A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant therapeutic effects in a mouse model of muscular dystrophy

Abstract

A resolutive therapy for muscular dystrophies, a heterogeneous group of genetic diseases leading to muscular degeneration and in the severe forms to death, is still lacking. Since inflammation and defects in nitric oxide generation are recognized key pathogenic events in muscular dystrophy, we have analysed the effects of a derivative of ibuprofen, NCX 320, belonging to the class of cyclooxygenase inhibiting nitric oxide donator (CINOD), in the α-sarcoglycan null mice, a severe mouse model of dystrophy. NCX 320 was administered daily in the diet for 8months starting 1month from weaning. Muscle functional recovery was evaluated by free wheel and treadmill tests at 8months. Serum creatine kinase activity, as well as the number of diaphragm inflammatory infiltrates and necrotic fibres, was measured as indexes of skeletal muscle damage. Muscle regeneration was evaluated in diaphragm and tibialis anterior muscles, measuring the numbers of centronucleated fibres and of myogenic precursor cells. NCX 320 mitigated muscle damage, reducing significantly serum creatine kinase activity, the number of necrotic fibres and inflammatory infiltrates. Moreover, NCX 320 stimulated muscle regeneration increasing significantly the number of myogenic precursor cells and regenerating fibres. All these effects concurred in inducing a significant improvement of muscle function, as assessed by both free wheel and treadmill tests. These results describe the properties of a new compound incorporating nitric oxide donation together with anti-inflammatory properties, showing that it is effective in slowing muscle dystrophy progression long term. Of importance, this new compound deserves specific attention for its potential in the therapy of muscular dystrophy given that ibuprofen is well tolerated in paediatric patients and with a profile of safety that makes it suitable for chronic treatment such as the one required in muscular dystrophies.

2011 Elsevier Ltd. All rights reserved.

Figures

Graphical abstract

Fig. 1

Chemical structure for NCX 320 4-(nitrooxy)butyl 2-(4-isobutylphenyl)propanoate, synthesized at the NicOx Research Institute (Bresso, Milan, Italy).

Fig. 2

NCX 320 inhibits COX-1 and COX-2 activities in RAW macrophages and induces vascular relaxation in rabbit aorta. (A) Inhibitory profile on COX-1 and COX-2 obtained by NCX 320 (filled triangles) or ibuprofen (open squares); the extent of drug-induced inhibition was estimated from the amount of prostaglandin E2 (PGE2) production. (B) Cumulative-concentration curve to NCX 320 (filled triangles) and ibuprofen (open squares) on methoxamine pre-contracted rabbit aortic rings. The vasodilating effect induced by NCX 320 was significantly reverted by the soluble guanylate cyclase inhibitor ODQ (open triangle). Data are expressed as mean ± SEM (n = 3). *p < 0.05 vs ibuprofen.

Fig. 3

NCX 320 improves muscle function in α-SG null mice. (A) Free wheel running to test spontaneous movement and (B) treadmill test to measure resistance to fatigue were performed in α-SG null mice treated for 8 months with ibuprofen or NCX 320 incorporated into the diet. Animals receiving the same diet without any drug were used as control. Data are expressed as mean ± SEM (n = 10). *p < 0.05 vs control animals and #p < 0.05 vs ibuprofen-treated animals.

Fig. 4

NCX 320 reduces skeletal muscle damage in α-SG null mice. (A) CK serum levels analysed in mice from 2nd to 4th month of age; (B) number of necrotic fibres quantified in sections of diaphragm muscles after 8 months of treatment ibuprofen or NCX 320 incorporated into the diet. Animals receiving the same diet without any drug were used as control. Data are expressed as mean ± SEM (n = 10). *p < 0.05 vs control animals and #p < 0.05 vs ibuprofen-treated animals. (C) and (D) Representative images of one out of 10 reproducible experiments for H&E and the Masson trichrome staining respectively; scale bars = 100 mm.

Fig. 5

NCX 320 reduces skeletal muscle inflammation in α-SG null mice. (A) Number of inflammatory infiltrates in diaphragm muscles; (B) levels of MCP-1, MIP-1α and TGF-β measured in homogenates from tibialis anterior muscles. Data were obtained from mice treated for 4 months with ibuprofen or NCX 320 incorporated into the diet. Animals receiving the same diet without any drug were used as control. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs control animals and #p < 0.05 vs ibuprofen-treated animals.

Fig. 6

NCX 320 increases muscle regeneration, myogenic precursor cells and regenerative potential. (A) Number of centronucleated-regenerating fibres quantified in sections of diaphragm and tibialis anterior muscles; (B) number of CD34/α7 integrin-positive cells isolated from tibialis anterior muscles was measured by flow cytometry; (C) expression of the differentiation markers myogenin (Myog) and myosin heavy chain (MHC) in isolated myogenic precursor cells. All parameters were evaluated in mice treated for 8 months with ibuprofen or NCX 320 incorporated into the diet. Animals receiving the same diet without any drug were used as control. Data are expressed as mean ± SEM (n = 10). *p < 0.05 vs control animals and #p < 0.05 vs ibuprofen-treated animals. (D) Representative images of the immunoblotting experiments.