Abstracts that will be presented on 58th Annual Meeting of American Academy of Neurology - San Diego , April,1-8, 2006

1) A CINRG Pilot Trial of Oxatomide in Steroid-Naive Duchenne Muscular Dystrophy

Gunnar M. Buyse, Diana Escolar, Nathalie Goemans, Erik K. Henricson, Marleen van den Hauwe, Alejandro Jara Vallejos, Leuven, Belgium, Cheng Shao, Kantilal M. Patel, Robert McCarter, Robert Leshner, Washington, DC, Julaine Florence, Saint Louis, MO, Jill Mayhew, Richmond, VA

OBJECTIVE: With CINRG (Cooperative International Neuromuscular Research Group), we have tested the hypothesis that oxatomide specifically targets the pathophysiological cascade of DMD by blocking the mast cell mediator cascade and inhibiting dendritic cell's release of cytokines, and hence may improve muscle strength in DMD patients. BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common and devastating type of muscular dystrophy. Mast cells have been implicated in the pathophysiology of DMD and its animal models. The mast cell stabilizer oxatomide has shown promise in a pre-clinical screening platform that uses the homologous mdx.jpg mouse model of DMD. DESIGN/METHODS: We tested the efficacy and safety of oxatomide in 14 ambulant steroid-naive DMD boys aged 5 - 10 years, in a pilot open-label two-center clinical trial with a 3 months medication-free lead-in period followed by a 6 months treatment period with oral oxatomide (30 mg/day). Drug efficacy was tested by measuring muscle strength quantitatively (QMT, 10 muscle groups) and manually (MMT, 34 muscle groups), and by timed functional tests. RESULTS: Repetitive measure analysis was applied using linear mixed-effects models, comparing intercepts and slopes of lead-in and treatment periods. Within the limitations of a smaller non-controlled trial, we conclude that 6 months oxatomide treatment does not improve muscle strength or function in a group of 5 to 10 year old DMD patients. There were no serious adverse events and overall oxatomide was well tolerated. Supported by: Janssen-Cilag.

2)  Adenovirus-Mediated Utrophin Gene Delivery as a Therapy for Duchenne Muscular Dystrophy

Jatinderpal R. Deol, Renald Gilbert, Mylene Bourget, Gawiyou Danialou, Joon-Shik Moon, Newark, NJ, Basil J. Petrof, Josephine Nalbantoglu, George Karpati, Montreal, QC, Canada

OBJECTIVE: To construct a fully deleted (gutted) adenovirus (AdV) capable of high expression levels of full-length isogenic utrophin (utr) in dystrophin (dys)-deficient muscle fibers. BACKGROUND: The introduction of gutted AdV vectors encoding full-length dys into muscles leads to significant mitigation of the dystrophic phenotype. However, dys is a neoantigen, causing deleterious immune responses. An alternative approach is to use utr, a functional homologue of dys, normally present only at the neuromuscular junction. DESIGN/METHODS: We have created a gutted AdV encoding full-length murine utr cDNA. The purified recombinant adenovirus was injected into tibialis anterior (TA) muscles of neonate and adult mdx.jpg mice for evaluation at 10, 30, 90, 180, and 360 days post-injection (pi). End points include utr expression, the restoration of the dys-associated protein complex, and the reversal of the physiological dystrophic phenotype. RESULTS: In neonates at 10 days pi, the mean number of extrasynaptic utr-positive fibers in vector vs control-injected TA was 1596 vs 114, corresponding to ~58 % transduction level. The levels in adults were 685 and 112 for vector and control injected TAs respectively, corresponding to ~23% transduction level. There was ~14 and 6 fold increase in utr expression over control levels in neonates and adults. In adults at 30 days pi, the mean number of utr positive fibers was 770 corresponding to ~35% transduction level. However, that number dropped substantially to an average of 80 at 60 days pi. At the same timepoint in neonates, the mean number was 794. CONCLUSIONS/RELEVANCE: There was a time-related decline of the initial high utr transduction of the TA muscles. We believe that this is mainly due to the fact that even the initial extrasynaptic utr level is not sufficiently high to prevent necrosis and loss of some of the transduced fibers. Further augmentation of the initial extrasynaptic utr should be helpful. Supported by: CIHR and l'AFM.

