A dual acting compound releasing nitric oxide

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Pharmacological Research xxx (2011) xxx–xxx
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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs
A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320,
shows significant therapeutic effects in a mouse model of muscular dystrophy
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Clara Sciorati a,d,∗ , Daniela Miglietta b,d , Roberta Buono a,c,d , Viviana Pisa d,e , Dario Cattaneo c,d ,
Emanuele Azzoni a,d,e , Silvia Brunelli a,d,e , Emilio Clementi c,d,e
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Q1
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San Raffaele Scientific Institute, Division of Regenerative Medicine, Via Olgettina 58, 20132 Milan, Italy
Nicox Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy
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Unit of Clinical Pharmacology – CNR Neuroscience Institute, Department of Clinical Sciences, University Hospital “Luigi Sacco”, Università degli Studi di Milano, 20157 Milan, Italy
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Dept. of Experimental Medicine, University of Milano-Bicocca, 20052 Milan, Italy
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E. Medea Scientific Institute, 23842 Bosisio Parini, Lecco, Italy
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a r t i c l e
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a b s t r a c t
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Article history:
Received 29 March 2011
Received in revised form 5 May 2011
Accepted 5 May 2011
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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 ␣-sarcoglycan null mice, a severe mouse model of dystrophy. NCX 320 was
administered daily in the diet for 8 months starting 1 month from weaning. Muscle functional recovery
was evaluated by free wheel and treadmill tests at 8 months. 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.
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Keywords:
Nitric oxide
Inflammation
Muscular dystrophy
Skeletal muscle regeneration
NCX 320
Ibuprofen
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1. Introduction
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Nitric oxide (NO) is involved in many mechanisms responsible for preserving muscle function [1] including both pre-synaptic
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Abbreviations: NO, nitric oxide; DMD, Duchenne muscular dystrophy; NCX
320, 4-(nitrooxy)butyl 2-(4-isobutylphenyl)propanoate; CINOD, cyclooxygenase
inhibiting nitric oxide donator; nNOS, neuronal nitric oxide synthase; LPS,
lipopolysaccharide from Escherichia coli; PGE2 , prostaglandin E2 ; IFN␥, interferon-␥;
COX-1, cyclooxygenase 1; COX-2, cyclooxygenase 2; ODQ, 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one; cGMP, cyclic guanosine monophosphate; CK, creatine kinase;
␣-SG, ␣-sarcoglycan; mdx, muscle dystrophin-deficient mice.
∗ Corresponding author at: San Raffaele Scientific Institute, Division of Regenerative Medicine, Via Olgettina 58, 20132, Milan, Italy, Tel.: +39 0226434814; fax: +39
0226434813.
E-mail address: [email protected] (C. Sciorati).
and post-synaptic neuromuscular transmission, energy supply and
mitochondrial biogenesis [1–4]. In addition, NO regulates repair of
the damaged muscle via specific actions on satellite cells, mononuclear progenitor cells found in mature muscle and located between
the basal lamina and sarcolemma which are normally quiescent,
but they can be activated in response to muscle injury [5–8].
Alteration in the amount and localisation of NO synthesis has
been implicated in the pathophysiology of skeletal muscle dystrophies. These genetic degenerative diseases of the muscle are
due to defects in muscle proteins, leading to structure alteration
and continuous damage of fibres during contraction [9]. Duchenne
muscular dystrophy (DMD) is the most severe form, with a paediatric onset and often leading to rapid paralysis and a premature
death, usually by respiratory and/or cardiac failure before 20th
year of age [9]. In DMD, as well as in the mouse models of muscular dystrophies, mdx or ␣-sarcoglycan (SG) null mouse, the splice
1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2011.05.003
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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variant of neuronal nitric oxide synthase (nNOS␮) is absent from
the sarcolemma, and relocated to the cytosol, with total muscle
NOS activity being thus reduced [10]. Overexpression of nNOS in
the mdx mouse was shown to yield substantial recovery of muscle
structure [11].
Histological analyses of dystrophic skeletal muscle in human
and in mice show that skeletal muscle myofibres are progressively
substituted by connective and adipose tissue [9]. This continuous
fibre damage is counteracted, at least initially, by sustained proliferation and activation of satellite cells leading to regeneration
of fibres; in later phases depletion of the pool of myogenic precursors, due to the repeated cycles of activation and proliferation,
leads to significant decrease of the muscle repair capacity [12,13].
Enhanced fibres destruction is also due to the extensive inflammatory response occurring within the muscle, which contributes
significantly to progression of muscular dystrophies [14]. Indeed,
DNA microarray and biochemical data show that inflammatory
mediators/effectors dominate the expression profile of muscles
from the mdx mouse model of dystrophy [15,16].
