Selected abstracts that will be presented in 13th International Congress of the World Muscle Society, Newcastle, September 2008

1) Optimization of antisense-mediated exon skipping in mouse models for Duchenne muscular dystrophy

Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). Here, antisense oligoribonucleotides (AONs) induce specific exon skipping during pre-mRNA splicing to restore the disrupted DMD open reading frame and allow generation of internally deleted, partially functional Becker-like dystrophins. Proof of concept has been achieved in cultured muscle cells from patients and in mouse and dog models. Recently, we confirmed exon 51 skipping and dystrophin restoration after a single intramuscular dose of AON in a local-administration clinical trial in four patients. Our current research focuses on optimizing systemic delivery of 2′-O-methyl phosphorothioate AONs and comparison of different routes of administration and dosing regimes. We show that after systemic injection, AONs are preferentially taken up by dystrophic muscle compared to healthy fibers. In addition, AON uptake by muscle is comparable for different routes of administration, while uptake by liver and kidney is highest after intravenous injection. Furthermore, we were able to induce exon skipping and dystrophin restoration in all muscles, including the heart after short term treatment with high AON doses and long term treatment with lower doses. This was accompanied by functional improvement and improved muscle integrity, without any apparent toxicity or effects on liver and kidney function. Finally, preliminary results suggest that 2′-O-methyl phosphorothioate AONs are highly sequence specific. These findings are encouraging for future clinical trials and eventual systemic application of this approach.

2) The systemic administration of a low dose of 2OMePS-AON combined with novel cationic polymethylmethacrylate nanoparticles induces the rescue of dystrophin expression in the mdx murine model

P. Rimessi¹, P. Sabatelli¹, M. Fabris¹, P. Braghetta², E. Bassi¹, P. Spitali¹, G. Vattemi³, G. Tomelleri³, L. Mari⁴, D. Perrone⁵, A. Medici⁵, M. Neri¹, M. Bovolenta¹, E. Martoni¹, N. Maraldi⁶, P. Bonaldo², F. Gualandi¹, L. Merlini¹, L. Tondelli⁷, K. Sparnacci⁸, A. Caputo², M. Laus⁸ and A. Ferlini¹

¹Section of Medical Genetics, University of Ferrara, Ferrara, Italy ²Histology, Microbiology and Medical Biotechnology, University of Padova, Padova, Italy ³Section of Clinical Neurology, University of Verona, Verona, Italy ⁴Chemistry, University of Ferrara, Ferrara, Italy ⁵Biology and Evolution, University of Ferrara, Ferrara, Italy ⁶IGM-CNR Unit of Bologna, Bologna, Italy ⁷ISOF-CNR Unit of Bologna, Bologna, Italy ⁸Enviromental and Life Sciences INSTM, University of Piemonte Orientale, Alessandria, Italy

For a subset of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious to restore the expression of dystrophin protein. Here we show that weekly intraperitoneal injections of 2′-O-methyl-modified full-length phosphorothioate (2OMePS) AON, recognising the splice donor of exon 23 in the dystrophin pre-mRNA, delivered by novel cationic polymethylmethacylate (PMMA+) nanoparticles (T1) induced the rescue of dystrophin expression in body-wide muscles, including the heart, of the mdx murine model. This novel non-viral vehicle allowed the administration of a low dose of AON, represented by 2.7 mg/kg, which was sufficient to induce significant levels of dystrophin rescue, although at variable degree among muscle tissues, both by Imuno- as Western-blotting. The same dose of naked AON did not produce dystrophin protein rescue. Quantitative transcription analysis showed increased levels of dystrophin messenger after T1-AON complexes treatment in the muscles analysed, including heart, while no effect was observed after naked AON administration. We conclude that the T1 nanoparticles have the capacity to bind and convey AONs in almost all muscles as well as to reduce the dose of AON required to give dystrophin rescue, therefore representing a promising therapeutic AONs delivery system for future systemic studies in DMD. In order to overwhelm possible disadvantages deriving from chronic administration and to improve the potential for such novel approach we are currently testing different nanoparticles formulations, varying in size, RNA binding affinity and biodegradation characteristics

3) Dystrophin expression after systemic delivery of morpholino antisense oligonucleotide in mdx mouse: A dose–response analysis

A. Malerba^a, G. Dickson^a and I. Graham^a

^aCentre for Biomedical Sciences, University of London, Royal Holloway, Egham, Surrey, United Kingdom

Duchenne muscular dystrophy (DMD) is the most common congenital muscular disorder. It is caused by mutations that create premature termination of dystrophin translation. One of the most promising approaches for the treatment of DMD is the administration of antisense oligonucleotide (AO). This approach applied to human muscle cells and in animal models of DMD restored the correct reading frame in the dystrophin transcript, thereby producing a shorter but partially functional protein. One of the most widely used AO chemistries is the phosphorodiamidate morpholino oligomer (morpholino). While it has been clearly demonstrated that administration of morpholino to mdx mice allows the skipping of the mutated exon 23, little is known about the pharmacokinetics and the best doses for the administration of this AO. It has been demonstrated that several injections of morpholino induce the expression of dystrophin in more fibres than after a single injection, but no data have been published that compare the administration of the same total dose over a set period of time using different numbers of administrations or different routes. Here we report that multiple injections of a low amount of morpholino show significantly more dystrophin positive fibres in a wide variety of muscle groups 8 weeks after administration than a single high dose with the total amount. Other histological features, such as the cross sectional area, the centronucleation index and the expression of the dystrophin-associated protein complex, showed a significant improvement in mice treated by repeated injection. These results show that it is feasible to restore dystrophin expression with high efficiency by repeated injections of low doses of morpholino.

4) Induced exon skipping in normal and mdx muscle

S. Fletcher^a, J.P. Steinhaus^a, C. Mitrpant^a, P.L. Meloni^a and S.D. Wilton^a

^aCentre for Neuromuscular & Neurological Disorders, University of Western Australia, Perth, Australia

Induced exon skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce exon skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin exon 23 skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing exon 23 in normal skeletal muscle, as detected by RT-PCR. However, exon 23 skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.

5) Restoration of dystrophin expression in mdx mouse by peptide-conjugated antisense oligonucleotide

H.F. Yin¹, Y.Q. Seow², H.M. Moulton³, P.L. Iversen³, J.K. Boutilier³ and M.J.A. Wood²

¹Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom ²University of Oxford, Oxford, United Kingdom ³AVI BioPharma Inc., Corvalis, United States

Duchene muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) or to tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs or to homing peptides and evaluated in mdx mice following systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles and in cardiac muscles. This is the first time that the successful body-wide restorations of dystrophin expression even in heart have been achieved at low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.

6) A model of human muscle regeneration in vivo to test potential therapies for DMD

C. Adkin^a, J. Meng^a, V. Arechevala-Gomeza^a, J. Morgan^a and F. Muntoni^a

At present there is no effective treatment for the lethal muscle wasting disorder Duchenne muscular dystrophy (DMD). Several potential therapies have been suggested and evaluated in some of the animal models available, but there has not yet been a systematic comparison of the various therapeutic methods. The aim of this project is to use cell culture and animal models of DMD to assess a number of potential therapies. The focus is on cell transfer based therapies and the use of antisense oligonucleotides (AONs) to correct out of frame mutations in the dystrophin gene by exon skipping. Myoblast cultures derived from normal and DMD patient muscle biopsies have been characterised in vitro and used as a model to test exon skipping strategies. In addition, these cells have been grafted into immunodeficient mouse models and progress has been made in finding the optimal method for transplanting human cells into mouse muscle. Alternative stem cell types have been isolated from human muscle, including pericytes, synovial MSCs and AC133+ cells. Work is ongoing to characterise these cells in vitro and asses their contribution to muscle regeneration in vivo. Further work is under way to improve muscle regeneration from different human stem cell types in vivo, to determine if these cells can produce functional satellite cells, to attempt to produce exon skipping in vivo in human muscle fibres and to examine the possibility of using a lentiviral vector to correct the mutation in DMD patient cells for use in an ex vivo autologous cell transfer therapy.

7) Murine embryonic stem cells injected into mdx mouse – In vivo myogenic capacity and immunogenic reaction

D. Ayub-Guerrieri¹, P.C.M. Martins¹, P.C.G. Onofre¹, V.F. Lopes¹, L.V. Pereira¹, C.M.C. Mori² and M. Vainzof¹

¹Human Genome Research Center, IB-USP, São Paulo, Brazil ²Department of Pathology, Veterinary Medicine and Zoology Institute-USP, São Paulo, Brazil

Duchenne muscular dystrophy (DMD) is the most common form of X-linked hereditary muscular dystrophy caused by the absence of dystrophin in muscle. How to replace dystrophin in the affected muscle is a huge challenge, and stem cells therapies are an increasing hope. The mechanism of embryonic stem (ES) cells contributing to the process of muscle regeneration and their in vivo immunogenicity is still not clear. Here we injected ES cells and differentiated embryonic bodies (EB) into mdx mouse legs, to verify their capacity of generate normal muscle, expressing dystrophin, in vivo. ES and EB cells injected without immunosuppression were not detected in the muscles after 1, 2 and 4 weeks. However, when ES were injected into the muscle of immunosuppressed mdx mice, they caused very intense inflammatory reaction and a significant increase in leg muscle mass after 2 weeks, with a possible teratoma formation. But, this new tissue did not contain cells with muscle fibers characteristics. Analysis of ES cell-specific polymorphic markers identified a concentration of at least 30% of those cells in the injected muscle, suggesting their maintenance in the affected muscle. Our results suggest the murine ES cells used are immunogenic, and their direction to muscle differentiation in vivo is not spontaneously done by the receptor muscle. Additional studies are necessary to increase the therapeutic potential of these cells in dystrophic murine models. Financial support: FAPESP-CEPID, CNPq, ABDIM-PETROBRAS.

8) Treatment with a soluble Activin type llB receptor increases muscle size and strength of dystrophin deficient muscle

J.L. Lachey¹, E.E. Pistilli², S. Bogdanovich², A.E. Pullen¹, R.S. Pearsall¹, T.S. Khurana² and J. Seehra¹

¹Acceleron Pharma, Cambridge, United States ²University of Pennsylvania, Department of Physiology, Pennsylvania Muscle Institute, Philadelphia, United States

Duchenne muscular dystrophy (DMD) patients fail to make functional dystrophin, a protein important in maintaining the integrity of muscle sarcolemma. Dystrophin deficient muscle undergoes cycles of degeneration and compensatory regeneration before ultimately succumbing to the damage. One therapeutic approach for DMD is to strengthen the existing muscle thereby making it less susceptible to damage. Myostatin and other negative regulators of muscle mass signal though Activin type llB receptor (ActRllB) to regulate muscle mass. Administration of a soluble form of ActRllB inhibits signaling at the endogenous receptor and results in dramatically increased muscle mass. Further studies determined that the increase was due to myofiber hypertrophy and not hyperplasia. In order to evaluate the potential benefit of increasing the size of dystrophic muscle, we treated dystrophin deficient mdx mice with the soluble ActRllB fusion protein RAP-031. Mdx mice treated with RAP-031 exhibit significantly increased body weight compared to vehicle controls. Body composition analysis by in vivo NMR scans determined the higher body weights to be due to a selective increase in lean tissue mass. For example, extensor digitorum longus (EDL) muscles from RAP-031 treated mdx mice were 42% heavier than the control group. We further demonstrated that increased muscle mass translated into increased muscle strength using a specially made in vitro muscle physiology testing unit. RAP-031 treatment increased both the absolute twitch and absolute tetanic contractile forces of EDL muscles by 52% and 31%, respectively. No changes in specific force were reported. Forelimb grip strength was used as a determinant of the overall neuromuscular function of the mice. Consistent with a functional improvement of the mdx muscle weakness, RAP-031 treated mdx mice pulled with 42% more force than the control cohort. These data support the idea that RAP-031-induced muscle hypertrophy provides functional benefit to dystrophic muscle.

