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Muscle fibers lacking dystrophin undergo a long-term alteration of Ca2+ homeostasis, partially caused by a leaky Ca2+ release ryanodine (RyR) channel. S48168/ARM210, an RyR calcium release channel stabilizer (a Rycal compound), is expected to enhance the rebinding of calstabin to the RyR channel complex and possibly alleviate the pathologic Ca2+ leakage in dystrophin-deficient skeletal and cardiac muscle. This study systematically investigated the effect of S48168/ARM210 on the phenotype of mdx mice by means of a first proof-of-concept, short (4 wk), phase 1 treatment, followed by a 12-wk treatment (phase 2) performed in parallel by 2 independent laboratories. The mdx mice were treated with S48168/ARM210 at two different concentrations (50 or 10 mg/kg/d) in their drinking water for 4 and 12 wk, respectively. The mice were subjected to treadmill sessions twice per week (12 m/min for 30 min) to unmask the mild disease. This testing was followed by in vivo forelimb and hindlimb grip strength and fatigability measurement, ex vivo extensor digitorum longus (EDL) and diaphragm (DIA) force contraction measurement and histologic and biochemical analysis. The treatments resulted in functional (grip strength, ex vivo force production in DIA and EDL muscles) as well as histologic improvement after 4 and 12 wk, with no adverse effects. Furthermore, levels of cellular biomarkers of calcium homeostasis increased. Therefore, these data suggest that S48168/ARM210 may be a safe therapeutic option, at the dose levels tested, for the treatment of Duchenne muscular dystrophy (DMD).-Capogrosso, R. F., Mantuano, P., Uaesoontrachoon, K., Cozzoli, A., Giustino, A., Dow, T., Srinivassane, S., Filipovic, M., Bell, C., Vandermeulen, J., Massari, A. M., De Bellis, M., Conte, E., Pierno, S., Camerino, G. M., Liantonio, A., Nagaraju, K., De Luca, A. Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

An absence of dysferlin leads to activation of innate immune receptors such as Toll-like receptors (TLRs) and skeletal muscle inflammation. Myeloid differentiation primary response gene 88 (MyD88) is a key mediator of TLR-dependent innate immune signalling. We hypothesized that endogenous TLR ligands released from the leaking dysferlin-deficient muscle fibres engage TLRs on muscle and immune cells and contribute to disease progression. To test this hypothesis, we generated and characterized dysferlin and MyD88 double-deficient mice. Double-deficient mice exhibited improved body weight, grip strength, and maximum muscle contractile force at 6-8 months of age when compared to MyD88-sufficient, dysferlin-deficient A/J mice. Double-deficient mice also showed a decrease in total fibre number, which contributed to the observed increase in the number of central nuclei/fibres. These results indicate that there was less regeneration in the double-deficient mice. We next tested the hypothesis that endogenous ligands, such as single-stranded ribonucleic acids (ssRNAs), released from damaged muscle cells bind to TLR-7/8 and perpetuate the disease progression. We found that injection of ssRNA into the skeletal muscle of pre-symptomatic mice (2 months old) resulted in a significant increase in degenerative fibres, inflammation, and regenerating fibres in A/J mice. In contrast, characteristic histological features were significantly decreased in double-deficient mice. These data point to a clear role for the TLR pathway in the pathogenesis of dysferlin deficiency and suggest that TLR-7/8 antagonists may have therapeutic value in this disease.

There is evidence that nitric oxide (NO) plays a critical role in skeletal muscle. In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to a variety of functional impairments such as muscle ischemia and compromised myogenesis. Previous studies have shown that NO donation associated with anti-inflammatory action showed beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of naproxcinod, an NO donating naproxen, on skeletal and cardiac muscle function in mdx mice. 4-week-old mdx mice were orally treated for 9 months with three different doses of naproxcinod (10, 21 and 41 mg/kg/day) compared with 0.9 mg/kg of prednisolone. Functional and behavioral parameters using a grip strength meter and open field digiscan were measured at 3, 6, and 9 months of treatment using SOPs developed by TREAT-NMD. Additionally, in vitro EDL force contraction, optical imaging of inflammation, echocardiography and blood pressure were evaluated at the 9 months prior to sacrifice. Naproxcinod treatment at 10 and 21 mg/kg resulted in significant improvements in hindlimb grip strength as well as approximately a 25–30% decrease in inflammation in fore and hind limbs measured by in vivo optical imaging in mdx mice. Naproxcinod induced significant improvements in heart function as evidenced by ameliorated fraction shortening and ejection fraction measured using echocardiography along with improvements in systolic blood pressure. Moreover, the long term detrimental effects of prednisolone typically observed in mdx skeletal and heart function were not observed at the effective doses of naproxcinod. In conclusion, naproxcinod seems to have significant potential as a safe therapeutic option for the treatment of muscular dystrophies.