Truten, BS, of MC2 for editorial support, Pharmaceuticals, Inc, and the National Institutes of Health (past grant funded by Alba Therapeutics. The first draft of the manuscript was prepared support). The remaining author discloses no conflicts. 1319.e1 Leffler et al Gastroenterology Vol. 148, No. 7

Online Supplementary Appendix Clinical Research Center, Bethlehem, PA), Dan Henry (J. Lewis Research, Inc, Salt Lake City, UT), Reed Hogan (Gastrointestinal Associates, Jackson, MS), Susan Hole Acknowledgments (Riverside Clinical Research, Edgewater, FL), Robert Larazotide Acetate Study Group Investigators consisted Holmes (PMG Research of Winston-Salem, Winston-Salem, of the following: United States, Atoya Adams (AB Clinical NC), Kiran Jagarlmudi (PMG Research of Salisbury, Salis- Trials, Las Vegas, NV), Stephen Amann (Digestive Health bury, NC), Rajeev Jain (Texas Health Research and Edu- Specialists, Tupelo, MS), Jeffrey Aron (California Pacific cation, Dallas, TX), Rajesh Jain (Rocky Mountain Clinical Medical Center, San Francisco, CA), Kamran Ayub (South- Research, Denver, CO), Ciaran Kelly and Daniel Leffler west Gastroenterology, Oak Lawn, IL), Natalie Bachir (Beth Israel Medical Center, Boston, MA), Thomas Klein (Essentia Health, Duluth, MN), Robert Barclay (Rockford (Heartland Research Associates, Wichita, KS), Richard Gastroenterology, Rockford, IL), Rudolph Bedford (Santa Krause (ClinSearch, LLC, Chattanooga, TN), Keith Laskin Monica Research Institute, Santa Monica, CA), Rajat (Pivotal Clinical Research, LLC, Perkasie, PA), Michael Bhushan (Metabolic Center of Louisiana, Baton Rouge, LA), LeVine (Atlanta Gastroenterology Associates, Marietta, Vicki Blumberg (Soundview Medical, Norwalk, CT), Wilmot GA), Nasrulla Manji (Pioneer Research Solutions, Inc, Sugar Burch (Franklin Gastroenterology, Franklin, TN), Khai Land, TX), Stephen Minton (Alexandria Clinical Research, Chang (Lakeview Medical Research, Summerfield, FL), Alexandria, VA), Vijay Narayen (Digestive Disease Associ- Shane Christensen (J. Lewis Research, Inc, Salt Lake City, ates, Baltimore, MD), Eric Newton (Omega Medical UT), Allan Coates (Gastroenterology Associates of Western Research, Warwick, RI), James Novick (Charm City Michigan, Wyoming, MI), Lawrence Cohen (Medical Research, Towson, MD), Robert Onder (Midwest Clinical Research Associates of New York, New York, NY), Anthony Research, St. Louis, MO), Daniel Pambianco (Charlottesville Colatrella (RPMS, Pittsburgh, PA), John Condemi (AAIR Medical Research, Charlottesville, VA), Craig Pepin (The Research Center, Rochester, NY), Clancy Cone (Montana Polyclinic-Madison Center, Seattle, WA), Joseph Murray Medical Research, Missoula, MT), Craig Curtis (Compass (Mayo Clinic, Rochester, MN), Ramon Reyes (The Iowa Research, Orlando, FL), Mark DeMeo (Rush University Clinic, West Des Moines, IA), Joseph Savon (South Jersey Medical Center, Chicago, IL), Douglas Denham (Clinical Gastroenterology, Marlton, NJ), Leah Schmidt (Genova Trials of Texas, Inc, San Antonio, TX), Christopher Dever- Clinical Research, Tucson, AZ), Purvee Shah (West Coast eaux (Alliance Clinical Research, Oceanside, CA), Anthony Clinical Trials, LLC, Costa Mesa, CA), Alex Sherman (Con- DiMarino (Thomas Jefferson University, Philadelphia, PA), corde Medical Group, PLLC, New York, NY), Helen Stacey Gerald Dryden (University of Louisville, Louisville, KY), (Diablo Clinical Research, Walnut Creek, CA), David Tessler Madeline DuPree (Consultants for Clinical Research of (Central Arizona Medical Associates, Mesa, AZ), Raymond South Florida, Boynton Beach, FL), Craig Ennis (Community Tobias (Birmingham Gastroenterology Association, Bir- Clinical Trials, Orange, CA), David Eskreis (New York Center mingham, AL), Douglas Trate (MediSpect,LLC,Boone,NC), for Clinical Research, Lake Success, NY), Beal Essink (Ore- Chung Tsai (Digestive Disease, Cleveland, OH), Martin van gon Center for Clinical Investigations, Inc, Portland, OR), Cleeff (PMG Research of Cary, Cary, NC), and Joseph Nelson Ferreira (Meritus Center for Clinical Research, Woolley (Chrysalis Clinical Research, St. George, UT); Hagerstown, MD), Deborah Flomenhoft (University of Ken- Canada, Premysl Bercik (McMasters University, Hamilton, tucky, Lexington, KY), Ronald Fogel (Clinical Research Ontario), Brian Bressler (GI Research Institute, Vancouver, Institute of Michigan, Chesterfield, MI), Keith Friedenberg British Columbia), Richard Fedorak (University of Alberta, (Great Lakes Gastroenterology, Mentor, OH), Peter Green Edmonton, Alberta), Sally Godsell (Okanagan Clinical Tri- and Christina Tennyson (Columbia University, New York, als, Ltd, Kelowna, British Columbia), Henryk Pluta NY), Michael Grossman (Clinical Research Associates, (Gastroenterology and Hepatology Clinic, Abbotsford, Oklahoma City, OK), Kumar Gutta (Texas Health Research British Columbia), and Ranjit Singh (PerCuro Clinical and Education, Fort Worth, TX), Ahmed Hasan (Northeast Research, Ltd, Victoria, British Columbia). June 2015 Larazotide Acetate in Celiac Disease 1319.e2

