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.

A new paramagnetically shifted imaging probe for MRI.

PURPOSE: To develop and characterize a new paramagnetic contrast agent for molecular imaging by MRI. METHODS: A contrast agent was developed for direct MRI detection through the paramagnetically shifted proton magnetic resonances of two chemically equivalent tert-butyl reporter groups within a dysprosium(III) complex. The complex was characterized in phantoms and imaged in physiologically intact mice at 7 Tesla (T) using three-dimensional (3D) gradient echo and spectroscopic imaging (MRSI) sequences to measure spatial distribution and signal frequency. RESULTS: The reporter protons reside ∼6.5 Å from the paramagnetic center, resulting in fast T1 relaxation (T1 = 8 ms) and a large paramagnetic frequency shift exceeding 60 ppm. Fast relaxation allowed short scan repetition times with high excitation flip angle, resulting in high sensitivity. The large dipolar shift allowed direct frequency selective excitation and acquisition of the dysprosium(III) complex, independent of the tissue water signal. The biokinetics of the complex were followed in vivo with a temporal resolution of 62 s following a single, low-dose intravenous injection. The lower concentration limit for detection was ∼23 µM. Through MRSI, the temperature dependence of the paramagnetic shift (0.28 ppm.K-1 ) was exploited to examine tissue temperature variation. CONCLUSIONS: These data demonstrate a new MRI agent with the potential for physiological monitoring by MRI. Magn Reson Med 77:1307-1317, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Simultaneous Triple Imaging with Two PARASHIFT Probes: Encoding Anatomical, pH and Temperature Information using Magnetic Resonance Shift Imaging.

The chemical shift of paramagnetically shifted resonances in lanthanide(III) complexes encodes information about temperature and has also been made to report pH in parallel, through introduction of a single phosphonate group adjacent to the reporter tert-butyl resonance. The enhanced sensitivity of this new probe has allowed the simultaneous triple imaging of the water signal and the shifted tert-butyl signals of thulium and dysprosium complexes of a common ligand, separated by over 160 ppm. In parallel spectral imaging experiments, the temperature and pH dependence of the frequency of the Tm and Dy signals has been deconvoluted, allowing the pH and temperature in the liver, kidney and bladder to be measured.

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.