Naproxcinod shows significant advantages over naproxen in the mdx model of Duchenne Muscular Dystrophy

Abstract

Background: In dystrophin-deficient muscles of Duchenne Muscular Dystrophy (DMD) patients and the mdx mouse model, nitric oxide (NO) signalling is impaired. Previous studies have shown that NO-donating drugs are beneficial in dystrophic mouse models. Recently, a long-term treatment (9 months) of mdx mice with naproxcinod, an NO-donating naproxen, has shown a significant improvement of the dystrophic phenotype with beneficial effects present throughout the disease progression. It remains however to be clearly dissected out which specific effects are due to the NO component compared with the anti-inflammatory activity associated with naproxen. Understanding the contribution of NO vs the anti-inflammatory effect is important, in view of the potential therapeutic perspective, and this is the final aim of this study.

Methods: Five-week-old mdx mice received either naproxcinod (30 mg/kg) or the equimolar dose of naproxen (20 mg/kg) in the diet for 6 months. Control mdx mice were used as reference. Treatments (or vehicle for control groups) were administered daily in the diet. For the first 3 months the study was performed in sedentary animals, then all mice were subjected to exercise until the sixth month. Skeletal muscle force was assessed by measuring whole body tension in sedentary animals as well as in exercised mice and resistance to fatigue was measured after 3 months of running exercise. At the end of 6 months of treatment, animals were sacrificed for histological analysis and measurement of naproxen levels in blood and skeletal muscle.

Results: Naproxcinod significantly ameliorated skeletal muscle force and resistance to fatigue in sedentary as well as in exercised mice, reduced inflammatory infiltrates and fibrosis deposition in both cardiac and diaphragm muscles. Conversely, the equimolar dose of naproxen showed no effects on fibrosis and improved muscle function only in sedentary mice, while the beneficial effects in exercised mice were lost demonstrating a limited and short-term effect.

Conclusion: In conclusion, this study shows that NO donation may have an important role, in addition to anti-inflammatory activity, in slowing down the progression of the disease in the mdx mouse model therefore positioning naproxcinod as a promising candidate for treatment of DMD.

Figures

Fig. 1

30 mg/kg of naproxcinod is an effective dose in mdx mice. In a first exploratory study, mdx mice were treated with two doses of naproxcinod (10 and 30 mg/kg) for 7 months. Locomotor function, assessed by treadmill running to exhaustion, was measured after 4 (a) and 7 months (b) of treatment. Quantification of inflammation in the tibialis anterior muscle of mdx mice treated with vehicle, 10 or 30 mg/kg naproxcinod was performed at the end of treatment. c Representative histological images of the tibialis anterior muscle after H&E staining and d quantification of inflammatory infiltrate area. Data are presented as mean ± SEM. *represents the comparison between vehicle and all the treatment groups. One-way ANOVA followed by Tukey post-hoc test. *P < 0.05. N = 8-10 mice/group. Bar = 100 μm

Fig. 2

Naproxcinod improves resistance to fatigue in exercised mdx mice. Resistance to fatigue was assessed by treadmill running to exhaustion following 6 months of treatment in exercised mdx mice with either vehicle (black bar), 20 mg/kg naproxen (grey bar) or 30 mg/kg naproxcinod (white bar). a Measurements made once a week for four consecutive weeks and b data obtained during the fourth week of running to exhaustion. Data are presented as mean ± SEM. # represents the comparison between each time point. Two-way ANOVA followed by Bonferroni post-hoc test. *represents the comparison between vehicle and all the treatment groups. One-way ANOVA followed by Tukey post-hoc test. * and # P < 0.05. N = 8-10 mice/group

Fig. 3

Naproxcinod improves skeletal muscle force in exercised mdx mice. a WBT5 and b WBT10 measured following 6 months of treatment in exercised mdx mice treated with vehicle (black bar), 20 mg/kg naproxen (grey bar), or 30 mg/kg naproxcinod (white bar). Data are presented as mean ± SEM. *represents the comparison between vehicle and treatment groups. # represents the comparison versus the naproxen-treated group. One-way ANOVA followed by Tukey post-hoc test. * and # P < 0.05, ***P < 0.001. N = 8-10 mice/group

Fig. 4

Naproxcinod significantly reduces inflammation in diaphragm of mdx mice. Quantification of inflammation in the diaphragm of mdx mice treated with vehicle (black bar), 30 mg/kg naproxcinod (white bar) or 20 mg/kg naproxen (grey bar) following 6 months of treatment. a Representative histological images of the diaphragm muscle after H&E staining and b quantification of the area of inflammatory infiltrates, expressed as a percentage of muscle cross section. Data are presented as mean ± SEM. *represents the comparison between vehicle and treatment groups. One-way ANOVA followed by Tukey post hoc test. ***P < 0.001. N = 4-5 mice/group. Bar = 100 μm

Fig. 5

Naproxcinod significantly reduces fibrosis in diaphragm of mdx mice. Quantification of fibrosis content assessed by Masson’s Trichrome staining in the diaphragm of mdx mice treated with vehicle (black bar), 30 mg/kg naproxcinod (white bar), or 20 mg/kg naproxen (grey bar) following 6 months of treatment. a Representative histological images of the diaphragm and b quantification of fibrotic area, expressed as a percentage of muscle cross section. Data are presented as mean ± SEM. *represents the comparison between vehicle and treatment groups. #represents the comparison versus the naproxen-treated group. One-way ANOVA followed by Tukey post-hoc test. ** P < 0.01; # P < 0.05. N = 4-5 mice/group. Bar = 100 μm

Fig. 6

Naproxcinod significantly reduces cardiac fibrosis. Quantification of the cardiac fibrosis assessed by Picro-Sirius staining in mdx mice treated with vehicle (black bar), 30 mg/kg naproxcinod (white bar), or 20 mg/kg naproxen (grey bar) following 6 months of treatment. a Representative images of the heart and b quantification of the area of fibrosis in the heart, expressed as a percentage of the whole heart. Data are presented as mean ± SEM. *represents the comparison between vehicle and the treatment groups. #represents the comparison versus the naproxen-treated group. One-way ANOVA followed by Tukey post-hoc test. **P < 0.01; # P < 0.05. N = 5-10 mice/group. Bar = 1 mm