Abstract
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.
Highlights
MATERIALS AND METHODSDuchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene and is inherited in an X-linked recessive fashion
All mice tolerated halofuginone treatment, vehicle and EP3533 administration with no side effects attributed to the drug and no significant weight difference was seen between treated and untreated groups
There were no significant differences between BL10 mouse groups in terms of weight, functional assessment, ex vivo measures of fibrosis or baseline and follow up R1 change
Summary
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene and is inherited in an X-linked recessive fashion. DMD is considered the most commonly inherited muscle disease in childhood, with an incidence of 1 in 3,500– 6,000 live male births [1,2,3]. Dystrophin, the protein product of the DMD gene, is a sub-sarcolemmal cytoskeletal protein present in all muscle [4]. The phenotype of patients with DMD is of progressive weakness of the skeletal, respiratory and cardiac muscles. Higher degrees of endomysial fibrosis in skeletal muscle has been associated with a worse prognosis in neuromuscular disease [8]. Sensitive and preferably minimally invasive techniques are increasingly important to evaluate putative therapies by quantifying fibrosis in vivo as a biomarker of disease progression
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