A variety of pharmacological and genetic approaches, aimed at
regulating NO supply to the muscle, have been used to ameliorate
the disease progression in both the ␣-SG-null and the mdx mouse
models [6,11,17–23].
We have recently reported that combining NO donation with
a non steroidal anti-inflammatory agent leads to a recovery of
muscle function which is both significant and persistent [18,24].
Specifically, chronic administration of the NO-donating flurbiprofen HCT 1026, belonging to the CINOD (cyclooxygenase-inhibiting
nitric oxide (NO)-donator) class [25], exerted significant therapeutic effects in two different mouse models of dystrophy [18].
HCT 1026 significantly slowed disease progression, maintaining the
functional capacity of muscles by reducing necrosis and inflammation, and preserving the regenerative potential in both the
␣-SG-null and the mdx mouse models. Since flurbiprofen is a potent
anti-inflammatory agent associated with marked gastro-intestinal
side effects and it is not approved for use in paediatric patients, we
have focused our attention on a chemically related drug, ibuprofen,
which is more widely used also in paediatric patients because of its
profile of safety [26–29].
In this study we have analysed the long-term efficacy of a derivative of ibuprofen, namely NCX 320, which targets two mechanisms:
NO donation and cyclooxygenase inhibition. We have defined its
pharmacological profile in terms of kinetics of ibuprofen and NO
release using biochemical and functional assays. We have then
examined the therapeutic potential of NCX 320 in the ␣-SG-null
mice, which are characterised by a severe dystrophic phenotype
with progressive damage and reduced regeneration capacity. NCX
320 induced persistent and significant reduction of both fibre damage and inflammation, thus preserving muscle integrity. Moreover,
NCX 320 significantly increased the myoblast precursor number
and differentiation capacity, maintaining the long-term regeneration capacity of muscle. The data with the prototype NCX 320 show
that a dual-acting compound possesses a potential for treatment of
muscular dystrophies.
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2. Materials and methods
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2.1. Materials
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Fig.
1. Chemical
structure
for
NCX
320
4-(nitrooxy)butyl
2-(4isobutylphenyl)propanoate, synthesized at the NicOx Research Institute (Bresso,
Milan, Italy).
and penicillin/streptomycin were purchased from Invitrogen
(Carlsbad, CA, USA). PGE2 EIA-kit was purchased from Cayman
Chemical (Ann Arbor, Michigan, USA). The protease inhibitor
cocktail was purchased from Roche (Basel, Switzerland). Elisa kits
for cytokines determinations were purchased from R&D System
(Minneapolis, MN, USA). Fluorescein isothiocyanate-conjugated
CD34 antibody was purchased from AbD Serotec (Oxford, UK)
and phycoerithrin-conjugated ␣7-integrin antibody from MBL
(Woburn, MA, USA). All other chemicals were purchased from
Sigma–Aldrich (St. Louis, MO, USA).
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2.2. In vitro profile of NCX 320
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2.2.1. Cyclooxygenase activity inhibition
To assess cyclooxygenase (COX)-inhibiting properties of the
compound, COX-1 and COX-2 activities were studied using resting
and LPS/IFN␥-stimulated RAW 264.7 cells respectively, as previously described [30]. The experiments were carried out in the
presence of 1 ␮M of each enzyme and porcine liver esterases (1.7
units per sample) were added to facilitate the release of ibuprofen
from NCX 320.
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2.2.2. Vasodilating effects
NO-donating activity was studied in the vascular relaxation
assay in rabbit aorta as previously described [31], Briefly, the
aortic rings were suspended in organ chambers containing
Krebs solution and connected to a force displacement transducer (Grass FT03) for the measurement of isometric force.
After equilibration, aortic segments were pre-contracted submaximally with methoxamine (3 ␮M) and, after obtaining a stable
tone, a cumulative concentration–response curve of test drugs
(0.01–100 ␮M) was established in the absence or presence of
the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 10 ␮M) to determine the vasodilating
effects dependent on NO/cyclic guanosine monophosphate (cGMP).
Data were expressed as percent of relaxation and plotted vs concentration.
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2.3. In vivo efficacy of NCX 320 in ˛-SG mice
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NCX
320
(Fig.
1),
4-(nitrooxy)butyl
2-(4isobutylphenyl)propanoate, was synthesized at the NicOx Research
Institute (Bresso, Milan, Italy). Ibuprofen was obtained from Albemarle Corporation (Baton Rouge, LA, USA). Interferon-␥ (IFN␥)
was from Roche Molecular Biochemicals (Mannheim, Germany).
