Therapies

Poster Presentations

Monday, July 8, 2002

national Workshops. Over 1300 scientist attended. Over 150 publications have resulted from collaborative work at ENMC Workshops. Extra funding by the European Union funding was obtained by 3 Consortia. Diagnostic criteria were established, several genes identified and treatment rationalised. Other regions are now trying to establish similar cooperative models. Other fields (Huntington, Parkinson) have approached ENMC for assistance to develop this approach to collaboration. Conclusions: ENMC is a successful, structural model for support of scientific exchange and cooperation. It is unique also because it is initiated and financed by European patient organisations and funding agencies in 9 European countries.

P-105 Neuromuscular Diseases and their Influence Over the Work Ability Ljubica Velickova*, Vildane Paloshi. Skopje, R. of Macedonia The neuromuscular diseases increase constantly in the last years. We thing that one of the reasons for that fact is the medicine devices became modern and either the early diagnostic of this kind of diseases. It is known that mostly the young people suffer from these diseases and we are especially interesting in how these diseases affect to the work ability, i.e. how they contribute to reduction and lost of it. There are 77,713 cases total that have been processed in a period since 1995 to 2001.37 patients of that number of patients with neuromuscular diseases have appeared at a commission medical checkup. 12 of the patients with neuromuscular diseases had Myasthenia Gravis, 6 patients had Guillain-Barre Syndrome, 12 patients were with Myotonic Dystrophy and 7 cases came with other polyneuropathia. 20 patients or 54% were females especially by the Myasthenia Gravis. Characteristic thing is that only 16% or 6 patients of 37 have had in 2-3 times reduced work ability with change of job and with a 4 hour halftime, but all the other cases were in difficult situation and were given a lost work ability status immediately. It should be considered that these diseases attack relatively the young people who have never established/begun a job.

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P-107 Molecular Mechanism of Action of the Glucocorticoid Defalazacort in Dystrophic Muscle S. Kolodziejczyk*, S. St-Pierre, L.A. Megeney. Ottawa, Canada

Objective: Duchenne Muscular Dystrophy (DMD) results from a mutation in the dystrophin gene, leading to a complete loss of the protein product. This complete absence of protein leads to severe skeletal muscle wasting and death in early adult hood. Currently, the only treatment intervention available to DMD patients is the administration of a subclass of glucocorticoids, specifically prednisone and deflazacort. Clinical trials have shown that prednisone/deflazacort treatment attenuates the progression of DMD, extending ambulation and maintenance of muscle strength. The mechanism(s) by which prednisone/deflazacort attenuates dystrophy progression remains unknown. Methods: Using biochemical and immunohistological approaches, we have tested the ability of Deflazacort to rescue the dystrophic phenotype in two model systems, a stable C2C12 cell line overexpressing the JNK 1 specific activator MKK7 as well as the m d x : M y o D - / - dystrophic mouse model, as well as ivestigate its probable mode of action. Results: MKK7 overexpressing cells treated with Deflazacort are able to differentiate and express differentiation specific markers. Furthermore, m d x : M y o D - / - mice injected interperitoneally with deflazacort (daily over three week period) display a significant decrease in myofiber damage compared to sham injected controls. Conclusions: Biochemical and immunohistological analysis suggest that deflazacort functions by translocating the transcription factor NF-ATcl from cytoplasm to nucleus, modulating differentiation and maintaining fiber integrity. P-108 Cranial Magnetic Resonance Imaging (MRI) Findings in Proximal Myotonic Myopathy (PROMM) and Myotonic Dystrophy (DM) C. Kornblum*, C. Grothe, N. Amanatidis, J. Reul, R. Schr der. Bonn,

Germany

Therapies P-106 Safety and Efficacy of Recombinant Acid Alpha-Glucosidase (rhGAA) in Patients with Classical Infantile Pompe Disease L. Klinge*, V. Straub, U. Neudoff, K. Goerlinger, E. Dittmar, T. Volt.

