Co-administration of ibuprofen and nitric oxide is an effective experimental therapy for muscular dystrophy, with immediate applicability to humans

Abstract

Background and purpose: Current therapies for muscular dystrophy are based on corticosteroids. Significant side effects associated with these therapies have prompted several studies aimed at identifying possible alternative strategies. As inflammation and defects of nitric oxide (NO) generation are key pathogenic events in muscular dystrophies, we have studied the effects of combining the NO donor isosorbide dinitrate (ISDN) and the non-steroidal anti-inflammatory drug ibuprofen.

Experimental approach: alpha-Sarcoglycan-null mice were treated for up to 8 months with ISDN (30 mg.kg(-1)) plus ibuprofen (50 mg.kg(-1)) administered daily in the diet. Effects of ISDN and ibuprofen alone were assessed in parallel. Drug effects on animal motility and muscle function, muscle damage, inflammatory infiltrates and cytokine levels, as well as muscle regeneration including assessment of endogenous stem cell pool, were measured at selected time points.

Key results: Combination of ibuprofen and ISDN stimulated regeneration capacity, of myogenic precursor cells, reduced muscle necrotic damage and inflammation. Muscle function in terms of free voluntary movement and resistance to exercise was maintained throughout the time window analysed. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination.

Conclusions and implications: Co-administration of NO and ibuprofen provided synergistic beneficial effects in a mouse model of muscular dystrophy, leading to an effective therapy. Our results open the possibility of immediate clinical testing of a combination of ISDN and ibuprofen in dystrophic patients, as both components are approved for use in humans, with a good safety profile.

Figures

Figure 1

Ibuprofen plus ISDN ameliorate muscle function over the long term, in α-SG-null mice. Free wheel running to test spontaneous movement (A) or treadmill test to measure resistance to fatigue (B) was carried out with mice treated with standard diet (STD) or a diet containing ibuprofen (IBU), ISDN or the two drugs combined. Parameters measured in matched wild-type animals (WT) are shown for comparison. *P < 0.01 for the effect of each treatment versus STD, #P < 0.01 for the effect of IBU plus ISDN versus that of each single drug; n = 7. α-SG, α-sarcoglycan; ISDN, isosorbide dinitrate.

Figure 2

Ibuprofen plus ISDN reduce skeletal muscle damage long term in α-SG-null mice. CK activity serum levels (A) and number of necrotic fibres quantified in sections of diaphragm muscles (B) obtained from mice treated with standard diet (STD) or a diet containing ibuprofen (IBU), ISDN or the two drugs combined. Parameters measured in matched wild-type animals (WT) are shown for comparison *P < 0.01 for the effect of each treatment versus STD, #P < 0.01 for the effect of IBU plus ISDN versus that of each single drug; n = 7. α-SG, α-sarcoglycan; CK, creatine kinase; ISDN, isosorbide dinitrate.

Figure 3

Histological analysis of dystrophic muscle in animals treated with standard diet, ibuprofen and/or ISDN. Representative haematoxylin and eosin-stained sections of diaphragms of mice treated with standard diet (STD) or a diet containing ibuprofen (IBU), ISDN or the two drugs combined, obtained after 4 (A) and 8 (B) months of treatment. ISDN, isosorbide dinitrate.

Figure 4

Ibuprofen and ibuprofen plus ISDN reduce skeletal muscle inflammation in α-SG-null mice. Inflammatory infiltrates were assessed by immunodetection with an anti CD11b antibody (green) (A). An anti laminin antibody was used to reveal the myofibres (red). The levels of CCL2/MCP-1, CCL3/MIP-1α, TGF-β and TNF-α were measured in homogenates of tibialis anterior muscles (B) obtained from mice treated with standard diet (STD) or a diet containing ibuprofen (IBU), ISDN or the two drugs combined. Parameters measured in matched wild-type animals (wt) are shown for comparison *P < 0.01 for the effect of each treatment versus STD, #P < 0.01 for the effect of IBU plus ISDN versus that of ISDN; n = 7. α-SG, α-sarcoglycan; ISDN, isosorbide dinitrate; MCP-1, monocyte chemoattractant protein-1; CCL2; MIP-1α, macrophage inflammatory protein 1α; CCL3; TGF-β, transforming growth factor β; TNF-α, tumour necrosis factor α.

Figure 5

Ibuprofen plus ISDN increase muscle regeneration, myogenic precursor cells number and regenerative potential. Number of centronucleated regenerating fibres quantified in sections of diaphragm and tibialis anterior muscles (A), number of CD34/α7 integrin-positive cells isolated from tibialis anterior muscles quantified by flow cytometry (B) and expression of the differentiation markers, myogenin (Myog) and myosin heavy chain (MHC), in myogenic precursor cells isolated and then cultured for 1 week (C) from mice treated with standard diet (STD) or a diet containing ibuprofen (IBU), ISDN or the two drugs combined. Parameters measured in matched wild-type animals (WT) are shown in panel A for comparison. *P < 0.01 for the effect of each treatment versus STD, #P < 0.01 for the effect of IBU plus ISDN versus that of each single drug; n = 7. ISDN, isosorbide dinitrate.