Dulbecco’s modified Eagle’s medium (DMEM), foetal calf serum,
2.3.1. Animals and drug treatment
␣-SG null mice were a kind gift of Dr. K Campbell (Iowa University, Iowa City, IA, USA). Animals were housed in the pathogen-free
facility at the San Raffaele Scientific Institute (Milano-Italy), and
treated in accordance with the European Community guidelines,
and with the approval of the Institutional Ethical Committee. Mice
(15 animals/group) were treated with 50 mg/kg/die of ibuprofen
or 65 mg/kg/die of NCX 320 (equimolar to ibuprofen) administered
daily into the diet (Mucedola, Milano, Italy) or with the same food
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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without drug (control group) starting at 1 month of age for up to
8 months. Animals were subjected to the following tests: serum
creatine kinase (CK) activity monthly for up to the 3rd month of
treatment (4 months of age), functional tests (free wheel running
and treadmill) after 8 months of treatment. At the 4th and 8th
month of treatment mice were sacrificed for histological analyses,
cytokine measurements and preparation of primary myoblasts. For
plasma measurements of the steady state concentrations of ibuprofen, nitrites or nitrates, separate groups of mice (7 mice/group)
were treated as above for 1 week.
2.3.2. Determination of ibuprofen, nitrites and nitrates in plasma
One-week following NCX 320 treatment, plasma was obtained
from the heart of animals and centrifuged 13,000 × g at 4 ◦ C for
5 min to remove cells. Plasma ibuprofen concentrations were
assessed using previously published methods [32,33]. The method
was linear from 2.5 to 250 ␮M, imprecision as well as inaccuracy
was below 10%.
For quantitative NO measurements, prior to the derivatization procedure, plasma (100 ␮L) were spiked with the 15 N-labeled
nitrite and nitrate as internal standards to achieve final concentrations of 0.2 ␮M for [15 N]nitrite and 2 ␮M for [15 N]nitrate.
Derivatization and quantification of nitrite and nitrate was performed as described [34].
2.3.3. Biochemical analyses and functional tests in dystrophic
muscle
Serum CK levels were measured in blood samples obtained by
tail vein withdrawal using an indirect commercially available colorimetric assay (Randox, UK) [17].
Functional muscle activity was measured using both the running wheel to assess free locomotor activity and the exhaustion
treadmill to assess resistance to fatigue [17]. For the running wheel,
three tests were performed on the same animal allowing 2 days
in between each measure, while the exhaustion treadmill test was
performed after an appropriate training period and three tests were
performed on the same animal allowing 1 week in between each
test.
2.3.4. Histological analysis
The animals were sacrificed by cervical dislocation and
diaphragm muscle was dissected and immediately frozen in liquid
N2 cooled isopentane. Haematoxilin–Eosin (H&E) or the Masson
trichrome stainings were performed in serial muscle sections following manufactures procedures (Bio Optica, Milan Italy). At least
10 random images for each muscle were taken at 10× magnification with a S100 TV microscope (Carl Zeiss MicroImaging Inc, Iena,
Germany) and analysed using a digitized imaging system (ImageJ
1.38× National Institute of Health) for counting necrotic and centronucleated fibres and for evaluating infiltrated inflammatory cell
number. Necrotic cells were identified by hypereosinophilia, thinning, waviness and eventually presence of many nuclei [35].
2.3.5. Cytokine determination in tibialis anterior muscle
Tibialis anterior muscles were isolated from sacrificed animals and rapidly homogenized in 20 mM Tris–HCl, containing
137 mM NaCl, 5 mM ethylenediaminetetraacetic acid and a protease inhibitor cocktail, pH 8.0, and centrifuged at 3000 rpm for
5 min at 4 ◦ C. After protein content determination, equal amounts of
protein were analysed using commercially available kits according
to manufacturer’s instructions.
2.3.6. Primary myoblast cells isolation and analysis
Primary myoblasts were isolated from quadriceps and gastrocnemius muscles as previously described [20]. Myoblasts were then
counted and stained with a fluorescein isothiocyanate-conjugated
3
CD34 antibody and a phycoerithrin-conjugated ␣7-integrinantibody according to manufacturer’s protocol. The number of
double positive cells was measured by flow cytometry analysing
10,000 events for each sample (FACScalibur, Becton Dickinson, San
Jose, CA, USA). Aliquots (1 × 105 ) of isolated cells were cultured
using matrigel-coated dishes in DMEM supplemented with 10%
foetal calf serum, 3% chick embryo, 100 U/mL penicillin, 100 ␮g/mL
streptomycin, and 50 ␮g/mL gentamycin for 1 week. To induce
myotube formation cells were then cultured for 24 h in differentiation medium (DMEM supplemented with 2% horse serum,
100 U/mL penicillin, and 100 ␮g/mL streptomycin). Cells were lysed
and analysed for differentiation proteins expression by sodiumdodecylsulfate electrophoresis as described [20].