Essen, Germany Objective: Pompe disease is an autosomal recessive muscle-wasting disorder caused by deficiency of the lysosomal enzyme GAA. Classical infantile Pompe disease is characterized by progressive cardiomyopathy, muscle weakness, respiratory insufficiency and death in early infancy. Methods: Four patients with classical infantile Pompe disease participate in two separate Phase 2 open-label, multinational multicenter studies. Two patients receive rhGAA derived from milk of transgenic rabbits 40 mg/kg IV weekly and two patients with CHO-cell derived rhGAA 10 mg/kg IV weekly. Safety is evaluated by recording adverse events, vital signs, physical examination and antibodies to rhGAA. Clinical efficacy endpoints include ventilator-free survival, left ventricular mass, motor and cognitive development, growth and muscle glycogen content. Two patients have been treated for more than one year and two for three months by now (mean age at enrollment 6,6 months, range 2,6-14,5). Results: Treatment is generally well-tolerated. There has been an overall improvement in left ventricular mass, cardiac function, skeletal muscle function and histologic appearance as evidenced by reduction of muscle glycogen. Younger patients and those with less advanced disease have shown the best clinical response. Conclusions: R h G A A appears to be well-tolerated and capable of improving cardiac status and skeletal muscle function in patients with classical infantile Pompe disease. Further long-term safety and efficacy data are required to assess the potenial of this therapy.

Objective: To determine whether brain MRI in PROMM differs from DM. Methods: 10 D M patients from 7 families (8 m/2 f; mean age 41.5; SD 413.7 y) and 7 PROMM patients from 5 families (3 m/4 f; mean age 52.6; SD 4- 11.3 y) were studied (T2-, TI-, FLAIR sequences). Results: Cranial MRI abnormalities were found in 9/10 D M and in all PROMM patients. Focal brain atrophy was seen in one D M patient, whereas diffuse brain atrophy was present in 5/7 PROMM patients (71%). Brain MRI revealed white matter hyperintense lesions (WMHL) without lobe predilection in 9/10 D M patients. 7/9 DM patients (78%) showed patchy lesions, whereas confluent periventricular and subcortical lesions were seen in 2/9 patients (22%). In 7 D M patients anterior bitemporal white matter lesions (ATWML) were found. In PROMM, 6/7 patients (86%) demonstrated predominantly frontoparietal WMHL. Confluent periventricular and/or subcortical lesions were seen in 4 of these patients (67%), whereas only 2/6 patients (33%) demonstrated mild patchy lesions. Conclusion: Brain MRI in PROMM more frequently revealed confluent W M H L with frontoparietal predilection that differed from predominantly patchy lesions in DM. ATWML were not present in PROMM in contrast to 70% of the DM patients. Brain atrophy was a prominent feature in PROMM. P-109 Creatine Monohydrate Does Not Improve Skeletal Muscle E n e r g y Metabolism in Mitochondrial Diseases C. Kornblum* 1, j. Eggers 1, T. Klockgether 1, R. Schr der t, M. Vorgerd 2, A. Papassotiropoulos 3, K. Fabian 4, J. Zange 5 , K. Mueller 5 . 1Bonn,

Germany; 2Bochum, Germany; 3Zurich, Switzerland; 4Dresden, Germany; 5K ln, Germany Objective: To determine whether creatine monohydrate (CM) can ira-

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Poster Presentations

Monday, July 8, 2002

prove skeletal muscle energy metabolism in patients with genetically proven chronic progressive external ophthalmoplegia (CPEO) or KearnsSayre Syndrome (KSS). Methods: 15 patients with single large-scale mtDNA deletions were treated with oral CM according to a double-blind, placebo-controlled crossover design. Each treatment phase with CM (150 mg/kg body weight/d) or placebo lasted 6 weeks with a wash-out period of 4 weeks. The in-vivo effect of CM on skeletal muscle was estimated at the end of each treatment phase by 31P-MRS in 12 patients. Clinical scores, laboratory investigations and tensiometric tests were performed in 15 patients. Results: 31P-MRS and clinical data before treatment with CM gave evidence of a severe impairment of mitochondrial function. There were no significant changes in the 31P-MRS parameters under CM. In particular, the intramuscular phosphocreatine (PCr) content did not increase, and there was no facilitation of postexercise PCr recovery with CM. Clinical scores, laboratory parameters and ergogenic tests did not alter significantly under CM, which was tolerated without major side effects in all patients. Conclusion: This is the first controlled study indicating that CM supplementation does not improve skeletal muscle energy metabolism and oxidative phosphorylation in mitochondrial disorders.

