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

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.

Induction of ATF4-Regulated Atrogenes Is Uncoupled from Muscle Atrophy during Disuse in Halofuginone-Treated Mice and in Hibernating Brown Bears

“…Subsequently, we then reported that pre-treatment with halofuginone mitigated the atrophy of the gastrocnemius muscle during hindlimb suspension. These positive effects of halofuginone treatment are consistent with reports showing that this dose (i.e., 0.25 μg/g) and frequency of administration (i) were very well-tolerated in mice for up to 3 months and (ii) improved muscle-cell survival, promoted membrane repair, and improved muscle performances in models of muscular dystrophies [31,33,[58][59][60]. However, none of these studies explored whether the potential effect of HF would involve the ATF4 pathway.…”

“…To evaluate the effects of a periodic halofuginone (HF) (#32481, Sigma, Saint-Quentin-Fallavier, France) administration, we performed a first protocol where mice received either HF (0.25 µg/g) or water (H 2 O) by gavage 3 times a week for 1 to 4 weeks (n = 6 animals per group). This dose was reported as well tolerated over longer periods [31,33,60]. Gastrocnemius muscle was sampled 6 h after the last HF/H 2 O administration at the end of each week.…”

Emerging MRI techniques for molecular and functional phenotyping of the diseased heart

“…The agent was a cyclic peptide with three gadolinium moieties, and the dense scar tissue of a mouse myocardial infarct was shown to enhance preferentially relative to surrounding healthy myocardium. Since the first demonstration in an infarction model, subsequent studies extended its application to mouse models of liver (17), pulmonary (18), and skeletal muscle (19) fibrosis. All studies confirmed superior sensitivity and specificity of collagentargeted agents for fibrotic tissue compared to non-targeted counterparts.…”

A Type I Collagen-Targeted MR Imaging Probe for Staging Fibrosis in Crohn’s Disease

“…Therefore, an accurate imaging technique for fibrosis analysis is desired. Recently, several studies have also shown that the use of ColI-targeted imaging probe in MR imaging can benefit the fibrosis diagnosis in Duchenne muscular dystrophy ( Murphy et al, 2021 ), nonalcoholic steatohepatitis ( Zhou et al, 2020 ), diffuse cardiac fibrosis ( Ezeani et al, 2021 ) and pancreatic cancer ( Polasek et al, 2017 ). In this study, we generated an CD fibrosis rat model to evaluate the strategy of targeting ColI for staging fibrosis in CD and to compare its efficiency with Gd-DTPA, which is a common MR imaging contrast medium.…”