9) Increased oxidative metabolism in mdx muscle treated by a combination of exon skipping and myostatin blockade

C. Hourdé^a, S. Marie^a, E. Mouisel^a, L. Garcia^a, J. Dumonceaux^a and H. Amthor^a

^aInstitut de Myologie - Inserm - Université Paris 6, UMR S 787, Paris, France

Myostatin blockade stimulates growth of skeletal muscle and has been suggested as a potential therapy for muscular dystrophies. Previously we observed a fibre type conversion towards glycolytic fibres in myostatin deficient mice, which was associated with a decreased number of mitochondria and reduction of oxidative enzymes SDH and COX, implicating a reduced oxidative metabolism of skeletal muscle as a possible adverse effect following lack of myostatin. Here we investigated the effect on muscle metabolism when myostatin blockade was used as a supportive therapy combined with skipping of dystrophin exon 23 in mdx mouse, the animal model for Duchenne muscular dystrophy. We used a AAV construct harboring a double vector which blocks myostatin signalling by RNA interference of the activin receptor IIb (sh-AcvRIIb-3) and which rescues dystrophin expression by using a modified exon skipping strategy (U7 snRNA targeting exon 23). Remarkably, here we demonstrate that combined exon skipping and myostatin blockade increased oxidative properties of treated mdx muscle. Intramuscular injection of U7/sh-AcvRIIb-inv vector into tibialis anterior muscles of mdx mice increased the activity of COX by 78% and of respiratory chain complexes II by 104% compared to the treatment with a control vector harboring scramble sh-RNA. Furthermore, we found an increase in citrate synthase activity of 13% compared to the controls. From these results we hypothesize that myostatin blockade has a different effect on muscle metabolism and subsequently on muscle function when used in combination with dystrophin rescue than myostatin blockade on its own. Therefore, side effects resulting from myostatin blockade may be reduced if applied as a supportive therapy. However, above demonstrated data do not permit to differentiate whether observed effects resulted from myostatin blockade or from exon skipping. Experiments to compare the effect of each strategy are on course.

10) Codon optimisation of microdystrophin results in improvements in expression and physiological outcome in the mdx mouse following AAV8 gene transfer

H. Foster¹, D.J. Wells², C. Trollet³, T. Athanasopoulos³, I. Graham³, K. Foster³ and J.G. Dickson³

¹Royal Holloway University of London, Egham, United Kingdom ²Imperial College London, London, United Kingdom ³Royal Holloway University of London, School of Biomedical Sciences, Egham, United Kingdom

Duchenne’s muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. DMD is caused by a lack of dystrophin protein in skeletal muscle. Lack of dystrophin compromises the integrity of the muscle cell membrane and results in muscle fibres that are highly prone to contraction induced injury. Consequently there are progressive rounds of degeneration and regeneration of the muscle. Such changes culminate in respiratory or cardiac failure in the third decade of life. Very efficient gene transfer of dystrophin is crucial if restoration of muscle function is to be achieved. AAV mediated strategies for gene transfer of dystrophin to muscle have been limited by the small cloning capacity of AAV vectors. As a result smaller versions of the gene or microdystrophins have been designed based on mutations observed in the milder allelic disorder Becker Muscular Dystrophy (BMD). The aim of the current study was to assess the impact of codon optimisation on microdystrophin (ΔAB,R3-R18/ΔCT) expression and function in the mdx mouse and to compare the function of two different configurations of codon optimised microdystrophin genes (ΔAB, R3-R18/ΔCT & ΔR4-R23/ΔCT), under the control of muscle restrictive promoter. Codon optimisation results in a significant improvement in the expression level of microdystrophin following intramuscular (IM) plasmid electrotransfer (25 μg) and systemic administration of AAV8 (3 × 1011vg). Specific force and force drop following eccentric contractions were analysed in the mdx mouse following IM injection of AAV8 (7.5 × 109vg) vectors into neonatal mice. Physiological parameters were assessed at 2 months post injection. Non codon optimised ΔAB, R3-R18/ΔCT did not improve any of the physiological parameters tested, codon optimised ΔAB, R3-R18/ΔCT resulted in significant improvement of specific force but did not improve resistance to eccentric contractions. However, following injection of codon optimised ΔR4-R23/ΔCT, specific force measurements and resistance to eccentric contractions were not significantly different to age matched wild type mice.

11) Evaluating the potential of AAV8 mediated intravenous transfer of myostatin propeptide to ameliorate the muscle pathology in mdx mouse

K. Foster¹, I. Graham¹, H. Foster¹, C. Trollet¹, P. Yaworsky², F. Walsh², P. Sharp³, D. Wells³ and G. Dickson¹

¹Royal Holloway University of London, School of Biological Sciences, Egham, United Kingdom ²Wyeth Research, Clinical Discovery, Collegeville, United States ³Imperial College London, London, United Kingdom

Duchenne’s muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. Lack of dystrophin within the muscle leads to muscle fibres which are highly prone to exercise induced injury, which consequently results in progressive rounds of degeneration and regeneration of the muscle, leading to respiratory or cardiac failure in the third decade of life. Myostatin is a member of the TGF-β super family and is a negative regulator of muscle mass, by affecting muscle cell proliferation and differentiation. Myostatin is produced almost exclusively in skeletal muscle tissue; like other TGF-β family members is first synthesised as a precursor protein (376 amino acids) that is processed at the cleavage site (R263SRR266) by calcium-dependent furin proteases to yield the N-terminal propeptide and the C-terminal mature peptide. This myostatin complex is secreted and circulates as a serum protein. The propeptide domain is activated by BMP-1/tolloid family of metalloproteases to release the mature peptide. In the canonical signalling pathway ‘free’ mature peptide first binds the cell surface activin receptor type IIb (ActRIIb), recruiting and phosphorylating of activin receptor type I (either activin-like kinase 4 (ALK4) or ALK5)); subsequently SMAD2 and SMAD3 are phosphorylated, bind the co-SMAD4 facilitating translocation to the nucleus, whereupon they act as a transcriptional modulator. Use of myostatin propetide has been shown to improve pathology in mdx mice. Here we evaluate whether a gene therapeutic approach of myostatin inhibition leads to improvement in the pathophysiology of the dystrophic phenotype in the mdx mouse. Recombinant myostatin propeptide was expressed in 6–8 week old mdx male and female mice following tail vein injections of AAV8. Gross body weights were monitored at weekly intervals up to 10 weeks post injection. At 10 weeks post injection the following muscles were recovered from treated and untreated groups; tibialis anterior, gastrocnemius, extensor digitorum longus, soleus, diaphragm and heart. Serum samples were also collected. Histological, morphometric, biochemical analyses and physiological data will be presented.

12) Immunosuppressive regimen fails to improve dystrophic phenotype and impairs muscle function in the dog model of Duchenne muscular dystrophy

I. Barthélémy^a, A. Uriarte^a, J.L. Thibaud^a and S. Blot^a

^aENVA, Laboratoire de Neurobiologie, Maisons-Alfort, France

Many therapies for DMD tested in dogs affected by Golden Retriever muscular dystrophy require immune response modulation. Cyclosporin and corticosteroids are known to improve dystrophic phenotype at anti-inflammatory dosages. However, the effects of this association at immunosuppressive levels must be characterized in GRMD dogs. In this aim, 5 GRMD dogs underwent CsA (20 mg/kg/d) and Prednisone (2 mg/kg/d) treatment starting at 2 up to 9 months of age. The consequences of the treatment on the dystrophic phenotype were assessed by clinical scoring and muscle biopsies. Effects on muscular function were examined using force measurements at 4, 6 and 9 months. Many side effects were observed, including growth retardation, overweight, extended papilloma, ectopic calcifications notably in the lungs, and luxations impairing locomotion. No evident influence of treatment on the clinical dystrophic phenotype was observed. Indeed, the clinical scoring evolution appeared heterogeneous, and superimposable to the range of scores of untreated GRMD dogs. Moreover, the treatment did not prevent hiatal hernias, a frequent consequence of diaphragmatic damages. The histological phenotype was not clearly improved, and intracellular calcifications were predominant. Force measurements showed that tetanic contraction force of the anterior compartment of the leg was decreased in comparison to untreated GRMD dogs. Moreover, the force decreased over time whereas it remained stable in untreated GRMD dogs, and increased in healthy dogs. This could be due to reversion of the type I predominance in treated dogs muscles, since the absence of dystrophin is known to impair type II more than type I fibers function. A muscle growth inhibition, due to calcineurin pathway blockade and corticosteroids could also be responsible for this loss of force. This study highlights deleterious repercussions of the association of CsA+Prednisone at immunosuppressive levels on GRMD dogs. These data are a keystone for the analysis of results in therapeutic trials. Thus, a force improvement can be interpreted with certainty as a gain of function due to the specific treatment, and not to the immunosuppressive regimen

13) Gastrostomy tube feeding in patients with severe Duchenne muscular dystrophy

To evaluate effects, risks and the indication of percutaneous endoscopic gastrostomy (PEG) for severe Duchenne muscular dystrophy (DMD), we reviewed clinical records of 21 patients with advanced DMD with gastrostomy in National Hospital Organization Nishitaga Hospital, Sendai, Japan. Mean age and mean body weight of the patients at the time of procedure were 29.3 years old and 26.2 kg, respectively. All patients were advanced and severe cases of DMD who had feeding problems such as swallowing difficulties, prolonged meal time, and weight loss. Ten patients received naso-gastric tube feeding in advance of PEG. All patients required respiratory support during procedure due to severe respiratory insufficiency: 4 patients with tracheostomy and the other 17 with non-invasive positive pressure ventilation (NPPV) with nasal masks. PEG was performed by Pull method at the affiliate hospital. In some patients, insertion of endoscope was difficult due to stiff jaws and enlarged tongues, requiring modification of bite blocks. During the procedure some patients with NPPV had air leaks after sedation resulting in insufficient ventilation and hypoxemia, which required oxygenation and disruption of sedation by benzodiazepine antagonist. No significant change was observed in body weight and laboratory data including serum albumin and total cholesterol levels after PEG on one-year evaluation, suggesting successful nutritional support by gastrostomy. Perioperative complications of PEG included intragastric bleeding in one patient, which needed an endoscopic intervention. Nine patients had minor cutaneous problems around the gastrostomy hole during follow-up period. PEG can be performed in severe DMD patients with NPPV or tracheostomy, and gastrostomy tube feeding benefits the patients in nutritional support. Indication and timing of PEG for DMD patients should be determined in the balance between benefits for quality of life in patients and potential risks of complications.

