Figure 4. Ex vivo analysis of SMT C1100 activity in the mdx mouse. (A) mdx mice were treated with SMT C1100 (50 mg/kg/day) or vehicle only (0.1% Tween-20/5% DMSO in PBS) via daily i.p. injection from two weeks of age for four weeks. Whilst contracting tetanically, EDL muscles were stretched at 15% of their fibre length. The difference in force produced between the first and fifth stretch is represented as an indicator of the resistance of the muscle to stretch-induced damage. *p,0.05; **p,1.061025; (B) levels of serum creatine kinase following oral gavage of mdx mice with 50 mg/kg SMT C1100 or vehicle from three weeks of age for four weeks (C) Muscles from the dosing described in (B) were processed to assess the percentage of centrally nucleated fibres *p,0.01; (C)***p = 0.0001; **p = 0.005; *p = 0.003. doi:10.1371/journal.pone.0019189.g004 values from the SMT C1100-dosed mdx were equivalent to those observed in sedentary mdx and wild type mice. Data with direct relevance to DMD treatment was generated using a fatigue assessment of the mice which underwent forced exercise. Fatigue was assessed in an acute endurance test and estimated as the maximal distance run before exhaustion. SMT C1100 Reduces mdx Dystrophic Symptoms Figure 5. Reduction in secondary pathological features. (A) Data demonstrates the reduction in overall skeletal muscle inflammation and fibrosis from mdx treated with SMT C1100 compared to vehicle only. SMT C1100 (50 mg/kg) or vehicle was delivered daily by oral gavage to groups of six mdx mice aged around 17 d for a total of 28 days. The TA, EDL, soleus, and diaphragm were removed and five sections from each muscle were stained and analysed blind by a board-certified veterinary pathologist for evidence of inflammation and fibrosis using a standard pathology scoring method described in the methods section. Scoring (0–3) was made for each section from each muscle then averaged for all muscles to give an overall assessment of improvement in the pathological effects of dystrophin deficiency; (B) Qualitative assessment of EDL muscle from SMT C1100-dosed mdx scored as 1 = mild - occasional mononuclear inflammatory cells in the inter-bundle connec- tive tissue with focal aggregations of mononuclear inflammatory cells. The arrows mark foci of inflammation. Qualitative assessment of EDL dosed with vehicle and scored as 2 = moderate - multiple foci of mononuclear inflammatory cell infiltration in the inter-bundle connec- tive tissue; occasional mononuclear inflammatory cells between individual muscle fibres. doi:10.1371/journal.pone.0019189.g005 Sedentary mdx mice, although run for a shorter distance than wild type [14], maintain the same exercise performance over time, whilst the exercised mdx demonstrate a dramatic increase in fatigability between the start and the fourth and fifth week of training (Fig. 6C). A partial restoration of the resistance to fatigue was observed in SMT C1100-dosed mice, with an increase in distance travelled of around 50% compared to vehicle only after 5 weeks of dosing. Interestingly, this effect was similar to that observed in the exercised mdx mice treated with PDN; which is currently the gold standard in clinical treatment for Duchenne patients. Significant synergy was observed when SMT C1100 was co-administered with PDN for five weeks. The forced exercise mdx were completely resistant to fatigue and were able to continue running as far as the sedentary mdx (Fig. 6C). This equated to an increase in distance travelled of around 350% compared to the vehicle-treated forced exercise mdx. Ex vivo analysis on isolated muscles from forced exercise mdx mice demonstrated that SMT C1100 exerted a significant amelioration of calcium-dependent functional parameters. These are typically modified in mdx muscles due to the altered calcium homeostasis, which in turn is believed to drive the rate of PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19189