P-110 Inhibition of Skeletal Muscle Fibrosis by Neutralizing TGF-betal Production Y. Li, W. Foster, Y.S.Chan, T. Horaguchi, N. Badlani, J. Huard.

Pittsburgh, USA Skeletal muscle undergoes recurrent cycles of degeneration and regeneration in patients with Duchenne's Muscular Dystrophy (DMD). This is similar to the repair processes of injured skeletal muscle. The injured muscle can promptly initiate regeneration, but the healing process is very slow and often results in incomplete functional recovery due to scar tissue formation. We have observed that TGF-betal is a central factor in the activation of the fibrosis cascade in injured muscle. We hypothesize that by neutralizing TGF-betal expression, the scar tissue formation will be inhibited, which will contribute to the functional recovery of the injured skeletal muscle. TGF-betal transfected myoblasts (C2C12) clone was subsequently transplanted into the skeletal muscle of SCID mice. We then investigated the histological changes associated with the treatment of different anti-fibrotic agents (decorin, r-INF, suramin and IGF-I). Various anti-fibrotic agents inhibited the proliferation of these TGF-betal clone cells, and also decreased TGF-betal-induced fibrotic protein (a-SMA, vimentin, fibronectin) expression in vitro. Delivery of certain anti-fibrotic proteins into the site of injury could help achieve a better muscle recovery. Conclusion: Functional anti-fibrotic therapy, based on neutralizing TGFbetal production, could decrease skeletal muscle fibrosis which was induced by traumatic injury or muscle disease, such as DMD.

P-111 The Application of Patients to Treat Duchenne Muscular Dystrophy by Aminoglycoside Antibiotics Toshihiko Miyagi*, Shigemi Kimura, Isao Fujii, Teruhisa Miike.

Kumamoto, Japan Objective: Duchenne muscular dystrophy(DMD) is caused by mutation in the dystrophin gene. A significant number of these mutations are premature stop codons. Recently, aminoglycoside antibiotics have been found to suppress premature stop codons located in the defective dystrophin gene in mdx mice. This suggest that aminoglycoside treatment may provide to this type of DMD patients. However, if this treatment was established, it is not easy to find applicable patients. Only 5 to 10% of DMD patients have these mutations and much effort are needed because the dystrophin gene is too long. Therefore brief screenig system is needed to hurry. We demonstrate an easy fibroblast curture system to find the patients for this therapy using adenovirus carrying the MyoD gene(AdMyoD).

Method: The fibroblast isolated from mdx mice were infected in vitro with AdMyoD and cukured with gentamicin. Result: At 14 days post infection, the fibroblasts began to form myotubes and dystrophin was detected in these myotubes by immnofluorescence staining. Conclusion: This fibroblast curture system is very easy and clear, moreover, laborios task of analyzing the sequence of dystrophin cDNA would not be necessary. Further investigation of this phenomenon is needed in DMD patients. This system may be useful for identifying patients with DMD caused by premuture stop codons. P-112 Positive Effect of Modal]nil on Daytime Somnolence on Patients with Myotonic Dystrophy K. Talbot*, J. Crosby, J. Stradling, D. Hilton-Jones. Oxford, UK Excessive daytime somnolence is an under recognised feature of patients with Myotonic Dystrophy and an important cause of morbidity. We used the Epworth Sleepiness score to assess degree of somnolence in 69 patients with Myotonic Dystrophy of varying degrees of clinical severity from a single Neuromuscular clinic. 72% of patients scored greater than 10, the standard cut off for excess daytime somnolence. No correlation was observed between the degree of sleepiness with the extent of the genetic mutation. Patients were randomised to receive 200mg of Modafinil per day or placebo in a double blind crossover trial. Each arm of the crossover study lasted for 4 weeks separated by a washout period of two weeks. Patients were assessed by overnight polysomnography, Maintenance of wakefulness test (MWT), a standardised driving test and the SF36 quality of life scale. No patients were found to be suffering from sleep apnoea. Positive effects were found in the MWT and in other measures. The pattern of abnormalities observed will be presented in detail and suggest that the aetiology of daytime somnolence in Myotonic Dystrophy, though it responds to treatment with Modafinil, has a different basis from that in other conditions which respond to the drug. P-113 Intermediate-Term Creatine Monohydrate Therapy Does Not Increase Strength or Body Composition in Patients with Muscular Dystrophy M.A. Tarnopolsky* 1, D. Biggar:, H. Naylor 1, T. Thompson 3, D. Mahoney 1, J. Vajsar 2, T.J. Doherty 3. 1Hamilton, Canada; 2Toronto,

