delivery of the antisense oligonucleotides. The efficacy of this approach was demonstrated using the dystrophin/utrophin knock- out mouse, where restoration of muscle function was demonstrated [6]. To treat more patients, different antisense sequences will need to be developed to target other exons and the regulatory authorities may treat each of these new constructs as a new drug. The ideal scenario would be to develop multi-exon skipping [7] but this may only be achieved using AAV delivery which faces immunological problems. We have taken an alternative pharmacological approach to DMD by developing an orally bioavailable small molecule which should be appropriate to treat all patients irrespective of their mutation and target both skeletal and cardiac muscle. Building on our work in the mdx mouse, which demonstrated that the loss of dystrophin could be compensated for by increasing the levels of the dystrophin-related protein, utrophin, we have developed novel small molecules which can transcriptionally upregulate the utrophin gene. The demonstration that increased utrophin can reduce the muscular dystrophy in the mdx mouse has been confirmed by others [8–11]. Our early data from the mdx mouse suggested that increasing the levels of utrophin over two-fold would be of great benefit [12]. SMT C1100 was the final product of an exhaustive chemical screening and optimisation campaign. In this paper we present evidence confirming an overall two-fold increase in both utrophin RNA and protein resulting in a significant reduction in dystrophic symptoms and increased muscle function in dystrophin-deficient mdx mice. This was a comprehensive study looking at the beneficial effects of daily dosing of SMT C1100 in both sedentary mdx and the more severely affected forced exercise model. If the results obtained here using SMT C1100 translated across to DMD patients then undoubtedly this would be a disease modifying therapy for DMD. Methods Ethics Statement All animal procedures were performed in accordance with UK Home Office regulations or in accordance with the Italian Guidelines for the use of laboratory animals, which conform with the European Community Directive published in 1986 (86/609/ EEC). The work performed in Oxford was performed under certificate of designation number 30/2306 and project license number 30/2652 following approval by the University of Oxford Departments of Physiology, Anatomy & Genetics and Experi- mental Psychology Joint Departmental Ethics Review Committee. The work performed by Covance Laboratories Ltd. was performed under certificate of designation number 50/8504 and project license number 60/3774 following approval by the Covance Ethical Review Process. The work in Bari was approved by the central review board under the Italian Minister of Health which authorizes that all animal studies conform to the ethical requirement and current law. Generation of a utrophin promoter screening cell line Murine H2K cells [13] were transfected with 8.4 kb of the human utrophin promoter including the first untranslated exon linked to a luciferase reporter gene and stable lines generated for screening (H2K-mdx utrnA-luc). Cell culture The H2K-mdx utrnA-luc cells were maintained in DMEM (Invitrogen) supplemented with 20% Fetal Bovine Serum Gold (PAA), 5% CEE (SLI), 2 mM L-Glutamine (Invitrogen), 1% SMT C1100 Reduces mdx Dystrophic Symptoms Penicillin Streptomycin (Invitrogen) and 2 mg/500 ml Mouse Interferon-c (Roche). Cells were maintained at 10% CO2 at 33uC. Normal human skeletal muscle cells (SkMc) were sourced from TCS cell works. Passaging was undertaken according to the supplier’s instructions including the use of specialist culture media. Cells were maintained at 5% CO2 at 37uC. In vitro assays For luciferase assays plates were seeded with H2K-mdx utrnA- luc at 5000 cells per well. These were incubated in 10% CO2 and 33uC for 24 h prior to dosing. All compounds were supplied as a 10 mM solution in dimethyl sulfoxide (DMSO). Cells were treated compounds dissolved in a final concentration of 0.3% DMSO. Assays were performed in triplicate and the compounds were dosed for 48 h. Luciferase levels were measured using the Steady-Glo Luciferase kit (Promega) and the plates were then read using a FLUOstar Optima plate reader (BMG Labtech). The means from the biological triplicates were used in a 4 Parameter Logistic Model to generate an EC50 and Hill slope. The software used to calculate this was XLfit version 4.2.2. Sedentary mice and drug treatment Three week-old male mdx (C57BL/10ScSn-Dmdmdx/J; Charles River Laboratories, MA, USA) littermates were randomly split between 2 groups and treated with SMT C1100 (50 mg/kg) or vehicle only (phosphate buffered saline (PBS), 0.1% Tween-20, 5% DMSO) via daily i.p. injection for four weeks. At the end of the drug treatment period mice were sacrificed by CO2 asphyxiation in accordance with Schedule I of the UK Animals (Scientific Procedures) Act 1986. C57BL/6 contractile properties were measured in EDL muscle dissected from eight week old untreated mice obtained at 4 week of age from Harlan (n = 5). All animal procedures were performed in accordance with UK Home Office regulations. In all other experiments described using the sedentary mdx mice dosing was by the oral gavage using a canula to deliver SMTC1100 or vehicle only on a daily basis for 28 days. At the end of this period the mice were sacrificed and muscle and blood samples were taken. Quantification of muscle and plasma levels of SMT C1100 was performed using CD1 mice. Exercised mice, treadmill running and drug treatment Most of the experimental procedures conform to the standard operating procedures for pre-clinical tests using mdx mice available at http://www.treat-nmd.eu/research/preclinical/SOPs/. A total of 24 mdx male mice of 4–5 weeks of age (Charles River- Italy), and homogeneous for body weight, underwent a 30 min running regime on an horizontal treadmill (Columbus Instru- ments) at 12 m/min, twice a week (keeping a constant interval of 2–3 days between each trial), for 4–6 weeks, according to a standard protocol [13,14]. Experimental groups were treated as follows: vehicle only (n = 7), SMT C1100 (50 mg/kg; n = 6), a- methyl prednisolone (PDN; 1 mg/kg; n = 5) and combination of SMT C1100 (50 mg/kg) and PDN (1 mg/kg) (n = 6). Age and gender-matched wild type C57/BL10ScSn) or non-exercised mdx mice were also used for specific experimental purposes, as indicated in the text. The dose of PDN has been chosen based on our previous studies [14]. The treatment started one day before the beginning of the exercise protocol, and continued until the day of sacrifice. SMT C1100 and the combination of PDN+SMT C1100 were dissolved in 5% DMSO, 0.1% Tween-20 in PBS, whilst PDN was dissolved in sterile water. Drugs were formulated for i.p. injection so that the correct dose was administered in 0.1 ml/10 g. Body weight was assessed weekly, as was fore-limb PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19189