Figure 7. Effect of SMT C1100 treatment on calcium-dependent functional parameters of exercised mdx muscles. (A) Strength- duration curves describing the mechanical threshold of treated and/or untreated EDL muscle fibers. The values are means 6S.E.M. (from 10–45 fibers from 3–5 muscles) of the membrane potential at which contraction occurs in response to a depolarizing voltage step of variable duration (5–500 ms) by means of two microelectrode ‘‘point’’ voltage clamp method. The goodness-of-fit has been estimated by x2 analysis. Error bars are sometime not detectable if smaller than symbol size. Although not shown for graphical reasons, the threshold values of C1100 treated myofibres are significantly different with respect to those of untreated ones, at each pulse duration (0.001,p,0.05 by Student’s t test); (B) ratio between twitch (sPtw) and tetanic tension, as mean 6S.E.M. from 5–7 EDL muscles. Significantly different *versus Exer mdx; p,0.05 and 1versus sedentary mdx; p,0.025. doi:10.1371/journal.pone.0019189.g007 References 1. Bogdanovich S, Perkins KJ, Krag TO, Khurana TS (2004) Therapeutics for Duchenne muscular dystrophy: current approaches and future directions. J Mol Med 82: 102–15. 2. Khurana TS, Davies KE (2003) Pharmacological strategies for muscular dystrophy. Nat Rev Drug Discov 2: 379–90. 3. McDonald CM, Henricson EK, Han JJ, Abresch RT, Nicorici A, et al. The 6- minute walk test as a new outcome measure in Duchenne muscular dystrophy. Muscle Nerve 41: 500–10. 4. Goemans NM, Tulinius M, Buyse G, Wilson R, de Kimpe R, et al. (2010) 24 week follow-up data from a phase I/IIa extension study of PRO051/ GSK2402968 in subjects with Duchenne muscular dystrophy. Neuromusc Disord 20: 639. SMT C1100 Reduces mdx Dystrophic Symptoms possible to treat all DMD and Becker patients, irrespective of their dystrophin mutation. In addition, it could also be used in combination with existing/novel strategies in the future, includ- ing utrophin stabilisation strategies such as biglycan. In choosing a dosing route, an orally bioavailable product to be taken at home would be the ideal preference. In short, SMT C1100 has the perfect profile - an oral drug suitable for treating all DMD patients. In the recent clinical trial sponsored by BioMarin, after repeat dosing SMT C1100 (BMN-195) achieved low plasma exposure. This is frequently a problem in Phase I trials and issues of low exposure can often be addressed by developing new formulations of the drug to increase bioavailability. From the data presented here, only modest plasma levels of around 0.5 mM SMT C1100 maintained over several hours are sufficient to generate enough utrophin for substantial benefit. This strongly supports the importance of retesting new formulations of SMT C1100 in new Phase I clinical trials with a view to progressing to DMD patient trials. Acknowledgments We thank Covance for designing and conducting the safety evaluation programme for SMT C1100. For the study on exercised mdx mice we thank Dr. Karina Litvinova and Dr. Valeriana Sblendorio for assistance with the physiology and in vivo experiments, respectively, Dr. Antonella Liantonio for fura-2 microspectofluometry and Prof. Beatrice Nico for supervision on immunofluorescence and morphological studies. We acknowledge Charles J. Sherr, M.D., Ph.D., a Howard Hughes Medical Institute Investigator at St. Jude Children’s Research Hospital, Memphis, USA, who developed the retroviral vector pSRalphaMSV-CDK4-tkneo used to generate the human DMD cell line provided by Vincent Mouly and Gillian Butler-Browne (Institute of Myology, Paris). We further acknowledge Prof. Steve Davies and Dr. Angela Russell (Chemistry Research Laboratories, Oxford University) who performed the initial pilot compound screens. We are grateful to the Medical Research Council, Muscular Dystrophy Campaign, Muscular Dystrophy Association USA and the Association Francaise contre les Myopathies for support of the work in Oxford. The support of Telethon-Italy (project GGP05130) for part of the work conducted in Bari is also gratefully acknowledged. Author Contributions Conceived and designed the experiments: JMT RJF KED AC RFC ADL RS FXW. Performed the experiments: RJF ACP SES DSP FJW AL SPW AC RFC. Analyzed the data: JMT RJF ACP SES DSP KED AC RFC ADL RS FJW FXW. Wrote the paper: JMT RJF ADL KED. Obtained permission to use the retroviral vector pSRalphaMSV-CDK4-tkneo used to generate the human DMD cell line: RJF. 5. Shrewsbury SB, Cirak S, Guglieri M, Bushby K, Muntoni F (2010) Current progress and preliminary results with the systemic administration trial of AVI- 4658, a novel phosphorodiamidate morpholino oligomer (PMO) skipping dystrophin exon 51 in Duchenne muscular dystrophy (DMD). Neuromuscular Disorders 20: 639–640. 6. Goyenvalle A, Babbs A, Powell D, Kole R, Fletcher S, et al. (2009) Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping. Mol Ther 18: 198–205. 7. Beroud C, Tuffery-Giraud S, Matsuo M, Hamroun D, Humbertclaude V, et al. (2007) Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy. Hum Mutat 28: 196–202. PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19189