2.4. Statistical analysis
Results are expressed as the mean ± SEM or median (interquartile range) according to their distribution based on results of the
Kolmogorov–Smirnov normality test. For the parameters with a
normal distribution, the Student’s t-test was used, whereas the
Mann–Whitney test was used for parameters with non-normal distribution. A p-value <0.05 was considered as statistically significant.
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3. Results
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3.1. In vitro profile of NCX 320
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3.1.1. NCX 320 inhibits COX-1 and COX-2 activities
To evaluate the anti-inflammatory effects of NCX 320, we
investigated its COX inhibitory activity in resting (COX-1) or
LPS/IFN␥-stimulated (COX-2) RAW 264.7 cells. NCX 320 inhibited
in a concentration-dependent manner the COX-1 and COX-2 activities (IC50 of 8.1 ± 1.1 and 21.6 ± 1.2 ␮M respectively) similarly to
ibuprofen, which displayed an IC50 of 11.7 ± 1.2 ␮M for COX-1 and
18.2 ± 1.5 ␮M for COX-2 (Fig. 2A). The experiments were carried
out in the presence of porcine liver esterases (1.7 units per sample)
to facilitate the release of ibuprofen from NCX 320.
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3.1.2. NCX 320 induces NO-dependent vasodilation
We evaluated whether NCX 320 releases bioactive NO, investigating NO-mediated vasorelaxation on
methoxamine-precontracted rabbit aortic rings. Cumulative
concentration–response curves to NCX 320 evoked concentrationdependent relaxation with an EC50 of 20.9 ± 3.9 ␮M, achieving
85% relaxation at the highest concentration tested of 100 ␮M
(Fig. 2B). These vasodilating effects depended on the activation
of the NO/cGMP pathway, as they were fully antagonized by
ODQ, a selective soluble guanylate cyclase (sGC) inhibitor (Emax :
22 ± 4%). Under the same conditions, ibuprofen (Emax : 35 ± 1%)
was completely inactive up to the highest concentration tested
(100 ␮M).
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3.2. In vivo efficacy of NCX 320 in ˛-SG mice
3.2.1. NCX 320 releases ibuprofen and NO following oral
administration in ˛-SG null mice
Following 1 week of treatment with NCX 320 incorporated into
the diet (65/mg/kg/die), we assessed the steady state plasma concentrations of ibuprofen and of nitrites or nitrates, as product of
NO metabolism. Plasma levels of ibuprofen were 22.0 ± 3.2 ␮g/mL,
a value similar to that found after a paediatric oral dose of
ibuprofen (150–300 mg/day) [36,37]. Nitrites and nitrates levels
were significantly (p < 0.05) increased when compared to control
animals (3.80 ± 0.10 ␮M and 39.7 ± 2.1 ␮M, respectively, for NCX
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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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.
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320-treated mice and 2.43 ± 0.13 ␮M and 25.9 ± 2.3 ␮M, respectively, for control mice).
3.2.2. Effect of NCX 320 on the amelioration of dystrophic muscle
function and structure in ˛-SG null mice
To study the therapeutic effect of NCX 320 in muscular dystrophy, we relied on ␣-SG null mice, in which the progressive muscle
degeneration with general impairment of locomotor function is
severe and resembles the one observed in human muscular dystrophy [18]. We evaluated the muscle functional activity by analysing
␣-SG null mice performance in both the free wheel (voluntary activity capacity) and the treadmill (resistance to forced exercise) tests
after 8 months of drug treatment. NCX 320-treated animals showed
significant amelioration in both tests when compared to control
␣-SG null mice treated with the standard diet. Conversely, ibuprofen alone induced a modest increase of voluntary activity and was
completely ineffective in ameliorating resistance to forced exercise
(Fig. 3A and B).
To investigate the mechanism of this effect, we analysed skeletal
muscle damage by evaluating serum CK levels, a hallmark of muscle
damage, as well as fibre degeneration and inflammatory reactions
at the level of the diaphragm that, as involuntary muscle, is one of
the most damaged in skeletal muscle dystrophy.
NCX 320 treatment significantly reduced CK activity (by 48%,
40% and 41% at 2, 3 and 4 months of age respectively, p < 0.05), an
effect significant greater than that observed with ibuprofen alone
(Fig. 4A).
