Use of EP3533-Enhanced Magnetic Resonance Imaging as a Measure of Disease Progression in Skeletal Muscle of mdx Mice.

As putative treatments are developed for Duchenne muscular dystrophy (DMD), sensitive, non-invasive measures are increasingly important to quantify disease progression. Fibrosis is one of the histological hallmarks of muscular dystrophy and has been directly linked to prognosis. EP3533 is a novel contrast agent with an affinity to collagen 1 that has demonstrated a significant and high correlation to ex vivo fibrosis quantification. Halofuginone is an established anti-fibrotic compound shown to reduce collagen skeletal muscle fibrosis in murine models of DMD. This experiment explored whether EP3533 could be used to detect signal change in skeletal muscle of mdx mice before and after a 12 week course of halofuginone compared to controls. Four age-matched groups of treated and untreated mice were evaluated: 2 groups of mdx (n = 8 and n = 13, respectively), and 2 groups of BL10 mice (n = 5 and n = 3, respectively). Treated mice received an intraperitoneal injection with halofuginone three times per week for 12 weeks, with the remaining mice being given vehicle. Both mdx groups and the untreated BL10 were scanned at baseline, then all groups were scanned on week 13. All subjects were scanned using a 7T Varian scanner before and after administration of EP3533 using a T1 mapping technique. Mice underwent grip testing in week 13 prior to dissection. Skeletal muscle was used for Masson's trichrome quantification, hydroxyproline assay, and immunofluorescent antibody staining. Untreated mdx mice demonstrated a significant increase in R1 signal from pre- to post-treatment scan in three out of four muscles (gastrocnemius p = 0.04, hamstrings p = 0.009, and tibialis anterior p = 0.01), which was not seen in either the treated mdx or the BL10 groups. Histological quantification of fibrosis also demonstrated significantly higher levels in the untreated mdx mice with significant correlation seen between histology and EP3533 signal change. Forelimb weight adjusted-grip strength was significantly lower in the untreated mdx group, compared to the treated group. EP3533 can be used over time as an outcome measure to quantify treatment effect of an established anti-fibrotic drug. Further studies are needed to evaluate the use of this contrast agent in humans.

Noninvasive quantification of fibrosis in skeletal and cardiac muscle in mdx mice using EP3533 enhanced magnetic resonance imaging.

PURPOSE: Duchenne muscular dystrophy (DMD) is a genetic condition caused by mutations in the DMD gene leading to muscle degeneration, fatty replacement of muscle cells and fibrosis. A major obstacle to advancing therapeutic research into muscular dystrophies is development of sensitive, noninvasive outcome measures. To date, no validated method to noninvasively quantify fibrosis within skeletal muscle exists. EP3533 is a gadolinium-based MRI contrast agent with an affinity to collagen-1. The purpose of this study was to determine whether EP3533-enhanced MRI could quantify fibrosis in a murine model of DMD (mdx) in muscle. METHODS: Mdx (n = 8) and control mice (BL10; n = 5) underwent contrast-enhanced MRI acquisitions with EP3533. T1 mapping pre- and postcontrast was performed in skeletal and cardiac muscle. Post-MRI the tibialis anterior (TA) and gastrocnemius (GCN) muscles and the heart were removed for fibrosis quantification by means of Masson's trichrome staining and the hydroxyproline assay. RESULTS: Significant differences in postcontrast R1 were demonstrated between mdx and BL10 mice using EP3533 (cardiac P = 0.02, GCN P = 0.04, TA P = 0.04). Change in R1 from baseline following EP3533 administration correlated strongly to hydroxyproline levels (GCN: r = 0.83, P = 0.001; TA: r = 0.73, P = 0.01). CONCLUSIONS: This study provides evidence for the suitability of EP3533 in the quantification of muscular fibrosis in mdx mice and demonstrated that EP3533-derived measurements correlated strongly to ex vivo fibrosis measurement.

The Classification, Natural History and Treatment of the Limb Girdle Muscular Dystrophies.

Over sixty years ago John Walton and Frederick Nattrass defined limb girdle muscular dystrophy (LGMD) as a separate entity from the X-linked dystrophinopathies such as Duchenne and Becker muscular dystrophies. LGMD is a highly heterogeneous group of very rare neuromuscular disorders whose common factor is their autosomal inheritance. Sixty years later, with the development of increasingly advanced molecular genetic investigations, a more precise classification and understanding of the pathogenesis is possible.To date, over 30 distinct subtypes of LGMD have been identified, most of them inherited in an autosomal recessive fashion. There are significant differences in the frequency of subtypes of LGMD between different ethnic populations, providing evidence of founder mutations. Clinically there is phenotypic heterogeneity between subtypes of LGMD with varying severity and age of onset of symptoms. The first natural history studies into subtypes of LGMD are in process, but large scale longitudinal data have been lacking due to the rare nature of these diseases. Following natural history data collection, the next challenge is to develop more effective, disease specific treatments. Current management is focussed on symptomatic and supportive treatments. Advances in the application of new omics technologies and the generation of large-scale biomedical data will help to better understand disease mechanisms in LGMD and should ultimately help to accelerate the development of novel and more effective therapeutic approaches.