Canada; 3London, Canada Objective: To determine the efficacy and safety of intermediate-term creatine monohydrate (CrM) supplementation in patients with muscular dystrophy. Methods: Ninety-six aduk and pediatric patients with muscular dystrophy completed a double-blind, randomized, cross-over trial with a four month CrM (aduk ~ 0.074 g/kg/d; pediatric ~ 0.102 g/kg/d), a 6 week wash-out, and a four month placebo (PL) phase. Pulmonary function, compound manual muscle strength testing, quantitative isometric strength testing of handgrip, dorsi-flexion and knee extension, handgrip and dorsi-flexion endurance (1 min), functional tasks, activity of daily living scales, body composition (total mass, fat-free mass, and total body water), serum creatine kinase activity, serum creatinine concentration and clearance, and liver function were measured. Several patients (N = 25) also had muscle PCr/ATP ratios determined in the forearm flexor muscles by 31 P-MRS. Results: Four months of CrM treatment did not improve muscle strength, body composition, functional tasks, or activities of daily living in patients with muscular dystrophy. There was no increase in skeletal muscle PCr/ATP ratio after CrM supplementation (CrM = 3.2 (1.5); PL = 3.3 (1.5), NS). Finally, there were no side effects reported or measured. Conclusions: CrM supplementation for four months was well tolerated but did not increase muscle strength or function in patients with muscular dystrophy.

Poster Presentations

Monday, July 8, 2002

P-114 A Double-Blind Randomised, Placebo Controlled Cross-Over Trial of Amantadine for the Treatment of Fatigue after the Guillain-Barre Syndrome P.A. van Doom 1, M.P.J. Garssen 1, ELM. Schmitz 2, I.S.J. Merkies 3.

Department of l Neurology and 2Statistics, Erasmus MC Rotterdam, The Netherlands; 3Department of Neurology, St. Elisabeth Hospitaal, Cura ao, Netherlands Antilles Objective: Severe fatigue is a major complaint in 80% of patients with immune-mediated polyneuropathies (IMP). Since amantadine is effective in treatment of fatigue in multiple sclerosis, a neuro-immunological disease, we started this trial. Methods: 80 GBS-patients. Primary endpoint: improvement of at least 1 point on the fatigue severity scale (FSS), a priorly evaluated scale in IMP. Study duration: 4 months; 2 weeks introduction and baseline measurements, 6 weeks amantadine or placebo and 2 weeks wash-out, followed by the reverse. Besides the FSS, other scales including the fatigue impact scale, SF-36 health survey and (para)clinical parameters were examined. Results: The study has recently been finished: median age 49 years, average 4.4 years after diagnosis, 50% male. 74 patients could be analysed; 46 patients did not improve on either amantadine or placebo, 9 patients improved (and reached primary endpoint) after amantadine, 16 patients after placebo and 3 patients improved on both therapies. Conclusion: This is the first controlled trial of a potential effective drug. Preleminary data show that amantadine is not effective in the whole group (p=0.23). Correlation studies and multivariate analyses to determine the strongest impact on fatigue and quality of life will be performed. The final results will be presented.

Utrophin P-115 Utrophin Expression Patterns and Implications for I)uchenne Muscular Dystrophy J. Ehmsen*, A. Weir, K. Davies. Oxford, UK Mdx mice transgenic for utrophin show marked improvement of the dystrophic phenotype and restoration of the sarcolemmal dystrophinassociated protein complex, endorsing utrophin as a realistic surrogate for dystrophin in patients with Duchenne muscular dystrophy. There are currently two known isoforms of utrophin, designated A and B, differing only in their first coding exon and splicing into a common 13 kb mRNA at exon 3. These transcripts are widely expressed from independently regulated promoters, offering two potential targets for pharmacological upregulation of utrophin in DMD. Both isoforms appear equally abundant in brain and skeletal muscle, while utrophin A is prevalent in the kidney and utrophin B in heart. It is possible, however, that examination of wholetissue expression may disguise more restrictive expression patterns at the level of specific cell types, as implicit in the more than 10-fold variation of promoter activity in various cell lines. Indeed, antibodies raised against the unique N-termini of both utrophin isoforms have recently revealed a more complex expression pattern than originally anticipated. We are therefore exploring the possibility of a unique functional role imparted to utrophin B by its unique N-terminus, using yeast two-hybrid analyses to search for alternative binding partners. Knock-in mice expressing separate reporter genes from the endogenous A and B promoters will also serve to refine our understanding of cell-specific utrophin expression patterns.