Evaluation of H&E-stained sections revealed a significantly
lower number of necrotic fibres (Fig. 4B and C) in diaphragm
muscles from animals receiving NCX 320 for 8 months (2.9 ± 0.6
fibres/mm2 ) compared to control, ␣-SG null mice treated with
the standard diet (10.7 ± 2.7 fibres/mm2 ). Moreover, NCX 320
decreased the accumulation of extracellular scar tissue as revealed
by blue in the Masson trichrome staining (Fig. 4D). On the contrary,
ibuprofen failed to significantly reduce necrotic fibres at 8 months
of age in diaphragm muscles (Fig. 4B).
In addition, animals treated with NCX 320 showed a reduced
inflammatory reaction compared to the control group as judged by
a significant decrease by 36% of inflammatory infiltrates observed
after H&E staining (Fig. 5A). NCX 320 inhibited the expression of the
pro-inflammatory cytokines TGF-␤, MIP-1␣ and MCP-1, measured
in tibialis anterior muscle homogenates (Fig. 5B). These results were
similar to those observed with ibuprofen (although it also reduced
TNF-␣ activity), consistent with the NSAID activity of NCX 320.
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3.2.3. NCX 320 induces preservation of muscle regeneration
capacity
The ability of muscle myogenic precursor cells to proliferate and
fuse to existing myofibres or to generate new myofibres is the key
event responsible for muscle repair after damage. In DMD and ␣SG null mice, this ability is significantly decreased because of the
exhaustion of the myogenic precursor pool due to excessive repair
[12].
We analysed sections of muscles isolated from mice after
8 months of treatment with NCX 320 or ibuprofen to measure
number of centronucleated/regenerating fibres. Analysis of both
diaphragm and tibialis anterior showed a significantly increased
number of newly-formed fibres in the muscles of animals treated
with NCX 320 with respect to control ␣-SG null mice while ibuprofen treatment was ineffective (Fig. 6A).
We also isolated the myogenic precursor cells from these
muscles, analysed their number by flow cytometry based on
co-expression of the markers CD34 and ␣7-integrin, and evaluated their ability to differentiate in vitro, a good proxy for their
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Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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Fig. 3. NCX 320 improves muscle function in ␣-SG null mice. (A) Free wheel running to test spontaneous movement and (B) treadmill test to measure resistance to fatigue
were performed in ␣-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 ␣-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.
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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Fig. 5. NCX 320 reduces skeletal muscle inflammation in ␣-SG null mice. (A) Number of inflammatory infiltrates in diaphragm muscles; (B) levels of MCP-1, MIP-1␣ and TGF-␤
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.
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regenerative potential [20]. Unlike ibuprofen, NCX 320 increased
significantly the number of myogenic precursor cells (Fig. 6B) as
well as their ability to express the differentiation markers myogenin and myosin heavy chain after 1 week of differentiation
(Fig. 6C and D).
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4. Discussion
335
The pharmacological approaches to human muscular dystrophies used in the clinic today are still based on glucocorticoids
administration, usually prednisolone or deflazacort, administered
according to various protocols, that delay the disease progression
however only temporarily and in the presence of severe side effects
[38]. Several alternative experimental pharmacological strategies
are being proposed, including administration of calcium antagonists and antioxidants, protease inhibitors, compounds that correct
dystrophin gene expression, modulate muscle growth, or stabilise
the link between cytoskeleton and extracellular matrix. None of
these therapies have so far yielded favourable outcomes in clinical trials [22]. Other approaches recently applied to dystrophy and
aimed at reducing inflammatory reaction by monoclonal antibodies or genetic techniques seem to be promising, however still far
from therapeutic application, because they are expensive and in
some cases target only subsets of patients [39,40].
In this study, we have characterised NCX 320, a member of
CINODs, a class of compounds designed to provide the antiinflammatory and analgesic efficacy of NSAIDs with additional
beneficial effects due to NO donation.
In particular, we have found that NCX 320 retains the same
anti-inflammatory effect of ibuprofen while releasing NO, and such
NO donation endows it with significant and persistent therapeu-
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tic effects in the ␣-SG null mouse model of dystrophy, preserving
muscle function, reducing muscle damage and maintaining muscle regeneration capacity. Indeed, those beneficial effects have not
been observed with ibuprofen alone which was effective only in
reducing inflammatory reaction.
The beneficial effects of NO on muscle function are well known
and the mechanisms of its action well understood [3]. Approaches
with NO-donors or l-arginine are indeed known to yield amelioration of the mdx dystrophic phenotype. However, these compounds
have no therapeutic potential since they yield only transient
effects and seldom have been shown to induce functional recovery
[21,41–45].
