DMD cells with SMT C1100 lead to a 2-fold increase in utrophin protein levels at an optimal concentration of 0.3 mM after 3 days of treatment (Fig. 1C). Plasma levels of SMT C1100 Significant plasma (Fig. 2A) and muscle (Fig. 2B) levels of compound were achieved for several hours following oral administration of SMT C1100 (50 mg/kg). From the EC50 data (Fig. 1A), taken together with the levels of utrophin upregulation achieved at various drug concentrations in both DMD and normal myoblasts, we can estimate that the effective concentration required for efficacy would be in the order of 0.5–1 mM. This means that therapeutic levels are achieved in muscle for at least eight hours following dosing. Toxicological evaluation of SMT C1100 In order to confirm that SMT C1100 had no obvious off target toxicological liabilities mice were dosed with high levels of compound. Overall no toxicologically significant changes in clinical condition, body weight, food consumption, haematology or clinical chemistry parameters were seen during the study. There were no microscopic findings within the comprehensive set of tissues analysed due to effects of SMT C1100. Conclusion from Covance Laboratories Ltd. confirmed that the study did not identify any toxicity that was attributable to dosing with SMTC1100. Based on the conditions of this study, it was considered that no toxicity was determined for SMT C1100 administered by oral gavage to the mouse up to dose levels of 2000 mg/kg/day for 28 days. This information was a key Figure 2. Plasma levels of SMT C1100 in the mouse. (A) SMT C1100 plasma levels were assessed over a 24 h period after oral gavage or i.p. delivery of 50 mg/kg of the compound into wild type CD1 mice. At set time points after administration groups of three animals were taken for blood samples at the times stated in the figure. (B) Thigh and diaphragm samples from CD1 dosed orally with 50 mg/kg were quantified for the presence of SMT C1100. doi:10.1371/journal.pone.0019189.g002 SMT C1100 Reduces mdx Dystrophic Symptoms component of the toxicology assessment which led to a successful clinical trial application and testing in healthy volunteers. This was only one component of a significant package of safety evaluation performed on SMT C1100. In vivo upregulation of utrophin To confirm the in vivo activity of SMT C1100, the dystrophin- deficient mdx mouse was used to monitor any changes in the dystrophic phenotype after chronic dosing for several weeks. To confirm increases in utrophin expression after repeated daily dosing with SMT C1100, muscle samples were taken for RNA and protein analysis. Fig. 3A demonstrates a two-fold increase in utrophin mRNA as determined by quantitative PCR from mdx mice dosed daily with SMT C1100 for 28 days compared to vehicle only. Fig. 3B demonstrates significant increases in utrophin protein quantified from western blots of heart; a muscle notoriously difficult to target with systemic administration of putative DMD therapeutics, and diaphragm; the skeletal muscle most affected in sedentary mdx mice. Fig. 3C illustrates a qualitative increase in sarcolemmal-bound utrophin in the tibialis anterior (TA) and EDL muscles after repeat dosing with SMT C1100 following muscle sectioning. A similar result has been observed in both diaphragm and hind limb muscles of forced exercise-treated mdx mice (data not shown), suggesting no impact of work load on drug action. This data confirms SMT C1100 drives increased utrophin transcriptional expression in vivo and, more importantly, demonstrates increased utrophin staining at the required site of action - the sarcolemma - and independently from muscle work load. Benefits of daily dosing of sedentary mdx mice with SMT C1100 In the case of the sedentary mdx mouse, there is a significant triggering of muscle degeneration at around 4 weeks which continues for a further 4 weeks where limb muscles then appear to reach stasis and levels of regeneration remain stable. One muscle where continued development of necrosis is seen is the diaphragm muscle. The dosing schedule for SMT C1100-treated mice was a single daily administration from day 21 for a further 28 days. This period of dosing encompassed the necrotic degenerative phase resulting from dystrophin deficiency. The hypothesis to protect myofibres from damage in the absence of dystrophin is that utrophin, if continually localised to the sarcolemma, will replace dystrophin function. If dystrophin negative fibres are protected from damage for longer by the continued presence of utrophin then the catastrophic secondary effects of regeneration, fibrosis and inflammation should be reduced and muscle should be able to function for longer. All of these endpoints are significantly improved in mdx dosed daily with SMT C1100 for several weeks compared to mdx dosed with vehicle-only. SMT C1100 addresses the primary cause of fibre loss by protecting the sarcolemma from damage as exemplified by increased resistance to eccentric contractions (Fig. 4A) and a reduction in serum creatine kinase levels (Fig. 4B). At the point where the muscle necrosis is at a maximum, SMT C1100 reduces the release of CK into the plasma by 75% compared to vehicle (Fig. 4B; 15 d after the start of dosing). When degeneration has stabilised there is still significant benefit seen as evidenced by continued lower levels of CK (Fig. 4B, 28 d after start of dosing). This data also demonstrates that beneficial effects of SMT C1100 driven utrophin upregulation must occur within a few days after the start of dosing. The resultant protection of dystrophin-deficient fibres by continued expression of utrophin resulted in a reduction in the PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19189