Patent Abstract:
the invention relates to a method of treatment for muscular dystrophies , including duchenne , becker , limb - girdle , facioscapulohumeral , congenital muscular dystrophies and the like using a combination of nitric oxide - releasing and anti - inflammatory compounds .

Detailed Description:
it is an object of the present invention a pharmaceutical composition comprising a therapeutically effective amount of at least one nitric oxide releasing compound and at least one anti - inflammatory compound as defined above and suitable diluents and / or excipients and / or adjuvants and / or emollients . the pharmaceutical composition is used for the prophylaxis and / or treatment of muscular dystrophy as defined above . these pharmaceutical compositions can be formulated in combination with pharmaceutically acceptable carriers , excipients , stabilizers , diluents or biologically compatible vehicles suitable for administration to a subject ( for example , physiological saline ). pharmaceutical composition of the invention include all compositions wherein said compounds are contained in therapeutically effective amount , that is , an amount effective to achieve the medically desirable result in the treated subject . the pharmaceutical compositions may be formulated in any acceptable way to meet the needs of the mode of administration . the use of biomaterials and other polymers for drug delivery , as well the different techniques and models to validate a specific mode of administration , are disclosed in literature . any accepted mode of administration can be used and determined by those skilled in the art . for example , administration may be by various parenteral routes such as subcutaneous , intravenous , intradermal , intramuscular , intraperitoneal , intranasal , transdermal , oral , or buccal routes . parenteral administration can be by bolus injection or by gradual perfusion over time . preparations for parenteral administration include sterile aqueous or non - aqueous solutions , suspensions , and emulsions , which may contain auxiliary agents or excipients known in the art , and can be prepared according to routine methods . in addition , suspension of the active compounds as appropriate oily injection suspensions may be administered . suitable lipophilic solvents or vehicles include fatty oils , for example , sesame oil , or synthetic fatty acid esters , for example , sesame oil , or synthetic fatty acid esters , for example , ethyloleate or triglycerides . aqueous injection suspensions that may contain substances increasing the viscosity of the suspension include , for example , sodium carboxymethyl cellulose , sorbitol , and / or dextran . optionally , the suspension may also contain stabilizers . pharmaceutical compositions include suitable solutions for administration by injection , and contain from about 0 . 01 to 99 percent , preferably from about 20 to 75 percent of active compound together with the excipient . compositions which can be administered rectally include suppositories . it is understood that the dosage administered will be dependent upon the age , sex , health , and weight of the recipient , kind of concurrent treatment , if any , frequency of treatment , and the nature of the effect desired . the dosage will be tailored to the individual subject , as is understood and determinable by one of skill in the art . the total dose required for each treatment may be administered by multiple doses or in a single dose . the pharmaceutical composition of the present invention may be administered alone or in conjunction with other therapeutics directed to the condition , or directed to other symptoms of the condition . usually a daily dosage of active ingredients is comprised between 0 . 001 to 100 milligrams per kilogram of body weight . the compounds of the present invention may be administered to the patient intravenously in a pharmaceutical acceptable carrier such as physiological saline . standard methods for intracellular delivery of peptides can be used , e . g . delivery via liposomes . such methods are well known to those of ordinary skill in the art . the formulations of this invention are useful for parenteral administration , such as intravenous , subcutaneous , intramuscular , and intraperitoneal . as well known in the medical arts , dosages for any one patient depends upon many factors , including the patient &# 39 ; s size , body surface area , age , the particular compound to be administered , sex , time and route of administration , general health , and other drugs being administered concurrently . it is a further object of the present invention a diet supplement comprising an effective amount of at least one nitric oxide releasing compound and at least one anti - inflammatory compound as defined above and diluents and / or adjuvants and / or eccipients . another object of the invention is a method of treating or preventing muscular dystrophy comprising administering the pharmaceutical composition or the diet supplement of the invention . the invention will be now described by means of non limiting examples referring to the following figures : fig1 : alpha - sarcoglycan ( sg )- null c57bl / 6 mice were treated for 30 days with the indicated drugs , or combinations of drugs incorporated into the diet as described in the methods . serum creatin kinase ( ck ) concentrations were measured from mouse blood samples obtained by tail vein withdrawal using the indirect colorimetric assay ( roche diagnostics ), following standard procedures . values shown are the results of 4 experiments ± s . e . m . * p & lt ; 0 . 