Post by David on Mar 9, 2008 15:28:57 GMT -5
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
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. Supported by: Muscular Dystrophy Association, NIH (NCRR).
Genotype Analysis Predicts Dilated Cardiomyopathy in Becker Muscular Dystrophy
OBJECTIVE: To identify dystrophin mutations predictive of dilated cardiomyopathy in patients with Becker muscular dystrophy (BMD) or X-linked dilated cardiomyopathy (XLDCM). BACKGROUND: BMD is caused by dystrophin gene mutations resulting in skeletal and cardiac muscle degeneration. When cardiac manifestations are out of proportion to skeletal muscle symptoms, the disorder is diagnosed as XLDCM. Dystrophin mutations predictive of XLDCM have been suggested, but representative examples from the literature are insufficient in number to establish with certainty. Defining the clinical presentations of specific mutations is crucial for risk classification, early detection and treatment to prolong survival. DESIGN/METHODS: BMD and XLDCM patients with dystrophin deletion/missense mutations and with cardiac histories were collected from databases at Nationwide Childrens Hospital, The Ohio State University, the Utah Dystrophinopathy Project, the Leiden Open Variation Database, and published case reports. Phenotype-genotype correlations were established based on age, skeletal muscle and cardiac manifestations, and echocardiograms. Statistical analysis was performed using Mann-Whitney U tests. RESULTS: Data from 126 patients were analyzed. Deletions in the N-terminus domain of dystrophin resulted in the earliest mean age of cardiac involvement (23 years) compared to mutations affecting the rod domain (41 years) or the hinge 3 region (39 years). Mutations affecting the spectrin repeats in the rod domain which preserve hinge 3 lead to an earlier onset (31 years) of cardiomyopathy than mutations which remove hinge 3 (47 years) (p<0.05). Mutations affecting the spectrin repeats preserving hinge 3 were subdivided based on whether they disrupted (out-of-phase) or preserved (in-phase) the spectrin repeats structure. Out-of-phase mutations predisposed to early onset (25 years) cardiomyopathy compared to in-phase mutations (37 years) (p<0.05). CONCLUSIONS/RELEVANCE: This genotype-phenotype analysis included the largest number of BMD patients to date. The results define dystrophin mutations predisposing to cardiomyopathy and enable clinicians to provide early intervention for at-risk patients that could improve the disease natural history.
Immediate Release Oral Pentoxifylline Is Poorly Tolerated in Duchenne Muscular Dystrophy Boys
OBJECTIVE: The objective of this open label pilot study of oral, immediate release pentoxifylline was to assess the tolerability, safety and efficacy on muscle strength and function in Duchenne Muscular Dystrophy (DMD). BACKGROUND: Severe to complete dystrophin deficiency produces the DMD phenotype. Untreated DMD boys demonstrate progressive, eventually fatal, muscle weakness. In relevant animal models, pentoxifylline slows disease progression. DESIGN/METHODS: This was an open label, prospective study with a 3 month lead in period followed by 12 months of treatment with a 20mg/kg/day dose of pentoxifylline (maximal dose 598 mg/day). Study subjects were 4-9 year old steroid-naive DMD boys. The primary efficacy measure was a total quantitative muscle testing (QMT) score. Secondary and exploratory endpoint measures were manual muscle strength (MMT), arm and leg QMT, timed function tests, muscle MRI, serum TNF- and TGF- levels and adverse event profiles. RESULTS: Of twenty patients screened, 17 enrolled and 9 completed the protocol. Age range was 4.3-8.5 years (mean 6.0, median 5.8). QMT and MMT measures did not show a significant change during the 3 month lead-in or 12 month treatment period. Five of 8 patient withdrawals were due to intolerable adverse events. Eleven of 17 patients experienced nausea and vomiting. Two patients experienced moderate (grade 3) to severe (grade 4) leucopenia. CONCLUSIONS/RELEVANCE: The immediate release oral formulation of pentoxifylline is not well tolerated in children with DMD. The 12% incidence of leucopenia in this study was higher than the reported incidence rate in adults (<1%). Leucopenia improved post drug withdrawal. The 65% incidence of vomiting was also higher than reported in adults (4.5%). The lack of deterioration in strength and function over 12 months in steroid naive DMD children (mean age 6 years) suggests a possible beneficial effect on disease progression and warrants further study with a different formulation of pentoxifylline.