14) Resting energy expenditure and nutritional inadequacy in Duchenne muscular dystrophy

H. Komaki¹, M. Shimizu¹, E. Nakagawa¹, M. Yoshimura², Y. Ohya², T. Fujisaki³, Y. Saida⁴, C. Kubota⁴, S. Itoh³, R. Shimazaki⁵, K. Sato⁵, T. Ishikawa⁵, H. Mochizuki⁶, T. Takanoha⁷, M. Konagaya⁸, T. Miyazaki⁹ and K. Tatara⁹

¹National Center of Neurology and Psychiatry, Department of Child Neurology, Kodaira, Tokyo, Japan ²National Center of Neurology and Psychiatry, Department of Neurology, Kodaira, Tokyo, Japan ³National Hospital Organization Matsue National Hospital, Matsue, Shimane, Japan ⁴National Hospital Organization Matsue National Hospital, Department of Pediatrics, Matsue, Shimane, Japan ⁵National Hospital Organization Nishibeppu National Hospital, Department of Neurology, Beppu, Oita, Japan ⁶National Hospital Organization Higashisaitama National Hospital, Department of Neurology, Hasuda, Saitama, Japan ⁷National Hospital Organization Higashisaitama National Hospital, Hasuda, Saitama, Japan ⁸National Hospital Organization Suzuka National Hospital, Department of Neurology, Suzuka, Mie, Japan ⁹National Hospital Organization Tokushima National Hospital, Department of Pediatrics, Yoshinogawa, Tokushima, Japan

To clarify nutritional status in Duchenne muscular dystrophy (DMD) patients, we evaluated resting energy expenditure (REE), body composition, blood nutritional markers. Seventy seven DMD patients 19.8 years of their mean age, and obtained informed consent were enrolled this study. Patients with tracheostomy, taking oxygen supplement, incapable of taking off non-invasive ventilation during REE measurement, and incapable of cooperation were excluded. All patients did not take tube feeding, gastrostomy and intravenous hyperalimentation was excluded in this study. REE was measured at least 2 times, and adopt average value using simple calorie meter in resting status at least 2 h, room temperature at 20–26 degrees. Body weight, height and vital capacity were measured in the standard methods. Blood tests including serum creatin kinase, creatinine, albumin, cholinesterase, prealbumin, transferrin, lymphocyte count, plasma amino-acid fraction were measured. Correlations with REE and other parameters were evaluated using Spearman correlation coefficient. REE and REE converted with weight showed low levels compared to that of normal subjects, and high variability among subjects. REE was correlated with age (P < 0.05), weight (P < 0.01), BMI (P < 0.01), prealbumin (P < 0.01), transferrin (P < 0.01), cholinesterase (P < 0.01), creatine kinase (P < 0.01), and vital capacity (P < 0.01). In the plasma amino-acid analysis, seven out of nine essential amino-acids including branch chain amino-acids (BCAA) were correlated with age. Our data suggests that REE of DMD patients was regulated with age (disease progression), nutritional status, and respiratory function. Correlation with REE and nutritional markers strongly indicate that DMD patients have latent nutritional inadequacy. Respiratory insufficiency may cause the up-regulation of REE and increase of energy demand. Active nutritional support especially including assessment and care of swallowing difficulty and introduction of gastrostomy was required in the management of DMD patients.

15) Cardiac involvement in manifesting female carriers of Duchenne and Becker muscular dystrophy

M.K. Al-raqad¹, A. Aboumousa¹, M. Guglieri¹, J.P. Bourke² and K. Bushby¹

¹Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, United Kingdom ²Cardiology Department, Freeman Hospital, NUTH Hospitals Trust, Newcastle upon Tyne, United Kingdom

Cardiac involvement, including the development of cardiomyopathy (DCM) has been reported in female carriers of Duchenne (DMD) and Becker muscular dystrophy (BMD). The incidence, especially amongst those manifesting symptoms of the disease, remains controversial. We assessed cardiac status in a group of manifesting DMD and BMD female carriers to define their incidence/risk of DCM. 18 manifesting female carriers were identified within the register of the Newcastle Muscle Centre (mean age 35 years; range 9–71). Manifesting carriers were defined as those with DNA confirmation of carrier status in whom clinical symptoms related to their carrier status had caused them to present to medical attention. ECG, 2-dimensional echocardiography and Doppler studies were performed in all. Follow up with ECG and Echo-assessments were available for 10 patients with a mean interval between assessments of 3.5 year (0.5–7.5 years). 12 (67%) were carriers of DMD and 6 (33%) BMD mutations - 15 (83%) had a positive family history. Presentation was with muscle weakness in 16, learning difficulties in 1 and cardiac failure in 1. Of the 18 manifesting carriers, 6 (33%) had at least one abnormality on 12-lead ECG and/or Echo and in 5 (28%) initial echo abnormalities confirmed a progressive DCM. One patient required cardiac transplantation, while four others stabilised or improved following initiation of cardioactive medications. Cardiomyopathy is part of the dystrophinopathies and is typically progressive in nature. The incidence is higher in manifesting than non manifesting carriers. Screening and early detection is important for planning prompt treatment and preventing progression of DCM.

16) Delayed development and learning difficulties as a predominant symptom in female carriers of Duchenne and Becker muscular dystrophy

M. Guglieri¹, A. Aboumousa¹, M. Eagle¹, J. Bourke², V. Straub¹ and K. Bushby¹

¹University of Newcastle upon Tyne, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom ²University of Newcastle upon Tyne, Department of Cardiology, Newcastle upon Tyne, United Kingdom

Duchenne and Becker muscular dystrophy (DMD/BMD) generally affect only males. Clinical signs of the condition have been reported in about 8% of female carriers, who may show slowly progressing motor disturbances such as delayed motor development, weakness, muscle pain, calf hypertrophy, and dilated cardiomyopathy. Cognitive and behavioural impairment occur in about one third of DMD/BMD patients but have not been reported so far as a typical feature in carriers. In this study we describe the phenotype of 5 DMD and 1 BMD carriers with a mean age of 13.83 years (age range: 7–20) who presented with motor and speech delay and learning difficulties. Two patients had no family history of DMD and were investigated because of hypotonia and developmental delay. Early history in all patients was characterized by delayed ability to walk independently (from 17 to 24 months) and speech delay. In childhood the clinical phenotype was predominantly characterized by learning difficulties and behavioural problems requiring educational support. A diagnosis of autism was made in one DMD carrier at the age of 5 years. All patients were described as clumsy however only 3 of them showed mild and slowly progressive proximal weakness on examination. Additionally, 3 patients reported muscle pain and cramps after exercise. Creatine kinase levels ranged from 195 to 3500 UI/l. Dilated cardiomyopathy was only detected in the BMD carrier at the age of 20 years. No patient showed respiratory involvement. All patients have a normal female karyotype and carry different mutations located in the distal portion of the dystrophin gene. In conclusion our data indicate that delayed development and learning difficulties may be the presenting features in DMD and BMD carriers and dominate the clinical picture in early childhood, which is an important point in highlighting the possibility of the diagnosis in sporadic cases.

17) Creatine kinase and transaminases in Duchenne muscular dystrophy

P.R. Fequiere¹, B.L. Wong¹ and P.S. Horn²

¹Cincinnati Children’s Hospital Medical Center, Department of Neurology, Cincinnati, OH, United States ²University of Cincinnati, Department of Mathematic Sciences, Cincinnati, OH, United States

Introduction: Duchenne muscular dystrophy (DMD) is the most common childhood muscle disorder, causing progressive weakness. Besides elevated creatine kinase (CK), DMD is also associated with increased transaminases (ALT, AST) which often prompt clinicians to look for liver disease. Aim: Determine expected ranges of CK/ALT/AST in DMD. Methods: Retrospective review (2002–2008) of 216 patients’ (386 samples) CK (less than 30,000 IU) and transaminases. Ninety-six patients had multiple results which were averaged. We also looked at a group of neuromuscular patients (n = 16) with no myopathies and normal values of CK and transaminases. Results: Two hundred and sixteen patients had confirmed DMD with no liver disease. Their age ranged from 3 to 25 years old. Mean CK was 12,217(SD: 7835, 95% CI: 11,166–13,267). Mean AST was 278.5 (SD: 159.1, 95% CI: 257–300).Mean ALT was 405.2 (SD: 240.5, 95% CI: 372–438). Mean CK/AST ratio was 41.6 (SD: 15.5, 95% CI: 39.5–43.7). Mean CK/ALT ratio was 31.8 (SD: 26.1, 95% CI: 28.3–35.4). CK levels correlated with transaminases: r = 0.88 for AST, r = 0.85 for ALT; p < 0.0001 for both. 86.1% of patients were noted to have ALT > AST (n = 186). For the patients with no myopathies (n = 16), mean CK was 147.3 (SD: 74.0, 95% CI: 107.6–187.0).Mean AST was 51.2 (SD: 13.5, 95% CI: 44–58.4). Mean ALT was 25.4 (SD: 15.8, 95% CI: 20.0–31.0).Mean CK/AST ratio was 3.0 (SD: 1.6, 95% CI: 2.1–4.0). Mean CK/ALT ratio was 6.1 (SD: 2.7, 95% CI: 4.7–7.6). Conclusion: We have provided estimates for the expected range and mean for AST and ALT elevation in DMD but no liver disease. ALT was more elevated in the majority of our patients. Elevation in both transaminases are highly correlated with CK elevation.This information can be used to determine whether concern for liver problems is warranted in DMD and in clinical trials for monitoring of liver dysfunction.

18) Predictive factors for progressive foot deformity in non-ambulant boys with Duchenne muscular dystrophy

C. Nicholson¹, M. Main¹, M. Kinali², F. Muntoni² and E. Mercuri²

¹Great Ormond Street Hospital for Children, Physiotherapy, London, United Kingdom ²Institute of Child Health, Dubowitz Neuromuscular Centre, London, United Kingdom

Equinus foot deformity is a common finding in non-ambulant boys with Duchenne muscular dystrophy (DMD). This is due to progressive TA contracture, weakness and imbalance of the muscles of the foot and ankle. This frequently leads to pain, pressure sores, difficulty with positioning and inability to wear footwear. The aims of this study were to investigate: (1) The frequency of severe and rigid foot deformity seen (2) Whether the use of orthotics (KAFO’s, day/night AFO’s) both in the ambulant and non-ambulant phase has an effect on the severity and rigidity of foot deformity (3) Whether those who have early TA release and/or serial casting have a better long term foot posture We undertook a prospective study on 20 non-ambulant boys with DMD (age range 9 year 1 month to 17 year 10 month, age range at loss of independent 7 year 4 month to 11 year 7 month) attending the neuromuscular clinic at Hammersmith Hospital. A specifically developed assessment was used to obtain subjective and objective data of foot posture and detailed history of orthotics, serial casting and surgery. Results showed that 8 boys had no foot deformity and 1 had unilateral foot deformity. Eleven boys had foot deformity, 8 of which were severe and could not be corrected to neutral. The boys who were compliant with daily use of orthotics (AFO’s and/or KAFO’s) had less severe and less rigid foot deformity. Of those boys who had TA releases (n = 5, 3 = prior to loss of ambulation, 2 = following loss of ambulation), those who were non-compliant with continued use of post operative orthoses went on to develop more severe and fixed foot deformities.