A step forward in terms of the use of NO for the therapy of muscular dystrophy came with CINODs. The clear advantage of these
compounds with respect to the classic NO donors is a sustained
release of low concentrations of NO, and its combination with an
anti-inflammatory activity [25]. Indeed, a recent study by our group
showed that another CINOD, the NO-donating flurbiprofen HCT
1026, was effective in slowing muscle dystrophy and that, at least in
animal models, it was superior to the glucocorticoid prednisolone
[18]. However, flurbiprofen produces severe gastrointestinal damage which makes it unsuitable for long-term treatment, especially
in paediatric patients [46]. Therefore, we have focused on NCX 320
because it is based on ibuprofen, whose profile of safety and tolerability is well established and consequently is accepted for paediatric
use [47]. NCX 320 is indeed cleaved to its active metabolites ibuprofen and NO and shows the same efficacy of ibuprofen in COX-1 and
COX-2 inhibition while donating bioactive NO, as demonstrated by
its ability to induce NO/cGMP dependent vasodilation.
The results obtained in the muscular dystrophy model are significant. The administration of NCX 320 in the ␣-SG null model of
dystrophy for 8 months reduced muscle damage through the multi-
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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7
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/␣7 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.
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ple beneficial effects of NO supply and the anti-inflammatory action
of ibuprofen. The ibuprofen moiety limited the generation of proinflammatory cytokines and the infiltration of inflammatory cells,
possibly in synergy with NO generation [48]. Conversely to other
therapeutic approaches, NCX 320 demonstrated a persistent and
beneficial effect in dystrophic mice in the absence of toxic side
effects.
It is also important to note that the drug was administered
starting from weaning until 10 months of age thus initiating when
animals show the first clear signs of pathology [49]. These signs
are consistent with those observed in dystrophic patients at the
time of diagnosis, which occurs usually between 2 and 6 years of
age when they develop delay in talking and walking [50]. Thus,
the drug regimen we applied in the mouse model translates reasonably well into humans initiating a therapy at the time of
diagnosis.
The important beneficial effects of combining the properties of
NO with those of anti-inflammatory drugs have been further confirmed by our recent study where the combination of the NO donor
isosorbide dinitrate (ISDN) and ibuprofen showed better therapeutic effects than ISDN or ibuprofen alone in the ␣-SG-null mouse
model [18]. However, an impressive effect of NCX 320 which was
not observed to such marked efficacy with the combination ISDN
plus ibuprofen was the capacity to preserve satellite cell number
and function, an important action in therapeutic perspective. Consequently, NCX 320 showed a better resistance to fatigue compared
to the combination.
For all the reasons mentioned above we think that NCX 320
and other compounds in the same class are an attractive promising
therapy for patients affected by muscular dystrophies.
5. Conclusion
420
NCX 320 showed significant and persistent therapeutic effects
preserving muscle function, reducing necrosis of fibres and
inflammatory reaction and maintaining regeneration capacity of
muscle. These data further confirm that NO donation together
with anti-inflammatory activity improves pathological markers
and locomotor function in a reference model. Given the wellestablished and wide use of ibuprofen, these findings indicate a
path forward for the development of new potential effective agent
for treatment of muscular dystrophy.
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Acknowledgements
430
This work was supported by Telethon Italia (GP007006,
to EC), the European Community’s framework programme
FP7/2007–2013 under grants agreements no. 241440 (ENDOSTEM)
and 223098 (OPTISTEM) (to EC and SB), Italian Ministry of Health
RC 2011 (to EC) and Association Française des Myopathies (13478,
to EC). The authors would also like to thank Dr. Ennio Ongini for his
scientific support and valuable comments and suggestions.
431
References
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[1] Kaminski HJ, Andrade FH. Nitric oxide: biologic effects on muscle and role in
muscle diseases. Neuromuscul Disord 2001;11:517–24.
[2] Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, et al. Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science
2003;299:896–9.
[3] Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol
Rev 2001;81:209–37.
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
422
423
424
425
426
427
428
429
432
433
434
435
436
437
439
440
441
442
443
444
445
G Model
YPHRS 2308 1–8
8
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
ARTICLE IN PRESS
C. Sciorati et al. / Pharmacological Research xxx (2011) xxx–xxx
[4] Brune B. Nitric oxide: a short lived molecule stays alive. Pharmacol Res
2010;61:265–8.
[5] Anderson JE. A role for nitric oxide in muscle repair: nitric oxide-mediated
activation of muscle satellite cells. Mol Biol Cell 2000;11:1859–74.
[6] Betters JL, Lira VA, Soltow QA, Drenning JA, Criswell DS. Supplemental nitric
oxide augments satellite cell activity on cultured myofibers from aged mice.
Exp Gerontol 2008;43:1094–101.
[7] Filippin LI, Moreira AJ, Marroni NP, Xavier RM. Nitric oxide and repair of skeletal
muscle injury. Nitric Oxide 2009;21:157–63.