Double-Blind Randomized Controlled Trial of SNT-MC17/Idebenone in Duchenne Muscular Dystrophy
OBJECTIVE: This 12-month study has evaluated the efficacy and tolerability of treatment with SNT-MC17/idebenone (450 mg/day) compared to placebo in children with Duchenne muscular dystrophy (DMD). BACKGROUND: Idebenone supports mitochondrial respiratory chain function and reduces oxidative stress, pathways that are involved in DMD pathogenesis. Previous long-term blinded controlled preclinical studies in the homologous mdx mouse have indicated that idebenone is cardioprotective and improves exercise performance in murine dystrophin-deficiency. DESIGN/METHODS: 21 DMD patients (8-16 y) with cardiac dysfunction were enrolled in the double-blind randomized placebo-controlled trial. Comedication with glucocorticoids was allowed at stable dosage; use of ACE-inhibitors was excluded. Thirteen patients received SNT-MC17 as a daily dose of 450 mg for 52 weeks, 8 patients were randomized to the placebo group. RESULTS: All subjects completed the study; the frequency and type of adverse events was comparable in the active and placebo treatment groups indicating good safety and tolerability of SNT-MC17. The primary endpoint was the change in peak systolic radial strain of the left ventricular inferolateral wall, the region of the heart which in DMD is affected early and most severely. For the observed cases, patients on SNT-MC17 improved by 17.313.2% from baseline (22.99.1%) to week 52, while patients on placebo improved by 7.512.0% from baseline (33.79.7%; p= 0.066 comparison SNT-MC17 vs placebo). Secondary outcome measures included respiratory function tests. Whereas SNT-MC17 treatment was associated with improvement in peak flow (30.854.4 L/min) and peak flow % predicted (2.813.8%) during the 52 week study period, patients on placebo deteriorated in peak flow (- 13.851 L/min; p= 0.039) and peak flow % predicted (- 8.513.8%; p= 0.042). CONCLUSIONS/RELEVANCE: This the first indication of clinical efficacy with SNT-MC17/idebenone on functional cardiac and respiratory parameters in DMD. The results provide the basis for the planning of further clinical development studies with SNT-MC17/idebenone in DMD.
Implementation of a Newborn Screening Program for Duchenne Muscular Dystrophy (DMD)
OBJECTIVE: Introduce a comprehensive newborn screening paradigm for Duchenne muscular dystrophy (DMD). BACKGROUND: The incidence of DMD is presumed to be 1:3500 newborn males, however, this has not been confirmed in an outbred population. Only small cohorts of newborns have been screened based on creatine kinase (CK) testing. The most useful, exacting and baby friendly paradigm includes testing both CK and DNA on the dried blood spots (DBSs) taken within the first 24 to 48 hours of life. DESIGN/METHODS: DBSs obtained within the newborn period were used to test for CK combined with a rapid direct method of sequence analysis of the dystrophin gene referred to as SCAIP sequencing. RESULTS: The first goal of this study was to establish a range of CK from 40,000 anonymous newborn male DBSs (250 + 111). Mutational analysis was tested on DBSs taken from 8 boys with known DMD mutations. Blood was obtained from the finger simulating heel stick samples obtained in the newborn period. Samples were analyzed blindly using modifications of SCAIP and matched successfully with known mutations. Once having established a CK testing range based on >3SD above the mean and a DNA confirmational method on DBSs, the second goal was achieved with implementing a newborn screening program in 6 birthing hospitals in central Ohio and Cincinnati areas. To date we have enrolled over 6000 newborn males. CONCLUSIONS/RELEVANCE: A successful paradigm for screening newborns for DMD has been established that will provide a definitive diagnosis of DMD in the newborn period. Survey of over 500 participants reveals a high degree of enthusiasm (>99%) for participation in this program. The long-term benefits include: 1) prevention of an unsuspecting second child with DMD; 2) establishing a registry for early intervention for clinical trials; and 3) determining the incidence of DMD in newborn males in an outbred population.
A Gene Therapy Approach To Treat Cardiomyopathy in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)
OBJECTIVE: At least 95% of DMD patients develop a cardiomyopathy, critical to their long-term prognosis. Most evolving strategies for replacing the missing dystrophin protein in skeletal muscle are not applicable to the heart. Our goal is to use adeno-associated virus (AAV) to replace the mutant gene. BACKGROUND: Dystrophinopathy victims historically died from respiratory complications while cardiac failure appeared catastrophically related to the stress of respiratory infections. Contemporary methods now protect the respiratory system related to improved equipment, antibiotics and vaccines; consequently, the progressive cardiomyopathy of DMD mandates attention. We have previously shown AAV serotype 8 to efficiently transduce skeletal muscle when delivered through the vasculature, and therefore chose to test its efficiency in transducing cardiac muscle of mdx mice, a model with similar lesions to DMD patients. DESIGN/METHODS: Using AAV8 carrying green fluorescent protein (GFP) as a marker gene we treated mdx mice at the neonatal stage (P2) [2 x 1011 vector genomes (vg)] by intraperitoneal injection, or at 8 weeks-old by intravenous tail vein injections (8 x 1011 vg). Hearts were harvested and stained for GFP four weeks post delivery. In addition, we developed a testing paradigm to serially measure in vivo cardiovascular function in mdx using echocardiography and electrocardiography (ECG). RESULTS: Gene delivery to mdx mice resulted in 73% gene expression in mdx mice treated with AAV8.GFP treated as adults and 65% expression in neonates. In addition, using our previously described methods, we observed a 52% reduction in left ventricular fractional shortening % (FS%) in the mdx mice as compared to the wild-type controls (52.05-2.77 vs. 27.513.06; Ctrl vs. mdx; p<0.05). CONCLUSIONS/RELEVANCE: Together these data support treatment of the cardiomyopathy of neonatal or adult mdx mice using rAAV8 to deliver a therapeutic dystrophin transgene while concurrently monitoring cardiac performance using serial, non-invasive outcome measures similar to those used in the clinic.