3) CINRG Open Label Pilot Clinical Trial of Coenzyme Q10 in Steroid Treated Duchenne Muscular Dystrophy

Diana Escolar, Erik Henricson, Carolina Tesi-Rocha, Robert McCarter, Washigton, DC, Heather Gordish, Washington, DC

OBJECTIVE: To evaluate the effect of Coenzyme Q10 (CoQ10) on muscle strength in steroid treated DMD. BACKGROUND: CoQ10 treatment in the mdx.jpg mouse increases strength similar to corticosteroids. This pilot study assessed whether CoQ10 improved strength in steroid-treated DMD to determine whether a randomized controlled study is warranted. DESIGN/METHODS: We conducted a 9-month, prospective open label study in ambulant DMD children. Each participant served as his own control. Enrollees included 13 ambulant steroid-treated DMD boys, aged 5-11 years, naive to CoQ10. Participants were followed for 3 months before beginning 6 months of CoQ10 therapy and maintained a therapeutic level of 2.5 ug/ml or higher. Participants underwent monthly strength evaluations using quantitative muscle testing (QMT) of elbow flexors and extensors, knee flexors and extensors and grip. Manual muscle strength testing (MMT), timed functional tests functional evaluation (FE) and left ventricular ejection fraction (LVEF) were also studied. CoQ10 levels and safety labs were monitored monthly. RESULTS: Eleven of 13 participants completed the trial (withdrawn = 1 noncompliance, 1 failure to reach therapeutic levels). Comparison of slopes between non-treatment and treatment periods showed improvement in right (p=0.011) and left (p=0..013) elbow flexion and elbow flexor/extensor average scores (p= 0.038). Similar non-significant trends were seen in grip score and total QMT score. There was no significant change by MMT, TFT and LVEF. One participant developed headaches attributed to high blood levels of CoQ10. CONCLUSIONS/RELEVANCE: CoQ10 increased strength in elbow flexors and extensors after 6 months of treatment. This significant effect was not reflected in other muscle groups, but increasing strength trends were observed in all muscle groups except knee flexor/extensors. CoQ10 failed to produce an improvement in LVEF. Our data shows association between CoQ10 and increased strength sufficient to recommend the design of randomized controlled trial. Supported by: CNMC GCRC NCRR 1MO1RR020359-01; Serving Up a Cure Committee; K-23 RR16281-01 from NCRR.

4) Biological Effects of a Potent, Muscle-Targeted, Orally Bioavailable Calpain Inhibitor (C101) for Duchenne Muscular Dystrophy (DMD)

Norman W. Barton, Phoenix, MD, Alfred Stracher, Marie A. Badalamente, Stony Brook, NY, Theodore Carver, Theresa Michele, Leslie DeVos, Hunt Valley, MD, Leo Kesner, Brooklyn, NY, Amy Deng, Kevin Wang, Monica Oli, Alachua, FL