01 vs untreated α - sg null control mice ; § p & lt ; 0 . 05 vs prednisolone - treated α - sg null mice . fig2 : alpha - sg - null c57bl / 6 mice were treated for 30 days with the indicated drugs , or combinations of drugs incorporated into the diet as described in the methods . locomotor performance was measured through the running wheel activity , to this end mice were housed individually in cages equipped with a trixie running wheel . each wheel revolution was registered through a magnetic switch , which was connected to a counter . the number of revolutions was recorded daily for 6 days . values shown are the results of 4 experiments ± s . e . m . * p & lt ; 0 . 01 vs untreated α - sg null control mice ; § p & lt ; 0 . 05 vs prednisolone - treated α - sg null mice . the results of 9 experiments ± s . e . m of running wheel activity measured in wild type ( wt ) c57bl / 6 mice are also reported . fig3 : alpha - sg - null c57bl / 6 mice were treated for 30 days with the indicated drugs , or combinations of drugs incorporated into the diet as described in the methods . tibialis anterior muscles were dissected and frozen in liquid nitrogen - cooled isopentane . the number of inflammatory infiltrates was measured on azan - mallory - stained serial muscle sections . values shown are the results of 4 experiments ± s . e . m . * p & lt ; 0 . 01 and ° p & lt ; 0 . 05 vs untreated α - sg null control mice . fig4 : alpha - sg - null c57bl / 6 mice were treated for 30 days with the indicated drugs , or combinations of drugs incorporated into the diet as described in the methods . diaphragm muscles were dissected , frozen in liquid nitrogen - cooled isopentane and stained with hematoxilin & amp ; eosin according to standard procedures . shown are the numbers of necrotic and centronucleated fibers in 4 animals per group ± s . e . m . * p & lt ; 0 . 01 vs untreated α - sg null control mice ; § p & lt ; 0 . 05 vs prednisolone - treated α - sg null mice . fig5 : alpha - sg - null c57bl / 6 mice were treated for 30 days with the indicated drugs , or combinations of drugs incorporated into the diet as described in the methods . shown are representative histological images of diaphragm muscles cross cryosections stained with in hematoxilin & amp ; eosin ( a ) or azan mallory ( b ) obtained as described in fig4 and 3 , respectively . arrows in panels a1 - 8 point to fiber necrosis , arrows in panels b1 - 8 point to inflammatory infiltrates . fig6 : satellite cells from alpha - sg - null c57bl / 6 mice were isolated from 3 - 5 days old mice and induced to differentiate in the presence of the indicated drugs as described in the methods . fusion index was determined at 48 and 72 h as the number of nuclei in sarcomeric myosin - expressing cells with more than 2 nuclei vs the total number of nuclei . reported are the results ± s . e . m in 4 experiments . * p & lt ; 0 . 01 and ° p & lt ; 0 . 05 vs untreated α - sg null control mice , § p & lt ; 0 . 05 vs prednisolone - treated α - sg null mice . the alpha - sg - null c57bl / 6 mouse , one of the most severe models of muscular dystrophy was used [ 10 , 11 ]. in some experiments wild type ( wt ) c57bl / 6 mouse were used as internal further control . aspirin ( sigma , a2093 ), ibuprofen , ( sigma , i7905 ), isosorbide - mononitrate ( ismn , science lab com sli1024 ), and isosorbide dinitrate ( isdn , alexis , 400 - 800 ), prednisolone ( sigma , p6254 ). in the in vivo experiments the drugs were administered daily in the diet to three months old animals for 30 days . the dose of aspirin was 80 mg / kg , the dose of ismn was 35 mg / kg and that of isdn was 10 mg / kg , while the dose of ibuprofen was 15 mg / kg . prednisolone was used as reference drug at the dose of 2 mg / kg . when combinations of drugs were used , the drugs were both incorporated in the same diet . control animals received the same diet without any drug incorporation . in the in vitro experiments , the drugs were applied to satellite cells at the concentration of 100 μm for ismn , 50 μm for isdn , 10 μm for ibuprofen , 50 μm for aspirin and 30 μm for prednisolone . when combinations of drugs were used , the drugs were administered at the same time . quantitative determination of creatine kinase activity in serum of control and drug treated - animals was measured using creatine kinase reagent ( randox , ck110 ), according to the manufacturer &# 39 ; s instructions . blood was collected from tail vein and serum obtained after centrifugation at 13000 rpm for 10 minutes was stored at − 80 ° c . before measurements [ 13 ]. voluntary wheel running was used as the exercise paradigm to avoid any physiological changes that may occur due to the stress of forced