Supplementary Figure 1. Study design and patient disposition. 1319.e3 Lefﬂer et al Gastroenterology Vol. 148, No. 7

Supplementary Figure 2. Tight junction structure and mechanism of action of larazotide acetate. Larazotide acetate is a TJ regulator octapeptide inhibitor of Vibrio cholera zonula occludens (ZO) toxin. TJs comprise more than 50 proteins, including the transmembrane proteins occluding claudin, junctional adhesion molecule (JAM), and cytoplasmic scaf- folding proteins ZO-1, ZO-2, and ZO-3. Larazotide acetate inhibits the cytoskeletal rearrangement and ZO-1 redistribu- tion caused by gliadin in epithelial cells regulates actin rearrangement and stabilizes TJ in vitro in response to various stimuli, including inﬂammatory cytokines, bacterial products, and gliadin. In addition, larazotide acetate inhibits trans- epithelial gliadin transport in vitro and decreases intestinal permeability and preserves barrier function in vitro and in vivo. Reprinted with permission from Martinez et al. Cellular and molecular basis of intestinal barrier dysfunction in the irritable bowel syndrome. Gut Liver 2012;6:305–315. © Korean Association of Medical Journal Editors. http:// gutnliver.org. June 2015 Larazotide Acetate in Celiac Disease 1319.e4

Supplementary Figure 3. No increase or worsening of serum antibody levels. 1319.e5 Lefﬂer et al Gastroenterology Vol. 148, No. 7

Supplementary Table 1.Treatment-Emergent Adverse Events Occurring in 3% or More of Patients in any Treatment Group

Larazotide acetate (n ¼ 258)

Placebo 0.5 mg TID 1.0 mg TID 2.0 mg TID (n ¼ 84), n (%) (n ¼ 85), n (%) (n ¼ 84), n (%) (n ¼ 87), n (%)