OBJECTIVE: To investigate the biological effects of C101 in muscle cell cultures in vitro and dystrophin deficient mdx.jpg mice in vivo. BACKGROUND: Calpain is a ubiquitous calcium-activated neutral protease. Dystrophin deficiency causes increased sarcolemmal permeability, calcium influx into muscle, calpain activation and initiation of a proteolytic cascade culminating in myonecrosis. C101 is a leupeptin analogue to which L-aminocarnitine is covalently bound. It is specifically designed to target muscle via the high affinity OCTN2 carnitine transporter. DESIGN/METHODS: The potency of C101 compared with leupeptin was assessed in human rhabdomyosarcoma cells that expressed high levels of OCTN2. Calpain activation was induced by addition of a calcium channel opener (maitotoxin) to cells grown to 80% confluence; 145- and 150-kD calpain-specific breakdown products of II-spectrin (SBDPs) were monitored over time by quantitative Western Blot analysis. The concentrations of C101 and leupeptin required to produce 50% reductions (IC₅₀) in the levels of SBDPs, which serve as unique biomarkers of calpain activity, were then determined under standard assay conditions. The mdx.jpg murine model (Jackson Labs, Bar Harbor, ME) was chosen to assess the inhibitory effects of C101 on myofiber degeneration associated with dystrophin deficiency. Thirty-day old mdx.jpg mice received daily intraperitoneal injections of C101 for 60 days at doses of 5 and 10 mg/kg; controls received physiologic saline. Selected muscles were harvested after 60 days for histological analysis and measurement of mean myofiber diameter. RESULTS: The IC₅₀ for C101 for prevention of 145-kD SBDP formation was 54-fold less than the value obtained for leupeptin (70 vs. 3750 M); a 130-fold reduction was observed for the 150-kD SBDP (400 vs. 51900 M). C101 preserved muscle morphology and significantly increased myofiber diameter in mdx.jpg mice compared with disease controls. CONCLUSIONS/RELEVANCE: C101 is a potent, oral, muscle-targeted calpain inhibitor that may be effective in treating DMD patients. Supported by: Ceptor Corporation, 200 International Circle, Suite 5100, Hunt Valley, MD 21030-1350.

5) Haploinsufficiency of Utrophin Gene Worsens Diaphragm and Quadriceps Muscle Dystrophy in mdx.jpg Mice

Lan Zhou, Jill A. Rafael-Fortney, Columbus, OH, Georgiana Cheng, Yuefang Zhou, Xiaohua Zhou, Bendi Gong, John D. Porter, Bethesda, MD, Henry J. Kaminski, Cleveland, OH

OBJECTIVE: To compare dystrophic changes of quadriceps and diaphragm muscles in mdx.jpg and mdx.jpg mice with utrophin haploinsufficiency (mdx.jpg/utr+/-).. BACKGROUND: Dystrophin deficient mice (mdx.jpg) display a mild muscular dystrophy (MD) phenotype as compared to Duchenne muscular dystrophy (DMD) patients. Mice lacking both dystrophin and utrophin (dko) suffer premature death from MD, mimicking human DMD. The muscle pathology of diaphragm, which manifests earlier in dko mice, is indistinguishable in mdx.jpg and dko mice around 10 weeks of age when dko mice die. It is thus thought that utrophin may fail to compensate for the effects of dystrophin loss on fibrosis in diaphragm. DESIGN/METHODS: Three pairs of mdx.jpg and mdx.jpg/utr+/- male littermates were sacrificed at 6 months of age. Muscle degeneration, inflammation, and fibrosis were evaluated by H&E staining and collagen III immunostaining. Muscle fibrosis was also quantified by measuring hydroxyproline content. RESULTS: At age 6 months, mdx.jpg quadriceps showed many small regenerated muscle fibers with increased central nuclei, and a few scattered endomysial inflammatory cells. There was no myofiber degeneration or endomysial fibrosis. In contrast, the quadriceps of mdx.jpg/utr+/- mice showed marked fiber size variation, multiple medium-sized foci of endomysial inflammatory cells, and notable increased endomysial collagen deposition. The diaphragm of both mdx.jpg and mdx.jpg/utr+/- mice showed prominent endomysial inflammation. The endomysial fibrosis, however, is much worse in mdx.jpg/utr+/- diaphragm as compared to mdx.jpg diaphragm. Consistent with these morphological findings, mean hydroxyproline content (m g/mg muscle tissue) was significantly increased in mdx.jpg/utr+/- quadriceps (1.16) and diaphragm (3.27) as compared to mdx.jpg quadriceps (0.53, p=0.02) and diaphragm (1.90, p=0.03), respectively. CONCLUSIONS/RELEVANCE: Our findings suggest that utrophin does compensate, although insufficiently for the effects of dystrophin loss in mdx.jpg diaphragm and quadriceps. With more severe muscle pathology, mdx.jpg/utr+/- mice may provide a better animal model than mdx.jpg mice for testing potential therapies for muscle fibrosis. Supported by: R24 EY 14837 and R01 EY-015306.