treadmill running . mice were housed individually in cages equipped with a trixie running wheel . each wheel revolution was registered through a magnetic switch , which was connected to a counter . the number of revolutions was recorded daily for 6 days [ 13 ]. diaphragm and tibialis anterior of untreated and drug - treated mice were isolated and included in killik ® frozen section medium , quickly frozen and cut into 8 - μm thick sections with the muscle fibres oriented transversely using a cryostat . sections were stained with either hematoxylin & amp ; eosin or azan mallory , to evaluate the number of inflammatory infiltrates and necrotic fibres ( 18 - 10 sections per tissue ), respectively [ 10 ]. satellite cells were isolated from 3 - 5 days old mice as described previously with some modifications [ 12 ]. in particular , after 3 days from isolation , proliferating myoblasts were harvested , counted , and plated on tissue culture plastic dishes coated with 1 mg / ml type i collagen ( sigma , c9791 ). after 2 days of proliferation in growth medium , myogenic cells accounted for more than 90 % of the cultures as revealed by anti - desmin immunostaining assay . preparations showing less than 90 % myogenic cells were discarded . myoblasts were shifted to differentiating medium in the presence / absence of drugs as indicated in the results . growth medium contained imdm medium supplemented with 20 % fetal bovine serum , 3 % chick embryo extract , 100 u / ml penicillin , 100 μg / ml streptomycin , 50 μg / ml gentamycin . differentiation medium contained imdm medium supplemented with 2 % horse serum , 100 u / ml penicillin , 100 μg / ml streptomycin . fusion index was determined as the number of nuclei in sarcomeric myosin - expressing cells with more than 2 nuclei vs the total number of nuclei [ 10 ]. student &# 39 ; s t test for unpaired variables ( two tailed ) was used . p & lt ; 0 . 05 was considered significant . the effects of the different drugs and their combination on the number of inflammatory infiltrates , the number of necrotic fibers per section , creatine kinase activity and free wheel distance ran are shown in table i . § p & lt ; 0 . 05 vs prednisolone , wt mice : 2 . 1 ± 0 . 3 km / 24 h in the free wheel run test . the authors found that administration of aspirin , ibuprofen , ismn and isdn alone did not have significant effects on any of the parameters measured . by contrast , the combination of aspirin and ismn and the combination of ibuprofen and isdn , were significantly effective in reducing the histological , functional and biochemical alterations observed in these animals ( table i and fig1 - 5 ). in particular , serum levels of creatine kinase , a hallmark of muscle damage , were significantly lower in combination - treated animals ( table i , fig1 ) when compared to control animals receiving the diet without any drug incorporation . consistently , animals treated by drug combination performed significantly better on the free - wheel running test ( table i , fig2 ) when compared to control and prednisolone - treated animals . in addition , the combined treatment aspirin plus ismn and ibuprofen plus isdn treatments on the free - wheel running test improved significantly free wheel running when compared to the reference compound prednisolone . moreover , animals treated with the drug combinations showed significantly reduced inflammatory infiltrates ( table i and fig3 , fig5 ). in addition , fibre necrosis was almost undetectable in animals treated with drug combinations ( table i and fig4 , fig5 ). fig5 reports representative images showing the reduction in inflammatory infiltrates ( panels b1 - 8 ) and fibre necrosis ( panel a1 - 8 ) induced by the drug combinations as indicated by the arrows . satellite cells isolated from newborn alpha - sg - null c57bl / 6 mice and plated at low density ( 6 × 10 3 cells / cm 2 ) were maintained for 48 h in growth medium , and then switched to the differentiating medium in the presence or absence of ismn , isdn , aspirin , ibuprofen , ismn plus aspirin or isdn plus ibuprofen . the fusion index was measured after 48 and 72 h , results are shown table ii and fig6 . effect of drugs and their combination on fusion index in satellite cells the authors found that the combinations of aspirin and ismn and of ibuprofen and isdn significantly increased the fusion index when compared to control cells ( table ii and fig6 ). ismn and isdn were effective on their own but less than when combined with aspirin or ibuprofen ( statistical significance values : * p & lt ; 0 . 01 vs control ; ° p & lt ; 0 . 05 vs ismn or isdn ). ismn and isdn alone or combined with aspirin and ibuprofen yielded fusion index values significantly greater than those obtained with prednisolone ( § p & lt ; 0 . 01 vs prednisolone ). to observe an enhanced satellite fusogenic properties is relevant in the therapeutic perspective to muscular dystrophies since enhanced fusion of satellite cells to damaged fibers implies a faster and more efficient repair of dystrophic damaged fibers . blake d j , weir a , newey s e , davies k e . 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