[8] Pisconti A, Brunelli S, Di Padova M, De Palma C, Deponti D, Baesso S, et al.
Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated
signaling pathway that controls myoblast fusion. J Cell Biol 2006;172:233–44.
[9] Emery AE. The muscular dystrophies. Lancet 2002;359:687–95.
[10] Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in
Duchenne muscular dystrophy. Cell 1995;82:743–52.
[11] Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol 2001;155:123–31.
[12] Jejurikar SS, Kuzon Jr WM. Satellite cell depletion in degenerative skeletal muscle. Apoptosis 2003;8:573–8.
[13] Morgan JE, Zammit PS. Direct effects of the pathogenic mutation on satellite
cell function in muscular dystrophy. Exp Cell Res 2010;316:3100–8.
[14] Spencer MJ, Tidball JG. Do immune cells promote the pathology of dystrophindeficient myopathies? Neuromuscul Disord 2001;11:556–64.
[15] Marotta M, Ruiz-Roig C, Sarria Y, Peiro JL, Nunez F, Ceron J, et al. Muscle
genome-wide expression profiling during disease evolution in mdx mice. Physiol Genomics 2009;37:119–32.
[16] Porter JD, Khanna S, Kaminski HJ, Rao JS, Merriam AP, Richmonds CR, et al.
A chronic inflammatory response dominates the skeletal muscle molecular
signature in dystrophin-deficient mdx mice. Hum Mol Genet 2002;11:263–72.
[17] Brunelli S, Rovere-Querini P, Sciorati C, Manfredi AA, Clementi E. Nitric oxide:
emerging concepts about its use in cell-based therapies. Expert Opin Investig
Drugs 2007;16:33–43.
[18] Brunelli S, Sciorati C, D’Antona G, Innocenzi A, Covarello D, Galvez BG, et al.
Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy. Proc Natl
Acad Sci U S A 2007;104:264–9.
[19] Colussi C, Gurtner A, Rosati J, Illi B, Ragone G, Piaggio G, et al. Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy.
FASEB J 2009;23:2131–41.
[20] Sciorati C, Galvez BG, Brunelli S, Tagliafico E, Ferrari S, Cossu G, et al. Ex vivo
treatment with nitric oxide increases mesoangioblast therapeutic efficacy in
muscular dystrophy. J Cell Sci 2006;119:5114–23.
[21] Voisin V, Sebrie C, Matecki S, Yu H, Gillet B, Ramonatxo M, et al. lArginine improves dystrophic phenotype in mdx mice. Neurobiol Dis 2005;20:
123–30.
[22] Wagner KR. Approaching a new age in Duchenne muscular dystrophy treatment. Neurotherapeutics 2008;5:583–91.
[23] Colussi C, Berni R, Rosati J, Straino S, Vitale S, Spallotta F, et al. The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac
arrhythmias in dystrophic mice. Cardiovasc Res 2010;87:73–82.
[24] Sciorati C, Buono R, Azzoni E, Casati S, Ciuffreda P, D’Angelo G, et al. Coadministration of ibuprofen and nitric oxide is an effective experimental
therapy for muscular dystrophy, with immediate applicability to humans. Br J
Pharmacol 2010;160:1550–60.
[25] Wallace JL, Viappiani S, Bolla M. Cyclooxygenase-inhibiting nitric oxide donators for osteoarthritis. Trends Pharmacol Sci 2009;30:112–7.
[26] Blandizzi C, Tuccori M, Colucci R, Fornai M, Antonioli L, Ghisu N, et al.
Role of coxibs in the strategies for gastrointestinal protection in patients
requiring chronic non-steroidal anti-inflammatory therapy. Pharmacol Res
2009;59:90–100.
[27] Brambilla G, Martelli A. Genotoxicity and carcinogenicity studies of analgesics, anti-inflammatory drugs and antipyretics. Pharmacol Res 2009;60:
1–17.
[28] Neubert A, Verhamme K, Murray ML, Picelli G, Hsia Y, Sen FE, et al. The
prescribing of analgesics and non-steroidal anti-inflammatory drugs in paediatric primary care in the UK, Italy and the Netherlands. Pharmacol Res
2010;62:243–8.
[29] Patrono C, Rocca B. Nonsteroidal antiinflammatory drugs: past, present and
future. Pharmacol Res 2009;59:285–9.
[30] Ronchetti D, Borghi V, Gaitan G, Herrero JF, Impagnatiello F. NCX 2057, a novel
NO-releasing derivative of ferulic acid, suppresses inflammatory and nociceptive responses in in vitro and in vivo models. Br J Pharmacol 2009;158:569–79.