6)  Recombinant AAV and Corticosteroid Therapy in mdx.jpg Mice: In Consideration for Clinical Trials

Christopher J. Shilling, Xiao Xiao, Pittsburgh, PA, R. Jude Samulski, Jade Samulski, Angelique S. Camp, Chapel Hill, NC, Jerry R. Mendell, Columbus, OH

OBJECTIVE: To evaluate the efficacy and safety of combining recombinant adeno-associated virus (rAAV) gene transfer and corticosteroids, in an acute and chronic paradigm, to simulate potential gene therapy for Duchenne muscular dystrophy (DMD). BACKGROUND: Corticosteroids represent the only known treatment to clinically improve boys with DMD. It is estimated that approximately 50% of the DMD population in the USA is treated with corticosteroids, mainly prednisone. Many consider it to be the standard of care since it is the only known clinically effective form of treatment. Gene therapy is on the horizon as a potential treatment option for some patients with DMD. It is important to establish potential interactions between viral gene transfer and corticosteroid use to satisfy issues raised by patients, clinicians, scientists and regulatory agencies. To address this issue we have combined rAAV-mini-dystrophin gene transfer in the mdx.jpg mouse with two regimens of corticosteroid use. DESIGN/METHODS: Six-week-old mdx.jpg mice on chronic (1mg/kg/day, up to 52 days) and acute (2mg/kg given 6-hours pre-rAAV-injection and 24- & 48-hours post-rAAV-injection) regimens of prednisilone (pred) and non-pred treated controls were injected unilaterally into tibialis anterior (TA) muscles with 1x10¹¹ vg/kg of rAAV-mini-dystrophin. Multiple time-points of evaluation extended over 90 days including transgene expression and inflammatory cell infiltration. RESULTS: Pred treatment did not preclude transgene expression compared to non-pred treated mdx.jpg mice. This is true for both acute pred administration, as well as chronic pred administration (more closely simulating the patient on long-term corticosteroids). Of particular importance, there was no evidence of toxicity or cytotoxicity from gene delivery in concurrence with corticoid steroid use as compared to controls. CONCLUSIONS/RELEVANCE: This data impacts future DMD gene therapy protocols and sets the stage for clinical trials including prednisone-treated patients. This will be of importance in regulatory and IRB approvals for gene therapy studies for this disease. Supported by: Muscular Dystrophy Association

7) Absence of T-Cells and B-Cells Does Not Improve Functional Outcome in mdx.jpg Mice

Paul T. Golumbek, Richard M. Keeling, Anne M. Connolly, Saint Louis, MO

OBJECTIVE: Does the absence of T-cells and B-cells (Ig) secondary to a genetic rag-/- mutation improve strength in mdx.jpg mice. BACKGROUND: mdx.jpg mice lack dystrophin protein and serve as our best animal model of Duchenne muscular dystrophy (DMD). Prednisone produces improvement in functional outcome/strength in both boys with DMD as well as the mdx.jpg mouse model. If prednisone acts by suppressing the immune function of T-cells and/or B-cells (Ig) or if they play an important negative role in the eitiology of DMD/mdx.jpg weakness then reducing their actions should improve functional outcome measures. DESIGN/METHODS: Mice harboring a recombinase-activating gene mutation (rag-) are unable to produce T-cells and B-cells (Ig) throughout their lifetime. Groups of twenty male mdx.jpg-rag- and mdx.jpg-Rag+ mutant mice were produced and longitudinal studies (36 weeks) of strength were performed. Two independent measures of strength, grip strength testing and hanging wire testing, were observed. Consistent with previous literature, two nadirs of strength (demonstrated by both measures) were observed. RESULTS: No improvement, by either strength measure, is demonstrated by the addition of the rag- mutation. CONCLUSIONS/RELEVANCE: This implies that T-cells and/or B-cells (Ig) while demonstrable in mdx.jpg-Rag+ mouse muscle by histology are playing a negligible role in functional outcome. Also, prednisone treatment acts via non T-cell/B-cell dependent mechanism(s) in mdx.jpg mice. Supported by: Muscular Dystrophy Association.