19) Identification and characterization of small molecules for the treatment of Duchenne muscular dystrophy

W. Friesen¹, S. Acharjee¹, J. Zhuo¹, R. Baiazitov¹, S. Lee¹, Y.C. Moon¹, H.L. Sweeney² and E. Welch¹

PTC Therapeutics, S. Plainfield, United States ²University of Pennsylvania, Philadelphia, United States

PTC Therapeutics, Inc. (PTC) and Parent Project Muscular Dystrophy (PPMD) are collaborating to discover new drugs to treat Duchenne muscular dystrophy (DMD). Several targets were selected to enter the drug discovery program based on functional validation from animal studies. The targets selected for high throughput screening (HTS) included: utrophin (UTRN), muscle-specific insulin-like growth factor (mIGF1), and alpha7 integrin (ITGA7). Using a proprietary drug discovery platform technology, referred to as GEMS (Gene Expression Modulation by Small-molecules), we sought to identify small molecules that upregulate the production of these protein targets to identify potential treatments for DMD. Constructs containing the firefly luciferase (fLuc) reporter gene flanked by the 5′ and 3′ untranslated regions (UTR) specific for each of the targets were stably transfected in human muscle (RD) or kidney (293H) cells and used in HTS. We identified hits that demonstrate concentration dependent activities in cell-based reporter assays and in assays that measure protein levels. Further, a number of molecules exhibit good pharmacological properties (e.g., low cytotoxicity and microsome metabolic stability). Presently, we are focused on optimizing the activity, potency and pharmacological properties of 2 chemical scaffolds for the mIGF1 program which exhibit up to 5-fold upregulation of mIGF1 and demonstrate structure-activity relationships. For UTRN and ITGA7, we have identified 2 chemical scaffolds for each target and are in the process of establishing structure–activity relationships for these chemical classes. The ultimate goal of this drug discovery and development effort is to identify small molecules that can specifically modulate the production of a number of proteins that can be used as monotherapy or as part of a combination therapy to treat Duchenne muscular dystrophy

20) Evaluation of novel compounds for upregulation of utrophin in animal models of Duchenne muscular dystrophy therapy

R.J. Fairclough¹, A. Potter¹, D. Powell¹, S. Squire¹, M. Bland¹, A. Bareja¹, J. Tinsley² and K.E. Davies¹

¹Oxford University, MRC Functional Genetics Unit, Oxford, United Kingdom ²Summit plc, Abingdon, United Kingdom

DMD is a severe X-linked recessive muscle wasting disease caused by mutations in the cytoskeletal protein dystrophin. With 1 in 3000 boys being affected, strategies including gene therapy, cell transfer and pharmacological screening are currently being pursued in attempts to alleviate the severe phenotype. Advantages of pharmacological screening include ease of systemic delivery of small compounds and the ability to evade immunological and/or toxicity issues. Our strategy of pharmacologically upregulating the dystrophin-related protein utrophin is designed to target the primary defect by restoring sarcolemmal stability in DMD. We have previously demonstrated that 3- to 4-fold upregulation is sufficient for complete prevention of pathology in transgenic mdx mice. Microarray analysis confirmed that overexpression of utrophin in mdx gives a gene expression profile comparable to wild type. Adenoviral delivery of utrophin demonstrated prevention of pathology in the GRMD dog model. Compounds carefully selected for structural diversity by Summit plc were screened in a high throughput utrophin transcriptional upregulation assay based on 8.3 kb of sequence upstream of the transcriptional start site of the neuromuscular junction isoform, utrophin A. Several compounds identified from this screen have since been assessed for their ability to alleviate muscle symptoms in vivo in mdx mice by in-depth analyses including utrophin localisation, expression levels, and improvement in muscle physiology. Timing and route of administration has been thoroughly investigated. Methods of monitoring drug efficacy in vivo are currently being developed. The region(s) defining promoter modulation by compound treatment are currently being determined in vitro using utrophin A promoter deletion constructs, and activity in human cells is being tested using an immortalised DMD cell line.

21) Diltiazem and verapamil protect dystrophin-deficient muscle fibers of mdx mice from degeneration: Potential role in calcium buffering and sarcolemmal stability

H. Santo Neto^a, C.Y. Matsumura^a and M.J. Marques^a

^aUniversidade Estadual de Campinas – Unicamp, Anatomia, Campinas, Brazil

The lack of dystrophin in dystrophin-deficient fibers of mdx mice and in Duchenne muscular dystrophy leads to sarcolemmal breakdown and enhanced calcium influx followed by muscle degeneration. We examined whether the calcium channel blockers diltiazem and verapamil could protect dystrophic muscles from degeneration. Mdx mice (n = 32; 18 days old) received daily intraperitoneal injections of diltiazem (n = 16; 72 mg/kg body weight) or verapamil (n = 16; 25 mg/kg body weight) for 18 days, after which the sternomastoid, diaphragm, tibialis anterior and cardiac muscles were removed. Control mdx mice (n = 16) were injected with saline. Both drugs significantly decreased the blood levels of creatine kinase (saline-treated mdx mice: 573 ± 245 U/l, diltiazem-treated mice: 161 ± 53^* U/l and verapamil-treated mice: 217 ± 57^* U/l; mean + SEM, ^*p < 0.05 vs. saline controls, Student’s t-test). The total calcium content, measured by plasma emission spectrometry, was 173–475% greater in mdx muscles compared to control C57Bl/10 muscles. Verapamil and diltiazem reduced the total calcium content only in diaphragm (diltiazem-treated mice: 229 mg calcium/kg vs. saline-treated mice: 295 mg calcium/kg; p = 0.06, Student’s t-test) and cardiac muscle (diltiazem/verapamil-treated mice: 10 mg calcium/kg vs. saline-treated mice: 16 mg calcium/kg; p < 0.05, Student’s t-test). Histological analysis showed that diltiazem significantly attenuated muscle degeneration only in diaphragm muscle (28% central nucleated fibers and 7% Evans blue-positive fibers in saline-treated mice vs. 12% central nucleated fibers and 1% Evans blue-positive fibers in diltiazem-treated mice; p < 0.05, Student’s t-test). Immunoblots showed a significant increase in the levels of calsequestrin and β-dystroglycan in some diltiazem- and verapamil-treated muscles. Diltiazem was more effective than verapamil in protecting against muscle degeneration in mdx mice, especially in the more affected muscles. These results indicate that calcium channel blockers protect against muscle degeneration in the absence of dystrophin. They also suggest that these drugs could be useful therapeutic alternatives in the treatment of Duchenne muscular dystrophy.

22) Tamoxifen improves the structure and the function of skeletal muscle in mdx mice

O.M. Dorchies^a, J. Reutenauer^a, O. Vuadens^a, S.A. Comyn^a and U.T. Ruegg^a

^aUniversity of Geneva, Laboratory of Pharmacology, Geneva, Switzerland

Data from the literature indicate that tamoxifen (Tam), a selective estrogen receptor modulator (SERM) used in the treatment of certain types of breast cancers, displays antioxidant, anti-apoptotic and anti-fibrotic properties and stabilises biological membranes. We hypothesised that Tam might improve both the structure and the function of dystrophic muscles. When applied to primary cultures of dystrophic myotubes, Tam and 4-hydroxy-Tam (10⁻⁸ to 10⁻⁶ M) were not toxic, reduced collagen content slightly, and exhibited a limited antioxidant activity against hydrogen peroxide. Mdx dystrophic mice, the most common model for Duchenne muscular dystrophy, were treated for about 15 months following weaning with 0.01% tamoxifen mixed into the food pellets (Tam intake approx. 10 mg/kg/day). Tam-treated mdx mice showed a significant decrease in body weight, likely due to the important reduction in white adipose tissue compared to untreated mice. The mass of the triceps surae (composed of soleus, plantaris and gastrocnemius muscles) was lowered to normal values, while the EDL and the diaphragm showed significant hypertrophy compared with both normal and untreated dystrophic animals. Using the horizontal wire test, the Tam-treated mdx performed as well as the normal mice and three times better than the untreated mdx mice. Isometric muscle force of the triceps surae was recorded. Remarkably, specific phasic and tetanic twitch tensions were increased by 100% and 70%, respectively, compared with untreated mdx, resulting in values higher than those of normal animals. The rates of contraction and relaxation were much slower than in the untreated mdx, and the force–frequency curve was significantly shifted to the left, suggesting a marked change toward a slower phenotype. In the Tam-treated mdx, the resistance to repetitive tetanisations was improved by 44% and creatine kinase levels were lowered by 50% compared with the untreated mdx. On-going histological analysis suggests a normalisation of the mean fiber diameter.

23) Urocortins improve skeletal muscle structure and function of mdx mouse via a cyclic AMP/PKA dependent pathway

J. Reutenauer¹, O.M. Dorchies¹, F.X. Boittin², O. Patthey-Vuadens¹ and U.T. Ruegg¹

1 University of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland ²University of Geneva, Department of Zoology & Animal Biology, Geneva, Switzerland

Urocortins are structurally related peptides of the corticotrophin releasing factor (CRF), which has been widely implicated in responses to physical, emotional and environmental stress. Recently, it has been reported that a CRF-receptor-2 (CRFR2) agonist increased muscle mass and slowed disease progression in the mdx muscle from necrosis and on the mechanism involved. Three-week-old mdx^5Cv mice were treated daily by sub-cutaneous injection for 2 weeks with either urocortin 1 at 300 μg/kg, urocortin 2 (30, 100 or 300 μg/kg), or vehicle. Isometric force recordings of the triceps surae (comprising soleus, plantaris and gastrocnemius muscles) showed that contraction and relaxation kinetics were shorter than in untreated mice. Moreover, the force frequency curve was shifted to the right, suggesting a change in the calcium homeostasis or in the distribution of fibres toward a faster phenotype. Interestingly, hindlimb muscles from mice receiving urocortins displayed a higher resistance to mechanical stress and muscle mass was significantly increased. Histology demonstrated that urocortins remarkably protected diaphragm, EDL and soleus muscles from necrosis. Finally, urocortins administration lowered plasma creatine kinase levels up to 49% compared with vehicle. In addition, basal calcium influx was measured in whole dystrophic muscle (EDL and soleus) and in diaphragm strips. Urocortin 2 concentration-dependently decreased the permeability of the dystrophic muscle to calcium by 20–40%. This effect was completely abolished by the selective CRFR2 antagonist astressin₂-B or an inhibitor of protein kinase A (PKA) while addition of forskolin, an activator of adenylate cyclase, mimicked the effect of urocortin 2. We conclude that urocortins improve both the structure and the function of dystrophic skeletal muscle via the stimulation of a cAMP/PKA-dependent signal transduction pathway. Thus, urocortins should be considered as potential candidates to counteract the impairment of calcium homeostasis observed in Duchenne Muscular Dystrophy.

