Azathioprine versus Beta Interferons for Relapsing- Remitting Multiple Sclerosis: A Multicentre Randomized Non-Inferiority Trial Luca Massacesi 1,2 *, Irene Tramacere 3 , Salvatore Amoroso 4 , Mario A. Battaglia 5 , Maria Donata Benedetti 6 , Graziella Filippini 3 , Loredana La Mantia 7 , Anna Repice 2 , Alessandra Solari 3 , Gioacchino Tedeschi 8 , Clara Milanese 3 1 Dipartimento di Neuroscienze, Psicologia, Farmaco e Salute del Bambino Universita ` di Firenze, Firenze, Italy, 2 Neurologia 2, Azienda Ospedaliero-Universitaria Careggi, Firenze, Italy, 3 Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy, 4 Dipartimento di Neuroscienze, Sezione di Farmacologia, Universita ` Politecnica delle Marche, Ancona, Italy, 5 Associazione Italiana Sclerosi Multipla (AISM), Fondazione Italiana Sclerosi Multipla (FISM), Genova, Italy, 6 Dipartimento Universitario di Neurologia, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy, 7 Unita ` di Neurologia - Multiple Sclerosis Center, I.R.C.C.S. Santa Maria Nascente Fondazione Don Gnocchi, Milano, Italy, 8 Clinica Neurologica, Universita ` di Napoli, Napoli, Italy Abstract For almost three decades in many countries azathioprine has been used to treat relapsing-remitting multiple sclerosis. However its efficacy was usually considered marginal and following approval of b interferons for this indication it was no longer recommended as first line treatment, even if presently no conclusive direct b interferon-azathioprine comparison exists. To compare azathioprine efficacy versus the currently available b interferons in relapsing-remitting multiple sclerosis, a multicenter, randomized, controlled, single-blinded, non-inferiority trial was conducted in 30 Italian multiple sclerosis centers. Eligible patients (relapsing-remitting course; $2 relapses in the last 2 years) were randomly assigned to azathioprine or b interferons. The primary outcome was annualized relapse rate ratio (RR) over 2 years. Key secondary outcome was number of new brain MRI lesions. Patients (n = 150) were randomized in 2 groups (77 azathioprine, 73 b interferons). At 2 years, clinical evaluation was completed in 127 patients (62 azathioprine, 65 b interferons). Annualized relapse rate was 0.26 (95% Confidence Interval, CI, 0.19–0.37) in the azathioprine and 0.39 (95% CI 0.30–0.51) in the interferon group. Non-inferiority analysis showed that azathioprine was at least as effective as b interferons (relapse RR AZA/IFN 0.67, one-sided 95% CI 0.96; p,0.01). MRI outcomes were analyzed in 97 patients (50 azathioprine and 47 b interferons). Annualized new T2 lesion rate was 0.76 (95% CI 0.61–0.95) in the azathioprine and 0.69 (95% CI 0.54–0.88) in the interferon group. Treatment discontinuations due to adverse events were higher (20.3% vs. 7.8%, p = 0.03) in the azathioprine than in the interferon group, and concentrated within the first months of treatment, whereas in the interferon group discontinuations occurred mainly during the second year. The results of this study indicate that efficacy of azathioprine is not inferior to that of b interferons for patients with relapsing-remitting multiple sclerosis. Considering also the convenience of the oral administration, and the low cost for health service providers, azathioprine may represent an alternative to interferon treatment, while the different side effect profiles of both medications have to be taken into account. Trial Registration: EudraCT 2006-004937-13 Citation: Massacesi L, Tramacere I, Amoroso S, Battaglia MA, Benedetti MD, et al. (2014) Azathioprine versus Beta Interferons for Relapsing-Remitting Multiple Sclerosis: A Multicentre Randomized Non-Inferiority Trial. PLoS ONE 9(11): e113371. doi:10.1371/journal.pone.0113371 Editor: Klemens Ruprecht, Charite - Universita ¨tsmedizin Berlin, Germany Received January 29, 2014; Accepted October 20, 2014; Published November 17, 2014 Copyright: ß 2014 Massacesi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The present study was funded by AIFA (Agenzia Italiana del Farmaco, www.agenziafarmaco.gov.it). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. Solari, Dr. Massacesi and Dr. Tedeschi have read the journal’s policy and have the following conflicts: Dr. Solari was a board member for Novartis, Biogenidec and Merck Serono, and has received speaker honoraria from Sanofi-Aventis. Dr. Massacesi has received reimbursements for meeting participation or educational grants from Biogen-Idec, Merk-Serono, Sanofi-Aventis and Novartis. In addition, he is a member of the Scientific Advisory Group Neurology of the European Medicine Agency (EMA) and of the Italian Medicine Agency (Agenzia Italiana del Farmaco, AIFA) Advisory Committee on Neurology, but the opinions included in this paper do not involve this activity. Dr. Tedeschi has received reimbursements for meeting participation or educational grants from Biogen-Idec, Merk-Serono, Sanofi-Aventis and Novartis. In addition, he was a member of the Italian Medicine Agency (Agenzia Italiana del Farmaco, AIFA) Advisory Committee on Neurology, but the opinions included in this paper do not involve this activity. All the other authors have declared that no competing interests exist. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. * Email: massacesi@unifi.it Introduction For almost three decades azathioprine (AZA) has been used in many countries to treat relapsing-remitting multiple sclerosis (MS) based on placebo controlled randomized clinical trials (RCTs) [1– 4]. Efficacy however was usually considered marginal [5,6], and following approval of b interferons (IFNs) AZA was no longer recommended as first-line therapy [7]. Lack of MRI evaluation, methodological weaknesses and the low power of the trials may have fostered perception of the poor efficacy of AZA, whereas PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e113371