P-116 NO and cGMP Preferentially Upregulate Utrophin a Transcripts in M D X Mouse Muscle Melissa A. Jones*, Mark A. Stocksley, Bernard J. Jasmin. Ottawa, ON

Canada One potential therapy for Duchenne muscular dystrophy is to replace

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the missing dystrophin with its autosomal homologue, utrophin. Previous studies suggested that nitric oxide (NO) regulates utrophin expression in skeletal muscle (Chaubourt et al., 1999). To investigate this possibility further and to define the molecular mechanisms involved, C2C12 myotubes were treated with 0.2mM L-arginine, 0.1mM SNAP and 101~M cGMP. Levels of utrophin A and B transcripts were assessed by semi-quantitative RT-PCR. Utrophin A transcripts increased significantly following all treatments. However, utrophin B transcripts were not detected in cukure. Interestingly, a 5-7 fold increase in utrophin protein was observed. In addition, aduk mdx mice were treated with 0.5% dietary L-arginine or 200mg/kg L-arginine IP injection daily for two weeks. Both groups displayed significant increases in the level of utrophin A transcripts. Immunofluorescence experiments demonstrated a robust increase in sarcolemmal utrophin. The effect appeared to be fiber type-specific, as not all fibers showed a similar increase. Furthermore, mdx mice were treated with a cGMP-specific phosphodiesterase inhibitor. These mice demonstrated even greater increases in the level of utrophin A transcripts and sarcolemmal utrophin. Thus, this study demonstrates NO and cGMP regulation of utrophin A transcripts in skeletal muscle in culture and in vivo. Supported by MDA (USA) and CIHR. P-117 Increase of Utrophin in Muscle Treated by L-Arginine: First Steps of the Way of Signalisation V. Voisin, P. Fossier, G. Baux, M. Isra 1, S. de la Porte. Gifsur Yvette,

France Objective: Duchenne Muscular Dystrophy (DMD) is a severe genetic disorder, due to a lack of dystrophin, a cytoskeletal protein. A potential way for treatment is to replace the lack of dystrophin by over-expression of utrophin, its homologous foetal form. In previous studies, we demonstrated that L-arginine (the substrate of the neuronal nitric oxide synthase (nNOS), associated with al-syntrophin in muscle fibres) induces the appearance of utrophin at the sarcolemma [1]. Methods and results: Here we demonstrate, after L-arginine treatment of mice, differential level of utrophin according to the studied muscle. The increase of utrophin is not limited to the muscle tissue but is also seen in other tissues such as brain and heart. First steps of transduction pathways to the expression of utrophin, induce cGMP synthesis and PKG activity. The over expression of utrophin is demonstrated by an increase in mRNA level. Conclusions: These results demonstrate that better manipulation of Larginine pathways could be useful in treating DMD through utrophin reexpression. References [1] Chaubourt E et a1.(1999). Neurobiol. Dis. 6, 499-507; (2000) C. R. Acad. Sci., Paris, Life Science 323 735-740; (2002) J. Physiol. (Paris) 96, 43-52.

Ventillatory Studies P-118 Quality of Life (QoL) in Families with a Ventilated Young Person who has Duchenne Muscular Dystrophy M. Eagle*, J. Swain, Kate Bushby. Newcastle, England

Objective: A pilot study was conducted to assess quality of life in families of ventilated patients with DMD. Methods: Semi-structured interviews and the SEIQoL assessment technique were used. SEIQoL is a standardised procedure that asks participants to choose five domains of life most important to them. Each area is rated for satisfaction/function using a visual analogue scale. The SEIQoL disc is used to give relative weight to each domain. Results: Family relationships were most important in determining satisfaction with QoL. Health was less important to QoL than family relation-