24) Green tea polyphenols and pentoxifylline stimulate dystrophic myotube formation and maturation in primary cultures

O.M. Dorchies^a and U.T. Ruegg^a

^aUniversity of Geneva, Laboratory of Pharmacology, Geneva, Switzerland

Mutations in the gene encoding dystrophin, a cytoskeletal protein normally stabilising muscle fibre membrane during contraction, are the cause of Duchenne muscular dystrophy (DMD). In DMD, the repetition of necrosis-regeneration cycles lead to exhaustion of the regenerative capability of muscle and progression of connective tissue, resulting in muscle wasting and finally to death. Here green tea polyphenols (GTP) among which (−)-epigallocatechin gallate (EGCg) is the major component, and pentoxifylline (PTX) a non-selective phosphodiesterase inhibitor also known as an anti-TNFα agent, were applied to primary cultures of dystrophic mouse (mdx) muscle cells at the onset of fusion and for up to 7 days thereafter. A single application of GTP or EGCg (approx. 2–25 μM) for the first 48 h increased fusion and myosin content. Upon EGCg (25 μM), the cytosolic and nuclear distribution of cyclins and members of the Myf family of muscle-specific transcription factors were altered, suggesting that exit from the cell cycle, a prerequisite to myoblast fusion, was facilitated. Also, the activation (phosphorylation) of the pro-myogenic p38 MAP kinase was enhanced while that of p44/42 (Erk1/2) and JNK1, known to transduce inhibitory signals for muscle differentiation was prevented. After 7 days of treatment, the structural proteins actinin and myosin heavy chains were increased by 2- to 3-fold, while desmin and utrophin, abundantly expressed in immature myotubes were decreased. GTP and EGCG promoted the redistribution of desmin from longitudinal filaments to its adult-type location in the Z discs. Preliminary findings suggest changes in the expression of proteins involved in calcium homeostasis (voltage-gated sodium channel, ryanodine receptor, calsequestrins). Most of these effects were reproduced with PTX. Our data demonstrate that GTP and PTX enhance myogenesis in vitro, which may explain our earlier reports on the improvement of muscle function in mdx mice fed GTP and PTX.

25) Green tea polyphenols as potential treatment of Duchenne muscular dystrophy

U.T. Ruegg^a, O.M. Dorchies^a, J. Reutenauer^a, O. Vuadens^a and E. Roulet^a

University of Geneva, Geneva, Switzerland

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. One of the consequences of the absence of dystrophin is elevation of cytosolic calcium concentration leading to muscle necrosis in DMD patients. Muscle wasting is aggravated by altered capability to regenerate and by fibrosis. We showed previously that glucocorticoids and creatine counteract this increased calcium influx in vitro and prevent muscle degeneration of dystrophic (mdx) mice, a murine model of DMD. As part of a strategy to identify drugs that are readily available and having an optimal cost benefit ratio, we investigated Green Tea Polyphenols (GTP) and their major component, (−)-epigallocatechin gallate (EGCG), with respect to their in vitro action and muscle function of dystrophic mice. Dystrophic mice were fed for 1 week, 5 weeks, or 15 months control chow or chow containing GTP or EGCG. Muscle histology showed that GTP and EGCG reduced necrosis after 1 week. Phasic and tetanic forces of the triceps surae muscles of treated dystrophic mice were increased up to 94%, close to values of normal mice. Muscles from treated dystrophic mice exhibited a 30–50% increase in resistance to repetitive tetanisation. These results suggest that administration of GTE or EGCG to dystrophic mice caused a delay in muscle necrosis, and improved dystrophic muscle function. In vitro studies confirmed the well-known antioxidant action, and showed a potent activity in enhancing muscle cell fusion and myotube maturation, as well as in inhibiting the fibrogenic process. Investigations regarding the mechanisms of action at the molecular level indicate that GTP and EGCG inhibit the expression of TNFα and TGFα1, decrease the activation of JNK1, a kinase promoting dystrophic muscle pathogenesis, and inhibit the expression and the activity of the calcium-independent isoform of phospholipase A₂, an enzyme overactive in DMD muscle and suspected to initiate abnormal calcium influx and therefore muscle cell death In view of these encouraging results, clinical studies with GTP in DMD patients are of high priority. We are looking for partners willing to perform such studies.

26)  In-vivo direct and indirect myocardial effects of captopril in the mdx mouse

R. Bauer¹, V.W. Straub¹, A. Blain¹, K. Bushby¹ and G.A. Mac Gowan²

¹Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom ²Freeman Hospital, Department of Cardiology, Newcastle upon Tyne, United Kingdom

The heart is almost always affected by a progressive cardiomyopathy in Duchenne muscular dystrophy (DMD). In heart failure, angiotensin converting enzyme inhibitors (ACE-I) are a standard therapy, though the role in DMD cardiomyopathy is controversial. Orally administered captopril was given for 8 weeks to 16 weeks old, male mdx mice, a mouse model for DMD. In-vivo cardiac function in treated and untreated mdx mice and age matched controls was assessed by conductance catheter investigations (pressure–volume loops). In addition, we analyzed myocardial fibrosis (Masson Trichrome) and in-vivo myocyte sarcolemmal injury (Evans blue dye). At baseline there is evidence of a compensated cardiomyopathy in the mdx mice with reduced myocardial contractility but preserved stroke volume. Treatment with captopril in mdx and wild type mice led to improved systolic function (stroke volume and cardiac output) due to reduced afterload (arterial elastance), though only in the mdx mice was improved myocardial contractility (endsystolic elastance and preload-recruitable-stroke-work) seen as well. Preload (maximum volume) was unchanged. Histopathologically, hearts from treated animals showed no differences in fibrosis (Masson trichrome) or sarcolemmal damage (uptake of Evans Blue dye) compared to untreated controls. In conclusion, ACE I in mdx mice lead to indirect myocardial effects through reduced afterload, and direct effects with increased contractility. However, myocardial injury is not reduced, suggesting that ACE inhibition is not sufficient to prevent the underlying pathophysiology of the cardiomyopathy.

27) Left ventricular remodeling after steroid therapy in the mdx mouse

R. Bauer¹, G.A. Mac Gowan², A. Blain¹, K. Bushby¹ and V.W. Straub¹

¹Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom ²Freeman Hospital, Department of Cardiology, Newcastle upon Tyne, United Kingdom

Duchenne muscular dystrophy (DMD) is associated with progressive cardiomyopathy and oral corticosteroids are an agreed gold standard for treatment of skeletal myopathy. The effects of steroids on cardiac function in these patients are not known. Orally administered prednisolone was given over 8 weeks to male mdx mice (the animal model for DMD), starting at 16 weeks of age. In vivo cardiac function was assessed with pressure–volume loops in treated and untreated mdx mice and age matched controls. Additionally, fibrosis (H&E) and in vivo myocyte sarcolemmal injury were assessed (Evans blue dye, EBD). At baseline, in mdx mice we found a cardiomyopathy with lower end systolic pressures, decreased myocardial contractility (lower maximum rate of left ventricular pressure development (dp/dtmax) and endsystolic elastance) and patchy myocardial fibrosis compared to healthy wildtype controls. After prednisolone there was ventricular dilatation (higher end-diastolic volumes) associated to diastolic dysfunction (prolonged relaxation (Tau) and higher end diastolic pressures). Histopathologically, hearts from treated mdx animals showed increased areas of fibrosis in a partially transmural pattern and increased myocardial cell damage reflected by higher Evans blue dye uptake (indicator of sarcolemmal injury). In conclusion, in our study, oral treatment of mdx mice with prednisolone leads to increased myocardial damage, left ventricular dilatation and diastolic dysfunction, suggesting that cardiac function must be carefully monitored in muscular dystrophy patients taking steroids.

28) Melatonin prevents oxidative-stress mediated mitochondrial permeability transition and death via enhancement of reduced pyridine nucleotides and glutathione in mouse skeletal muscle cells

Y. Hibaoui^a, E. Roulet^a and U.T. Ruegg^a

^aUniversity of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases including muscle disorders. Protecting mitochondrial function, therefore, is vital for cells to survive. In this study, we demonstrate that melatonin, the main secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic/necrotic events and death in C57BL/6J myotubes. In particular, melatonin potently prevented myotube death induced by tert-butylhydroperoxide (t-BHP) in a concentration-dependent manner (10⁻⁴–10⁻⁶ M). This protective effect was more potent than that of N-acetyl-l-cystein, a well known antioxidant that increases cellular pools of free-radical scavengers. Moreover, melatonin maintained plasma membrane integrity after t-BHP exposure and prevented t-BHP-induced fissions of the long mitochondrial filaments and mitochondrial swelling. To determine if the mitochondrial protection provided by melatonin was due to the inhibition of the formation of reactive oxygen species (ROS), intracellular ROS levels were measured using fluorescence imaging. Application of t-BHP produced a rapid and significant increase in ROS generation in myotubes. This effect was concentration dependently prevented by pretreatment of the myotubes with melatonin. Considering that t-BHP cytotoxicity was also prevented by cyclosporin A, a mitochondrial permeability transition pore (mPTP) inhibitor, we investigated the effect of melatonin on mPTP. Melatonin prevented t-BHP-induced mitochondrial depolarization and protected the pyridine nucleotides and glutathione (two regulators of mPTP opening under conditions of oxidative stress) against t-BHP-induced stress. Using isolated mitochondria, we found that melatonin (10⁻⁸–10⁻⁶ M) desensitized the mPTP to Ca²⁺ and prevented t-BHP-induced mitochondrial swelling, pyridine nucleotide and glutathione oxidation, and enhanced mitochondrial function. In conclusion, our findings suggest that inhibition of the mPTP may essentially contribute to the protective effect of melatonin against oxidative stress in myotubes.

29) Effect of calpain and proteasome inhibition on calcium-dependent proteolysis and muscle histopathology in the mdx mouse

A. Briguet^a, M. Erb^a, I. Courdier-Fruh^a, P. Barzaghi^a, G. Santos^a, H. Herzner^a, C. Lescop^a, H. Siendt^a, M. Henneboehle^a, P. Weyermann^a, J. Dubach-Powell^a, G. Metz^a, T. Meier^a and J. Magyar^a

^aSanthera Pharmaceuticals (Switzerland) Ltd., Liestal, Switzerland

The progressive muscle weakness as observed in Duchenne muscular dystrophy (DMD) is a consequence of dystrophin deficiency. Loss of this functional protein leads to elevated levels of intracellular calcium, a key step in the cellular pathology of DMD. The cysteine protease calpain is activated in dystrophin-deficient muscle, and its inhibition is regarded as a potential therapeutic approach. In addition, previous work has shown that the ubiquitin/proteasome system also contributed to muscle protein breakdown in dystrophic muscle and, therefore, also qualifies as a potential target for therapeutic intervention in DMD. The proteolysis induced by increased calcium levels was characterized in cultured muscle cells and revealed two distinguishable phases, an initial calcium influx-dependent calpain activity and a subsequent calcium-independent activity of the ubiquitin/proteasome system. Next, novel small-molecule inhibitors that inhibit both calpain as well as the 20S proteasome in a cellular system with impaired Ca2+ homeostasis were optimized. Upon administration of such inhibitors to mdx mice significant improvement of quantitative histological parameters was observed in particular with compounds strongly inhibiting the 20S proteasome. In order to investigate the role of calpain inhibition without interfering with the ubiquitin/proteasome system, we crossed mdx mice with transgenic mice overexpressing the endogenous calpain inhibitor calpastatin. As expected, proteolysis by calpain is strongly inhibited in the transgenic mdx mouse; surprisingly this calpain inhibition did not ameliorate muscle histology. Our results indicate that inhibition of the proteasome rather than calpain is required for histological improvement of dystrophin deficient muscle. In conclusion, our inhibitors qualify as potential candidates for pharmacological intervention in muscular dystrophy mainly due to the inhibitory activity against the ubiquitin/proteasome system.

