In vitro activity: Ezutromid (also known as BMN-195 and SMTC-1100) is a first-in-class, orally bioavailable, small molecule modulator of the utrophin's translation with EC50 of 0.4 uM. It is currently in development to treat Duchenne muscular dystrophy (DMD) which is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle. Ezutromid demonstrated significant disease modifying effects in DMD models and was safe and well tolerated with plasma concentrations achieved sufficient to cause a 50% increase in concentrations of utrophin in cells in vitro.

Kinase Assay:

Cell Assay: Ezutromid induces increased levels of utrophin RNA in human muscle cells. Treatment of human DMD cells with SMT C1100 lead to a 2-fold increase in utrophin protein levels at an optimal concentration of 0.3 uM after 3 days of treatment. Ezutromid wassafe and well tolerated with plasma concentrations achieved suf?cient to cause a 50% increase in concentrations of utrophin in cells. Ezutromid led to a 30% increase in Utrn mRNA level and resulted in a 2.0-fold increase in UTRN protein level . Cells were treated with compound for 48 h in standard growth medium containing 0.3% DMSO. The chart shows relative luminescence (RLU) in relation to five different doses of SMT C1100. A Four Parameter Logistic Model was used to generate an EC50. Points represent a mean ±S.E. of three experiments and are typical of the results for all batches of SMTC1100. The structure for SMTC1100 is shown; (B) SMT C1100 significantly increased mRNA copy number of the utrophin transcript in SkMC cells. In this assay Gene Expression Assay 4326315 was used for β-actin detection and Gene Expression Assay Hu01125984_m1 was used for utrophin transcript detection (both Applied Biosystems). Cells were exposed to SMT C1100 in standard media with 1% DMSO (vehicle) for 72 hours with six biological replicates. *p = 0.01 relative to vehicle only; (C) Utrophin protein levels in human DMD cell line treated with SMT C1100 (1 μM) or vehicle (0.1% DMSO). Blots were stained with anti-utrophin (MANCHO3; 1∶100) and ECL HRP-conjugated anti-mouse antibody (GE Healthcare). Bands were quantified using Image J and arbitrary units represent utrophin levels corrected for equal loading by α-actinin immunostaining. Results represent a mean ± S.E based on n = 3. +p = 0.00683; §p<0.001; #p<0.005 relative to vehicle-treated cells.