Figure 6. Effect of SMT C1100 on in vivo parameters of exercised mdx mice. (A) Maximal fore limb strength after 4 weeks of either exercise and/or drug treatment. The values are mean 6 SEM from the number of animals shown in each bar (B) Normalized force increment, i.e. difference between the mean values of normalized fore limb strength at time 4 and at time 0. Normalized force values have been calculated for each mouse as the ratio of maximal fore limb strength to mouse body weight. The values are mean 6 SE. The SE of DNF has been calculated as detailed elsewhere [14,21]. For (A) and (B) statistical significance between groups was evaluated by ANOVA test SMT C1100 Reduces mdx Dystrophic Symptoms for multiple comparison (F-values) and Bonferroni t-test post hoc correction. Significantly different versus *sedentary mdx and 1Exer mdx; p,0.05; (C) Resistance to treadmill running, calculated as the maximal distance the mouse can run when undergoing a single bout of treadmill exercise. The values are mean 6 SEM from 3–7 mice and show the maximal distance run (in meters) at T0 (start of forced exercise and dosing) and after four (T4) and five weeks (T5). Statistical significance between groups was evaluated by ANOVA test for multiple comparison (F-values) and Bonferroni t-test post hoc correction. Highly significant differences were observed between groups and within groups at the different ages (F.10; p,0.005). The symbols show statistical differenc- es *versus sedentary mdx at T4 and #versus vehicle-treated exercised mdx at either T4 or T5 (p,0.05 and less). doi:10.1371/journal.pone.0019189.g006 degeneration. In SMT C1100-treated EDL muscle fibres the strength-duration curve describing the mechanical threshold was significantly shifted toward the more positive membrane potential values and almost overlapped with that observed in wildtype myofibres (Fig. 7A). The rheobase value of SMT C1100-treated muscles approached the wild type value (269.360.4 mV), and was approximately 5 mV less negative than that of non-treated exercised group (270.561.2 mV vs. 27561.5 mV: p,0.05 by Student’s t test). Interestingly, this parameter is not ameliorated by a partial increase in dystrophin expression by gentamicin treatment [21]. Similarly, the ratio between twitch and tetanic tension was significantly reduced in SMT C1100-treated exercised mdx EDL muscles with respect to untreated counterparts, again demonstrating that SMT C1100 treatment generates similar values to those typically found in wild type EDL muscle (Fig. 7B). The amelioration of calcium-dependent parameters was paralleled by a partial, although significant, 18% decrease in the cytosolic Ca2+ level, as determined by fura-2 microspectrofluorimetry [14] (data not shown), thus corroborating that the sarcolemmal bound utrophin stimulated by SMT C1100 treatment can improve calcium-mediate mechanotransduction signalling. Discussion This manuscript illustrates the effectiveness of dosing a well- established mouse model of DMD with a novel oral utrophin upregulator for several weeks. SMT C1100 induces increased levels of utrophin RNA in human muscle cells and significantly reduces dystrophin-deficient muscle pathology to such an extent that significant benefit on whole body strength and endurance is observed. Currently PDN and deflazacort are the only drugs approved by the regulatory authorities for the treatment of DMD. We believe that fatigue testing of mdx after a regime of forced exercise is a good surrogate for the primary clinical endpoint which will be used in DMD trials, i.e. in the 6MWD. Dosing with SMT C1100 alone demonstrated significant benefit in this surrogate model, and the 50% increase in the distance walked would have achieved the required efficacy endpoint if translated over to the 6MWD in DMD trials. Combining doses of SMT C1100 and PDN for several weeks completely prevented fatigue in this model. Thus, the combination of the two drugs with presumed different modes of action protect the muscle from fatiguing with exercise, thereby allowing for significantly increased ambulation. High levels of long term steroid use have unwanted side effects, however a steroid sparing therapy (either reducing dose or frequency to alleviate the unwanted side effects) working synergistically with a utrophin upregulator, has the potential to become the new standard of care for all DMD patients. These results show proof-of-principle for the development of small molecules able to increase levels of utrophin for the therapy of DMD. The great advantage of this approach is that it will be PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19189