30) Carbonic anhydrase inhibitors’ therapeutic effects on dystrophin-deficient mouse muscle

C.P. Pertl¹, L.S. Segalat², H.L. Lochmueller³ and C.T. Thirion¹

¹Friedrich-Baur-Institute, Ludwig-Maximilians-University, Neurology, Munich, Germany ²Centre de Genetique Moleculaire et Cellulaire, Universite Lyon 1, UMR 5534, Lyon, France ³University Newcastle upon Tyne, Newcastle Muscle Centre, Newcastle upon Tyne, United Kingdo

Duchenne muscular dystrophy (DMD) is an X-linked degenerative muscular wasting disease. Invertebrate animal models allow for high-throughput screening of a multitude of drugs in a physiological context. In Caenorhabditis elegans, mutations of the dys-1 gene in association with a mild MyoD mutation result in a muscular degenerative phenotype. This model shows a nearly immobilized phenotype and was used to screen for molecules able to rescue this phenotype. A screen of about 1000 compounds identified several substances that rescued the C. elegans dys-deficient phenotype. Two of these effective substances, methazolamide and dichlorophenamide, are carbonic anhydrase inhibitors used for anti-glaucoma, benign intracranial hypertension and hypocalcemic periodic paralysis therapy. Carbonic anhydrase catalyzes the rapid conversion of carbon dioxide to bicarbonate and protons. We performed feeding experiments for duration of 120 days with food containing methazolamide and dichlorophenamide in mdx mice. The mdx mouse presents the most common murine model for DMD. A point mutation in the dystrophin gene at position 3185 results in a premature stop codon. Apart from a small number of revertant fibres, skeletal muscle fibres are dystrophin-negative and the phenotype is subtle if compared to humans with most prominent degradation of the diaphragm muscle. Therefore, we examined histological appearance of three muscles M. tibialis anterior, diaphragm and M. soleus of treated and control animals. Furthermore, muscle force was measured in M. extensor digitorum longus (EDL). Dichlorophenamide reduces the number of centrally nucleated and round fibres in diaphragm significantly. In TA and Sol we observed a shift of the fibre perimeter variation towards larger hypertrophic fibres. Specific tetanic muscle force increased significantly in the right treated EDL muscle group, however the treatment failed to increase resistance of muscle fibres to eccentric contractions.

31) Phase 2b Study of PTC124 in Duchenne/Becker muscular dystrophy (DMD/BMD): Demographic and other baseline data

L.A. Atkinson¹, A.L. Reha¹, G.L. Elfring¹, R. Finkel², B. Wong³, K. Flanigan⁴, C.M. McDonald⁵, K. Bushby⁶, T. Voit⁷, G. Spinella⁸, V. Cwik⁹ and L. Miller¹

¹PTC Therapeutics, South Plainfield, New Jersey, United States ²Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States ³Cincinnati Children’s Hospital, Cincinnati, Ohio, United States ⁴University of Utah, Salt Lake City, Utah, United States ⁵University of California, Davis Medical Center, Sacramento, California United States ⁶Institute of Human Genetics, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom ⁷Myology Institute, Paris, France ⁸Parent Project Muscular Dystrophy, Middletown, Ohio, United States ⁹Muscular Dystrophy Association, Tucson, Arizona, United States

Background: PTC124 is an orally bioavailable, small molecule with potential clinical utility in treating genetic disorders through induction of readthrough of nonsense mutations and production of full-length, functional proteins. A prior Phase 2a study showed PTC124 is active and generally well tolerated in boys with nonsense-mutation-mediated DMD. These data supported the initiation of this long-term Phase 2b, international, multicenter, randomized, double-blind, placebo-controlled, dose-ranging, efficacy and safety study. Methods: This study is enrolling 165 ambulatory boys   5 years of age with nonsense-mutation-mediated DMD/BMD at   35 investigational sites. Patients are randomized in a 1:1:1 ratio to either a higher dose of PTC124, a lower dose of PTC124, or placebo. Subjects receive study drug orally 3 times per day for 48 weeks and are evaluated at clinic visits every 6 weeks. The primary objective is to evaluate the effect of PTC124 on ambulation as assessed by change in 6-minute-walk-distance. The study also assesses at-home activity as measured by pedometry; evaluates timed tests of standing, stair climbing, and running; measures strength; determines the frequency of accidental falls; assesses patient/parent-reported health-related quality of life, verbal memory and attention, and treatment satisfaction; and follows serum creatine kinase values as a measure of whole body muscle fragility. Clinical results will be placed in context with change in biceps muscle dystrophin expression in pre-treatment vs on-treatment muscle biopsy samples. Results: Subject enrollment is ongoing. Available demographic and other baseline data will be presented. Conclusion: This study constitutes the first pivotal, registration-directed trial in DMD/BMD. In the subset of subjects whose disease is mediated by a nonsense mutation, clinical development of PTC124 may offer a treatment approach that targets the underlying cause of the disease. The available cross-sectional data will provide insight into the study population and augment understanding of the natural history of nonsense-mutation-mediated DMD/BMD.

32) Pentoxifylline treatment fails to rescue muscle strength and function deterioration in prednisone-treated Duchenne muscular dystrophy (DMD)

D.M. Escolar¹, K. Gorni², A.C. Tesi-Rocha¹, J. Mah³, Y. Nevo⁴, A. Korengberg⁵, H. Kolski⁶, T. Bertorini⁷, A. Connolly⁸, N. Kuntz⁹, A. Zimmerman¹, L. Morgenroth¹⁰, A. Arrieta¹, J. Mayhew¹¹, J. Florence¹¹, L. Nei¹², F. Hu¹, E. Henricson¹³, R. Leshner¹ and A. Dubrovsky¹⁴

¹Children’s National Medical Center, Washington, United States ²IRCCS Fondazione Istituto Neurologico, Pavia, Italy ³Alberta Children’s Hospital, Calgary, Canada ⁴University of Tel Aviv Sourasky Medical Center, Tel Aviv, Israel ⁵Children’s Hospital, Melbourne, Australia ⁶Glenrose Rehab Hospital, Edmonton, Canada ⁷University of Tennessee, Memphis, United States ⁸Washington University at St. Louis, StLouis, United States ⁹Mayo Clinic, Rochester, United States ¹⁰Children’s Hospital of Pittsburgh, Pittsburgh, United States ¹¹Washington University, StLouis, United States ¹²Georgetown University, Washington, United States ¹³University of California Davis, Davis, United States ¹⁴Fundacion Alfredo Thompson, Buenos Aires, Argentina

Objective: This clinical trial was conducted to evaluate the additive effect of Pentoxyfylline to prednisone by stabilizing or improving muscle strength and function in DMD. Background: Treatment of DMD with prednisone results in slowing, but not prevention, of deterioration of muscle strength and function. Pentoxyfylline is an anti-inflammatory and anti-fibrotic drug that prevents muscle strength deterioration by 51% in the exercised mdx mouse model of the disease. If these effects translate to human, a combination therapy could halt disease progression, or produce a steroid sparing effect. Desing/Methods: This was a randomized, controlled, double-blind 12 months study of Pentoxyfylline in steroid-treated DMD boys over the age of 7. The primary objective of the study was to determine whether daily treatment with PTX could significantly improve total QMT score at 12 months compared to patients receiving placebo. Secondary endpoints were QMT arm and leg scores, grip, MMT score, timed function, quality of life and exploratory surrogate outcome measurements of levels of TGF-β and TNF-α. Safety laboratory assessments and adverse events were collected. Results: Based on preliminary results, seventy three patients were enrolled, 64 randomized and 57 completed the protocol. Mean and median age were 10.07 and 9.5 respectively. Baseline characteristics between groups were not statistically significant. Total QMT score changes at 12 months was not significantly different between groups (p = 0.5). None of the secondary strength and functional endpoints differed between groups at 12 months. The PTX group presented with increase incidence of coagulation, skin and mild gastrointestinal side effects. Discussion: This study has shown that Pentoxyfylline treatment, although well tolerated over a year, has no effect in slowing disease progression over prednisone in DMD boys.

33) Equipoise concerning corticosteroid use in boys with Duchenne muscular dystrophy: persistent wide variations in practice

R.C. Griggs¹, B.E. Herr¹, M. Eagle², E. McColl², G. Bell², R. Tawil¹, S. Pandya¹, M. McDermott³, K. Bushby², TREAT-NMD Investigators⁴ and MSG Investigators⁵

¹University of Rochester, Neurology, Rochester, NY, United States ²University of Newcastle, Neurology, Newcastle upon Tyne, United Kingdom ³University of Rochester, Biostatistics, Rochester, NY, United States ⁴University of Newcastle, Newcastle upon Tyne, United Kingdom ⁵University of Rochester, Rochester, NY, United States

At least 4 double blind, randomized placebo controlled trials over the past 20 years have established that prednisone 0.75 mg/kg/day is of short-term benefit in terms of muscle strength in boys with Duchenne Muscular Dystrophy (DMD). This benefit was shown in these trials to be maintained for up to 18 months. Despite this evidence, at the start of a collaborative project in 2003 bringing together investigators in Europe and the U.S. to develop standards for care in DMD and to design a randomized trial comparing corticosteroid regimens a survey established that this regimen was not in use in the majority of centers and that intermittent and other corticosteroid regimens were more frequently used. In 2004 both a Cochrane review and an American Academy of Neurology practice parameter concluded that the prednisone regimen 0.75 mg/kg/day was of established short-term efficacy. Four years on, to investigate if there is a continued need for a trial of corticosteroid regimens, this group of investigators have again surveyed clinicians to see if there remains equipoise concerning corticosteroid use. A survey of 105 DMD clinics in January 2008 documented continuing wide variations in practice with a total of 29 different corticosteroid regimens reported. The 4 most frequently used regimens are: 0.75 mg/kg/d prednisone, 60 centers; 0.9 mg/kg/d deflazacort, 31 centers; 75 mg/kg/d prednisone 10 days on/10 days off, 32 centers; 5 mg/kg/d weekends, 10 centers. Although less than 10% of Centers now do not routinely prescribe corticosteroids, there remains no consensus on the optimum regimen for their use in DMD.