Subjects with 1 AE 53 (63.1) 54 (63.5) 52 (61.9) 54 (62.1) System organ class Preferred term Gastrointestinal disorders 21 (25.0) 20 (23.5) 28 (33.3) 27 (31.0) Diarrhea 7 (8.3) 7 (8.2) 5 (6.0) 8 (9.2) Nausea 9 (10.7) 4 (4.7) 7 (8.3) 7 (8.0) Constipation 2 (2.4) 3 (3.5) 7 (8.3) 5 (5.7) Abdominal pain 4 (4.8) 1 (1.2) 5 (6.0) 6 (6.9) Vomiting 6 (7.1) 2 (2.4) 2 (2.4) 3 (3.4) Abdominal pain–upper 2 (2.4) 3 (3.5) 4 (4.8) 2 (2.3) Abdominal distention 1 (1.2) 2 (2.4) 5 (6.0) 1 (1.1) Flatulence 0 2 (2.4) 4 (4.8) 3 (3.4) Gastroesophageal reﬂux disease 0 2 (2.4) 1 (1.2) 6 (6.9) Dyspepsia 3 (3.6) 1 (1.2) 2 (2.4) 2 (2.3) Infections and infestations 25 (29.8) 23 (27.1) 23 (27.4) 18 (20.7) Sinusitis 5 (6.0) 5 (5.9) 5 (6.0) 4 (4.6) Nasopharyngitis 4 (4.8) 4 (4.7) 5 (6.0) 3 (3.4) Inﬂuenza 4 (4.8) 3 (3.5) 4 (4.8) 2 (2.3) Urinary tract infection 2 (2.4) 1 (1.2) 1 (1.2) 5 (5.7) Gastroenteritis, viral 2 (2.4) 1 (1.2) 3 (3.6) 2 (2.3) Upper respiratory tract infection 0 2 (2.4) 1 (1.2) 3 (3.4) Pharyngitis streptococcal 0 3 (3.5) 1 (1.2) 0 Nervous system disorders 10 (11.9) 8 (9.4) 6 (7.1) 12 (13.8) Headache 8 (9.5) 2 (2.4) 5 (6.0) 6 (6.9) General disorders and administration site conditions 2 (2.4) 4 (4.7) 5 (6.0) 8 (9.2) Fatigue 0 1 (1.2) 3 (3.6) 6 (6.9) Musculoskeletal and connective tissue disorders 5 (6.0) 6 (7.1) 3 (3.6) 5 (5.7) Fatigue 0 1 (1.2) 3 (3.6) 6 (6.9) Back pain 3 (3.6) 1 (1.2) 1 (1.2) 2 (2.3) Skin and subcutaneous tissue disorders 5 (6.0) 5 (5.9) 3 (3.6) 3 (3.4) Respiratory, thoracic, and mediastinal disorders 4 (4.8) 4 (4.7) 2 (2.4) 5 (5.7) Cough 3 (3.6) 0 0 0 Investigations 3 (3.6) 6 (7.1) 1 (1.2) 4 (4.6) Low serum ferritin level 1 (1.2) 3 (3.5) 0 0 Psychiatric disorders 1 (1.2) 3 (3.5) 1 (1.2) 2 (2.3)

NOTE. Patients were counted only once per category. A treatment-emergent AE (TEAE) was deﬁned as an AE that occurred during the double-blind treatment phase any time between the ﬁrst dose and 7 days after the last dose of study drug. TEAEs were coded using MedDRA version v.14.1. June 2015 Larazotide Acetate in Celiac Disease 1319.e6

Supplementary Table 2.Adverse Events Leading to Withdrawal From the Study

Dosing group Patient no. TEAE? Preferred term Drug related?

Placebo 1 Yes Diarrhea Probably Nausea Probably Abdominal pain–upper Probably 2 Yes Colitis–microscopic No 3 Yes Headache Probably 0.5 mg TID 1 Yes Basedow’s disease Unlikely 2 Yes Migraine Possibly 3 No Myalgia No Musculoskeletal stiffness No Asthenia No Arthritis No 4 Yes Eructation Probably Flatulence Probably Abdominal pain–upper Probably 5 Yes Gastroesophageal reﬂux disease No 1.0 mg TID 1 Yes Diarrhea No 2 Yes Vertigo Probably 3 Yes Abdominal distension Unlikely Fatigue Unlikely Fecal incontinence Unlikely 4 Yes Celiac disease Unlikely Gastroenteritis viral No 5 Yes Diarrhea Possibly 2.0 mg TID 1 Yes Anxiety No 2 Yes Headache Yes 3 No Arthralgia No Myalgia No Headache No Asthenia No Nausea No 4 No Headache No 5 Yes Memory impairment Unlikely Disturbance in attention Unlikely Constipation Possibly Nausea Possibly

NOTE. The Medical Dictionary for Regulatory Activities coding system was used. TEAE, treatment-emergent AE; TID, 3 times daily.

Supplementary Table 3.Adverse Events Related to Iron Status in the Placebo and 0.5-mg Groups

Subjecta Treatment Baseline ferritin level Week 12/end of treatment ferritin level

1 0.5-mg Larazotide acetate 8.5 25.3 2 0.5-mg Larazotide acetate 7.0 6.8 3 0.5-mg Larazotide acetate 10.7 7.3a 4 0.5-mg Larazotide acetate 5.9 9.8 5 Placebo 10.2 40.6 6 Placebo 11.7 8.0 aEarly study termination.