[31] Momi S, Impagnatiello F, Guzzetta M, Caracchini R, Guglielmini G, Olivieri R,
et al. NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits
cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic
properties. Eur J Pharmacol 2007;570:115–24.
[32] Farrar H, Letzig L, Gill M. Validation of a liquid chromatographic method for the
determination of ibuprofen in human plasma. J Chromatogr B Analyt Technol
Biomed Life Sci 2002;780:341–8.
[33] Zhao X, Chen D, Li K, Wang D. Sensitive liquid chromatographic assay for the
simultaneous determination of ibuprofen and its prodrug, ibuprofen eugenol
ester, in rat plasma. Yakugaku Zasshi 2005;125:733–7.
[34] Tsikas D. Simultaneous derivatization and quantification of the nitric oxide
metabolites nitrite and nitrate in biological fluids by gas chromatography/mass
spectrometry. Anal Chem 2000;72:4064–72.
[35] Ontell M. Muscle fiber necrosis in murine dystrophy. Muscle Nerve
1981;4:204–13.
[36] Lotsch J, Muth-Selbach U, Tegeder I, Brune K, Geisslinger G. Simultaneous fitting
of R- and S-ibuprofen plasma concentrations after oral administration of the
racemate. Br J Clin Pharmacol 2001;52:387–98.
[37] Steen AE, Reeh PW, Geisslinger G, Steen KH. Plasma levels after peroral and
topical ibuprofen and effects upon low pH-induced cutaneous and muscle pain.
Eur J Pain 2000;4:195–209.
[38] Manzur AY, Kuntzer T, Pike M, Swan A. Glucocorticoid corticosteroids for
Duchenne muscular dystrophy. Cochrane Database Syst Rev 2008:CD003725.
[39] Han R, Frett EM, Levy JR, Rader EP, Lueck JD, Bansal D, et al. Genetic ablation of
complement C3 attenuates muscle pathology in dysferlin-deficient mice. J Clin
Invest 2010;120:4366–74.
[40] Lerario A, Cogiamanian F, Marchesi C, Belicchi M, Bresolin N, Porretti L, et al.
Effects of rituximab in two patients with dysferlin-deficient muscular dystrophy. BMC Musculoskelet Disord 2010;11:157.
[41] Barton ER, Morris L, Kawana M, Bish LT, Toursel T. Systemic administration of larginine benefits mdx skeletal muscle function. Muscle Nerve 2005;32:751–60.
[42] Benabdellah F, Yu H, Brunelle A, Laprevote O, De la PS. MALDI reveals membrane
lipid profile reversion in MDX mice. Neurobiol Dis 2009;36:252–8.
[43] Hnia K, Gayraud J, Hugon G, Ramonatxo M, De la PS, Matecki S, et al. l-Arginine
decreases inflammation and modulates the nuclear factor-kappaB/matrix metalloproteinase cascade in mdx muscle fibers. Am J Pathol 2008;172:1509–19.
[44] Marques MJ, Luz MA, Minatel E, Neto HS. Muscle regeneration in dystrophic
mdx mice is enhanced by isosorbide dinitrate. Neurosci Lett 2005;382:342–5.
[45] Wang G, Burczynski FJ, Hasinoff BB, Zhang K, Lu Q, Anderson JE. Development of
a nitric oxide-releasing analogue of the muscle relaxant guaifenesin for skeletal
muscle satellite cell myogenesis. Mol Pharmacol 2009;6:895–904.
[46] Holzer P, Jocic M, Cabre F, Mauleon D. Estimation of acute flurbiprofen and
ketoprofen toxicity in rat gastric mucosa at therapy-relevant doses. Inflamm
Res 2001;50:602–8.
[47] Autret-Leca E. A general overview of the use of ibuprofen in paediatrics. Int J
Clin Pract Suppl 2003:9–12.
[48] De Palma C, Di Paola R, Perrotta C, Mazzon E, Cattaneo D, Trabucchi E, et al.
Ibuprofen-arginine generates nitric oxide and has enhanced anti-inflammatory
effects. Pharmacol Res 2009;60:221–8.
[49] Duclos F, Straub V, Moore SA, Venzke DP, Hrstka RF, Crosbie RH, et al. Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. J Cell Biol
1998;142:1461–71.
[50] Boland BJ, Silbert PL, Groover RV, Wollan PC, Silverstein MD. Skeletal, cardiac,
and smooth muscle failure in Duchenne muscular dystrophy. Pediatr Neurol
1996;14:7–12.
Please cite this article in press as: Sciorati C, et al. A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant
therapeutic effects in a mouse model of muscular dystrophy. Pharmacol Res (2011), doi:10.1016/j.phrs.2011.05.003
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