34) Randomized, double-blind, controlled study to compare efficacy and tolerability of standard daily prednisone regime with a novel intermittent high dose regime in ambulant boys with Duchenne muscular dystrophy

D. Escolar¹, C. McDonald², A. Korengberg³, T. Bertorini⁴, T. Lotze⁵, M. Ryan⁶, P. Clemens⁷, R. Leshner¹, A. Pestronk⁸, L. Morgenroth⁷, A. Arrieta⁹, E. Henricson², J. Mayhew⁹, J. Florence⁸, T. Duong⁹, L. Nei¹⁰, F. Hu⁹, A.C. Tesi-Rocha¹ and A. Connolly⁸

¹Children’s National Medical Center, Washington, United States ²University of California Davis, Davis, United States ³Children’s Hospital, Melbourne, Australia ⁴University of Tennessee, Memphis, United States ⁵Children’s Hospital, Houston, United States ⁶Children’s Hospital at Westmead, Sydney, Australia ⁷Children’s Hospital of Pittsburgh, Pittsburgh, United States ⁸Washington University, StLouis, United States ⁹Children”s National Medical Center, Washington, United States ¹⁰Georgetown University, Washington, United States

Objective: This study evaluated the hypothesis that high dose prednisone given over 2 days of the week has similar efficacy but fewer side effects than the standard daily prednisone regime. Background: DMD is a progressive lethal muscle disorder. Daily prednisone treatment at 0.75 mgs/day results in increased function and prolonged ambulation. Weight gain, behavioral problems and growth deceleration have hindered its acceptance as standard treatment. Design/Methods: Double-blind, controlled, randomized 12 month study comparing prednisone 0.75 mgs/kg/day with weekly 10 mg/kg divided over two days. Participants were steroid naive DMD boys, ages 4 to 10. Co-primary outcome measures for efficacy and tolerability were quantitative total muscle strength and body mass index. Secondary outcomes included QMT arm and leg scores, grip, MMT, timed function, FVC, height, weight, bone density, cataract formation, blood glucose, blood pressure and behavioral changes. Primary analysis was comparison of QMT score improvement and of BMI changes between groups at 12 months. Results: Seventy six patients were enrolled, 64 randomized and 55 completed the protocol. Mean age was 7.296 (sd = 1.78). There were no significant differences in baseline characteristics between groups. After 12 months of treatment the improvement on the total QMT score did not differ between groups (−0.05 (CI = −0.15, 0.05) p = 0.34) nor did all other efficacy measures. BMI increased in both groups, with a significant difference between groups (higher in daily) at 3 months (p =  0.04), that lost significance at 12 months (p = 0.07). There was a significantly greater increase in growth in the intermittent dose regime group (p = 0.004) and no difference in weight. Conclusions/Relevance: This study confirms that an intermittent dose regimen of prednisone can be equally efficacious to daily steroids, with a lesser effects on growth retardation and BMI. Other results on adverse events, including behavior, cataract formation and bone density will be presented.

35)  Benefits of initiating prednisone treatment in non-ambulatory patients with Duchenne muscular dystrophy (DMD)

S. Pandya¹, D. Fox², K. Fox², C. Westfield², Y. Su², K. Campbell², D. Guntrum¹, E. Ciafaloni¹ and R.T. Moxley¹

¹University of Rochester, Neurology, Rochester, United States ²NY State Department of Health, Troy, United States

Objective: To document the effects of prednisone therapy in patients who were non - ambulatory at initiation of treatment. Background: The Clinical Investigation in Duchenne Dystrophy(CIDD) group reported one open trial and three randomized controlled trials that documented the benefits of Prednisone treatment in DMD. Each of these trials included boys between the ages of 5-15 years- about 20% of them non-ambulatory. Yet clinicians remain concerned about offering or initiating corticosteroid treatment in their non - ambulatory patients. Methods: We retrospectively reviewed data on all patients:1) born between 1970-1989, 2) started on prednisone after 1991, 3) followed at our site for at least 3yrs. Results: We identified 72 patients who met the above criteria. Of these 23 were non -ambulatory at initiation of prednisone. The mean age at loss of ambulation in this cohort of 23 boys was 10.4 yrs (sd +/- 1.9). The mean age at initiation of treatment was 13.2 yrs (sd +/-3.0). The forced vital capacity (FVC) at initiation of treatment was 1.8 L (sd +/- 0.8) and one year after treatment 2.0 L (sd +/- 0.6). The non-ambulatory cohort reached a peak FVC of 2.2 L (sd +/- 0.7) at age 15.2 yrs (sd +/-3.0). Fourteen of these 23 patients required use of night time BiPap at a mean age of 22.7 yrs (sd +/- 4.5) after their FVC had declined to 1.0 L (sd +/-0.6). Discussion: Our long term prednisone treatment data confirm and extend the observations from the CIDD study that non -ambulatory patients who receive prednisone show an increase in pulmonary function. Conclusions: Clinicians should recommend and offer corticosteroids treatment to their non-ambulatory patients with DMD.

36) Two excited Duchenne muscular dystrophy brothers: The late use of steroid

A. Brassanini^a, C.A. Meloe Souza^a, A.J. Godoy^a and M.B. Magario^a

^aMedical School of UNAERP, Ribeirão Preto, Brazil

Several reports have shown the benefits of using steroids for Duchenne muscular dystrophy patients. If the prescription should be initiated in the cases of older patients is still controversial. We report the effects of a late steroid therapy for two Duchenne boys, brothers, severely disabled. ESJ, 30, presented frequent falls and difficulty to rise from the sitting position at the age of 5. Three years later a muscle biopsy pointed to the diagnosis of DMD. At 11 he stopped walking. After 3 years he had difficulty to move the upper limbs. In 2004 he first came to our clinic. He was under no medication; had been using ventilatory assistance for years and at 26 he needed it every night, for the whole sleeping time. In the end of 2007, the parents finally agreed giving steroids to their boys. We started predinisone, 40 mg per day (0.75 mg/Kg/day). JPS, 22, at the age of 3 falled frequently and showed difficulty to rise. At nine, stopped walking and at 18 had to use ventilator every night. We first saw him in 2004. He was in a bad shape. We also started the steroid therapy in the end of last year: prednisone, 15 mg per day. In the end of March the patients were interviewed. It was very impressive their excitement: both reported reduction of the muscle pain and increased overall motivation. They could remain in the sitting position for a longer time, had a stupendous appetite. Regarding cognitive functions, their concentration improved, as well as the libido. They gained weight and muscle volume (pectorals). It was possible to reduce the time they used the ventilator, with no pulmonary symptoms. Their performance during the sessions of physical therapy also improved. They noticed an enhancing in the quality of their sleep. No side effects were reported. Of course we want to see the long term effects, but their excitement was pretty much contagious and we strongly recommend the introduction of steroids even for very disabled patients.

37) Long bone fractures and bone healing in boys with Duchenne muscular dystrophy treated with daily, long-term Deflazacort

D. Biggar¹, V.A. Harris¹, B. Alman² and J. Vajsar²

Bloorview Kids Rehab, Toronto, Canada ²Hospital for Sick Children, Toronto, Canada

A major side effect of long-term deflazacort treatment is osteoporosis and an increased risk of long bone fractures. Objective: To document the occurrence of long bone fractures and subsequent bone healing in boys with DMD who were treated with long-term, daily deflazacort (starting dose 0.9 mg/kg/day) and supplemental vitamin D (1000u/day) and calcium (750-1000mg/day). Design: A prospective collection of fracture data from 93 boys with DMD treated with daily deflazacort from 1997 to 2007. Main Results: Nineteen of 93 boys had a long bone fracture; 5 of 19 had a subsequent second fracture at a different site. Mean Lumbar spine (L1-4) bone mineral density (BMD) for the 19 boys was Z = −3.1 ± 0.9. There was no correlation of BMD L1-4 Z scores with the number of years on deflazacort (mean 6.8 ± 2.6 yrs) or the cumulative lifetime dose. The femur fractured in 13 boys, the tibia and/or fibula in 10 boys and patella in 1 boy. Six boys had fracture stabilization by internal fixation with normal bone healing and no postoperative infection. Nineteen of 24 fractures were fragility fractures. Seventeen of 19 boys (ages 11.4 ± 3 yrs) were ambulators at the time of their first fracture; 10 of 17 resumed walking after fracture healing; the remaining 7 boys (ages 13 ± 2.2yrs) stopped walking. Five of the 6 boys treated by internal fixation were weight bearing and in a physiotherapy program within the first week after surgery. Conclusion: We conclude that approximately 20% of boys on long-term daily deflazacort have long bone fragility fractures. Fracture healing appears normal despite deflazacort treatment and reduced BMD. There is a significant risk (40%) of losing ambulation after a lower extremity fracture. While long-term fracture data comparing prednisone and deflazacort treatments are needed, deflazacort, with its bone-sparing property, might be the corticosteroid of choice for boys with DMD who are at risk for osteoporosis secondary to reduced mobility.

38) Vertebral fractures in corticosteroid-treated boys with Duchenne muscular dystrophy

G.P. Ambegaonkar¹, M. Kinali², F. Muntoni³, S.A. Robb¹ and A.Y. Manzur¹

¹Great Ormond Street Hospital, Department of Paediatrics, London, United Kingdom ²Institute of Child Health, Department of Paediatrics, London, United Kingdom ³Institute of Child Health, Department of Paediatrics, London, United Kingdom

Corticosteroids increase muscle strength, prolong ambulation and preserve respiratory function in DMD. Adverse effects include vertebral fractures (VF), which are not otherwise a feature of DMD. We describe the presentation of vertebral fractures and their relation to corticosteroid treatment. We carried out a retrospective study of the dosage, schedule and duration of corticosteroid treatment at time of fracture. We recorded the presentation of the fracture, treatment used and the outcome. Five boys suffered VF, whilst ambulant and with no previous history of fractures. One had concomitant hydrocephalus limiting mobility. Mean age SD of starting prednisolone (0.75 mg/kg/day) was 8.50.89 yrs. Mean age at fracture was 11.31.71 yrs. Four boys were initially on the intermittent regime (10 days on, 10 days off), followed by daily regime and developed fracture within 10.8 ± 10.7 months of changing the regime. At the time of VF, mean prednisolone dose was 0.67 mg/kg/day (range 0.53-0.75).Two boys presented with sudden onset back pain on forward flexion, two had precipitating trivial falls and one chronic back pain. Lateral spinal X-rays showed osteopenia and either a reduction of vertebral body height or wedging/crushing. Single vertebra was affected in 2/5 and multiple vertebrae in 3/5 patients. Lumbar vertebrae were fractured in 4/5, and thoracic in 1/5 patients. At the time of fracture, DEXA z score ranged from −0.9 to  − 1.7, and no patient was Vit D deficient. IV Palmidronate therapy afforded rapid resolution of pain. All patients elected to continue with prednisolone despite the VF. In this cohort, bone density z scores were not in the osteoporotic range at the time of VF, suggesting that DEXA scans may not necessarily predict VF. Though effective VF treatment with palmidronate is available, an active debate about optimal surveillance of vertebral bone health, and evidence based prophylactic treatment of VF is needed.

39) Carvedilol can reduce cardiac events in Duchenne muscular dystrophy

T. Matsumura¹, T. Tamura², S. Kuru³, Y. Kikuchi⁴ and M. Kawai²

¹National Hospital Organization Toneyama National Hospital, Neurology, Toyonaka, Japan ²National Hospital Organization Higashi-Saitama National Hospital, Neurology, Hasuda, Japan ³National Hospital Organization Suzuka National Hospital, Neurology, Suzuka, Japan ⁴Nagasaki University School of Health Science, Occupational Therapy, Nagasaki, Japan

Cardiac dysfunction is one of the most serious complications in Duchenne muscular dystrophy (DMD). Beta-blocker is known to improve prognosis of chronic cardiac failure of adults, but its efficacy and safety for the patients with muscular dystrophy has not been fully assessed. Thus we made a multicenter trial of carvedilol to investigate the efficacy and safety for DMD patients. Fifty-four DMD patients participated to this study and forty-one patients received carvedilol and thirteen patients were without carvedilol. The hazard ratio of cardiac events was much lower in patients receiving carvedilol. Mortality was also lower in patients with carvedilol, though not significant. In patients with carvedilol, the heart rate was decreased after introduction of carvedilol, while patients without carvedilol showed no significant changes. The reduction of heart rate by carvedilol was correlated to the dosage of carvedilol. Patients with cardiac events showed higher mean HR and lower dose of carvedilol. There were no significant changes in ejection fraction and brain natriuretic peptide during study period in both groups. Although serious adverse events were rare during the introduction of carvedilol, both the longer period for up-titration of carvedilol and the minor complaints were common for patients with impaired cardiac function. It seemed that carvedilol is relatively safe and can improve prognosis of cardiac dysfunction of DMD.

40) Growth hormone may improve growth, neuromuscular and pulmonary function in Duchenne muscular dystrophy

M.M. Rutter¹, S.R. Rose¹, M. Kalra² and B.L. Wong³

¹Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Cincinnati, United States ²Cincinnati Children’s Hospital Medical Center, Division of Pulmonary Medicine, Cincinnati, United States ³Cincinnati Children’s Hospital Medical Center, Division of Pediatric Neurology, Cincinnati, United States

Background: Glucocorticoids prolong ambulation and survival in Duchenne muscular dystrophy (DMD), but cause growth failure, obesity, pubertal delay and osteoporosis, negatively impacting quality of life. Growth hormone (GH) may offer potential benefits, but data regarding safety and efficacy in DMD are lacking. Anecdotal reports suggest that GH may cause neuromuscular deterioration and be detrimental in DMD. Objective: To report preliminary experience of GH safety and efficacy in DMD boys with glucocorticoid-induced growth failure. Design/methods: A case series of DMD boys with severe steroid-induced growth failure treated with GH. Outcomes included growth velocity, weight, BMI, neuromuscular and pulmonary function, and side effects. Results: Of 21 boys started on GH, eight have been treated for 6–20 (mean 13.1) months. They had received deflazacort or prednisone for 3–11 years. Mean pre-treatment height and growth velocity were −4.0 (range −2.5 to −5.0) SDS and 0.5 (0–1.8) cm/year, respectively. Peak stimulated GH levels were low (1.2 –7.8, mean 3.9 ng/ml) in the six patients tested. Average height velocity during the first year on GH therapy was 5.4 (3.2–8.4) cm/year. Mean BMI decreased by 2.6 (−6.0 to +0.9) kg/m². Neuromuscular function improved in four younger boys (ages 9.4–13.6 years), with mean decreases timed Gower tests by 20% (14–27%) and 30-foot runs by 9% (unchanged to 18%), during the first year. Three older boys (ages 14.3–17.5 years) had stable neuromuscular function at a stage of expected decline. Median forced vital capacity changed by +29% (−9% to +33%). Two patients had mild progression of scoliosis; no other side effects were noted. Conclusions: GH treatment of DMD boys with steroid-induced growth failure appears safe, and may improve growth velocity, body composition, neuromuscular and pulmonary function.

41) The rationale for immunosuppressive treatment in DMD

R. Korinthenberg^a

^aUniversity Hospital Freiburg, Paediatric, Neurology and Muscular, Disorders, Freiburg, Germany

Corticosteroids, currently the gold standard treatment for Duchenne muscular dystrophy (DMD), have renewed interest in immunological studies for DMD. Despite their positive effects on muscle strength, respiratory function and overall life expectancy, corticosteroids have a broad spectrum of side effects. Optimized care and alternative treatment regimes aim to reduce and improve the side effects of long-term daily steroid application. The precise molecular mechanism of steroids is still not entirely understood, but immunosuppression is one possible explanation. The inflammatory reaction in DMD is not an unspecific reaction as in traumatic injury. CD4⁺- and CD8⁺-cells promote the pathology even before histological changes, and congenital absence of T-cells has been shown to prevent fibrosis in the mdx diaphragm. Activated macrophages also promote muscle damage and their elimination prevents membrane lysis. In DMD, TGF-β, which inhibits muscle regeneration, is strongly upregulated. Corticosteroids downregulate genes involved in inflammatory responses and reduce infiltrating cells, collagen and cytokines, including TGF-β. They also reduce CD8⁺ cells, infiltrated muscle fibres and connective tissue proliferation in DMD. Not all immunosuppressants show positive effects in dystrophin deficiency: azathioprin is just one example that does not. In contrast, Cyclosporin A (CsA), which is used in cell transplantation and as a corticosteroid-sparing drug, improved voluntary and tetanic force in two DMD trials. CsA acts via inhibition of calcineurin and the mitochondrial permeability transition pore. Overexpression of calcineurin leads to upregulation of utrophin and amelioration of the phenotype in mdx. At a dosage of 10 mg/kg, CsA gave rise to histological and functional improvements in mdx mice. High-dose CsA (>25 mg/kg) on the other hand inhibits regeneration of atrophic muscles and deteriorates the mdx phenotype. To define the role of CsA in DMD we have begun a randomized, placebo controlled trial with 4 mg/kg CsA, the first results of which will be presented.

42) Re-establishment of dystrophin expression by exon skipping and/or trans-splicing approaches

L. Garcia^a

^aUPMC-Inserm UMRS787, Institut de Myologie, Paris, France

Dystrophin, with its modular structure, and in particular its central domain made up of 24 spectrin-like repeat motifs, is a protein that can support the deletion of certain internal areas provided that the final coding sequence is preserved. Taking into account the organization of exons in the DMD gene, one can imagine that a sequence eliminated by a mutation (deletion or point mutation) can be restored by the exclusion of one or more exons if the local sequence allows it. This is called therapeutic exon skipping (TES). Exon skipping is executed during the splicing process and can be induced by antisense oligonucleotide (modified to resist RNases) complementary to key-sequences, which lead the splicing of primary transcripts. It is possible to combine splicing therapy using antisense oligonucleotide with a traditional gene therapy technology, in order to obtain, with a single injection, an endogenous synthesis of antisense and a durable therapeutic effect. The practical application of a clinically relevant gene therapy for DMD requires the treatment of the whole skeletal and cardiac musculature. Only a systemic injection would allow to approach this objective, as demonstrated by initial trials in mice. Such a method is not without risk and requires long and expensive developments. First of all, it is essential to be able to produce the required quantity of vector, then, the systemic mode of administration needs to be developed and its safety should be guaranteed. We are currently testing the feasibility of a systemic administrations of an AAV(U7) vector in the GRMD dog model, in order to anticipate, on a rational basis, the adaptation of such protocols in man. On the other hand, TES is possible only in patients affected by genetic diseases in which (i) it restores a truncated but functional protein, or (ii) it destroys a target downstream of the cascade of events provoked by the mutation. Many pathological conditions escape these prerequisites. Then, we anticipate the development of a more universal strategy of RNA repair where the mutated exon is replaced by its normal form. This approach also makes it possible to reintroduce missing exons.

43) SNT-MC17/idebenone in Duchenne muscular dystrophy: long-term blinded controlled preclinical study in the mdx mouse followed by a 12 month double-blind randomized controlled trial in humans

G.M. Buyse¹, N. Goemans¹, G. Van der Mieren¹, M. Erb², J. D’hooge¹, P. Herijgers¹, E. Verbeken¹, A. Jara³, A. Van Den Bergh¹, I. Courdier-Fruh², P. Barzaghi², M. den Hauwe van¹, D. Thijs¹, I.J.M. Groot de⁴, U. Schara⁵, B. Ceulemans⁶ and L. Mertens¹

¹University Hospitals Leuven, Leuven, Belgium ²Santhera Pharmaceuticals, Liestal, Switzerland ³Biostatistical Center KU Leuven, Leuven, Belgium ⁴UMC St. Radboud, Nijmegen, Netherlands ⁵Lukaskrankenhaus GmbH, Neuss, Germany ⁶University Hospital Antwerp, Antwerp, Belgium

Background and strategy. In Duchenne muscular dystrophy (DMD) cardiac and respiratory complications are the predominant causes of death. There is a strong scientific rationale for SNT-MC17/idebenone as a therapeutic approach for DMD, based on disease pathogenesis and the drug’s potential to improve mitochondrial respiratory chain function and reduce oxidative stress. We first performed a preclinical blinded controlled study with SNT-MC17/idebenone in the dystrophin-deficient mdx mouse. The mouse model allowed presymptomatic initiation and veritable long-term administration of treatment, as well as gold standard in vivo invasive cardiac contractility assessments. Second, we performed a phase II double-blind randomized placebo-controlled trial to evaluate efficacy and tolerability of treatment with SNT-MC17/idebenone compared to placebo in children with DMD. Results. (A) Preclinical study: compared to wild-type mice, placebo-treated mdx mice showed cardiac hypertrophy, diastolic dysfunction, reduced contractile reserve with systolic failure and 58% mortality during low-dose dobutamine stress, cardiac inflammation and fibrosis, and reduced voluntary wheel running performance. SNT-MC17idebenone treatment significantly protected left ventricular contractility, significantly reduced cardiac inflammation and fibrosis, and significantly improved voluntary running performance in mdx mice. (B) Clinical study: SNT-MC17/idebenone showed good safety and tolerability. There was a significant treatment effect favouring SNT-MC17/idebenone on the primary cardiac endpoint (myocardial function at left ventricle inferolateral wall), as well as on secondary cardiac and respiratory outcomes. No significant differences were detected for quantitative upper limb muscle strength. Conclusions. We have identified a novel potential therapeutic strategy for DMD. Early-initiated and long-term SNT-MC17/idebenone was cardioprotective and improved exercise performance in the mdx mouse. The phase II clinical data are the first indication ever of clinical efficacy with SNT-MC17/idebenone on functional cardiac and respiratory parameters in DMD, and these results provide the basis and guidance for phase III studies with SNT-MC17/idebenone in DMD. Our findings also illustrate that the mdx mouse provides unique opportunities for long-term controlled prehuman therapeutic studies.