Pentoxifylline Treatment Fails To Rescue Muscle Strength and Function Deterioration in Prednisone-Treated Duchenne Muscular Dystrophy
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. DESIGN/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- and TNF-. 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. CONCLUSIONS/RELEVANCE: 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.
Imatinib Attenuated Skeletal Muscle Dystrophy in mdx Mice
OBJECTIVE: To test anti-inflammatory and anti-fibrotic effects of imatinib on mdx mice, a mouse model of Duchenne muscular dystrophy (DMD). BACKGROUND: DMD is the most common genetic muscle disease. DMD is lethal and currently untreatable. Muscle necrosis, inflammation, and fibrosis are prominent pathological features, and pharmacotherapy to ameliorate these pathological changes may represent an important therapeutic approach for DMD. Imatinib is an anti-neoplastic therapy that selectively inhibits the tyrosine kinase activity of c-abl, c-kit, and PDGF receptors (PDGFRs). It is also a promising anti-inflammatory and anti-fibrotic therapy as demonstrated by its substantial inhibition of inflammation and fibrosis of liver, kidney, lung, or skin in various animal models of disease. DESIGN/METHODS: We treated mdx mice with intraperitoneal injection of imatinib at the peak of limb muscle inflammation and at the onset of diaphragm fibrosis; controls received PBS vehicle or were left untreated. Muscle necrosis and inflammation were quantified by measuring serum CK activity and percentage of CD45 immunoreactive area, respectively. Muscle fibrosis was evaluated by collagen III immunostaining. Muscle function was assessed by measuring hindlimb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was studied by Western blot. RESULTS: Compared with control mdx mice, imatinib-treated mdx mice showed striking pathological and functional benefit. Serum CK level, diaphragm and quadriceps inflammation areas, and diaphragm fibrosis were markedly reduced. Hindlimb grip strength was significantly improved. Reduced clinical disease was accompanied by suppression of TNF-alpha and IL-1beta mRNA expression, and inhibition of c-abl and PDGFR phosphorylation. CONCLUSIONS/RELEVANCE: 1) Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis. 2) Imatinib likely exerts its therapeutic effects on mdx mice by inhibiting c-abl and PDGFR signaling and downstream inflammatory cytokine and fibrotic gene expression.
How Frequently Are Corticosteroids Used in Boys with Dystrophinopathy in the USA: Data from the MDSTARnet
OBJECTIVE: To review the use of corticosteroids for the treatment of Duchenne Muscular Dystrophy (DMD) in clinical practice from 1991 to 2005 in a large population- based cohort in the USA. BACKGROUND: Corticosteroids are the only pharmacological treatment that has been shown to have positive effect on muscle strength and function in DMD in three randomized controlled trials and a 3 year follow up open study. These studies were published in 1991. DESIGN/METHODS: The Muscular Dystrophy SurveillanceTracking and Research Network (MD STARnet) is a population-based surveillance system funded by the Centers for Disease Control to identify all cases of Duchenne /Becker Muscular Dystrophy (DBMD) born since 1982 and/or residing within Arizona, Colorado, Iowa and Western New York State. A pooled database containing clinical information abstracted from medical records on 433 individuals was analyzed. RESULTS: The proportion of boys receiving corticosteroids increased from 27% in 1991 to 44% in 2005. The proportion of boys receiving corticosteroids in 2005 by state is: NY: 57 % (clinic A= 75%; clinic B= 5 %), CO: 29%, AZ: 38%, IA: 50 %. Mean age when corticosteroids are started is 7.4 years (median= 7; range 3-18). Mean duration of treatment is 57 months. Most common reasons for discontinuing corticosteroids are: weight gain, behavioral side effects and becoming wheelchair bound. Most common reasons for changing corticosteroids dose are: weight gain, behavioral side effects and parents request. CONCLUSIONS/RELEVANCE: The proportion of DMD boys taking corticosteroids has increased in the past 15 years but is overall still less than 50%. Large variability in practice remains between States and between Neuromuscular Clinics. We plan a repeat analysis in 2 years to assess the impact of the AAN Practice Parameter on the use of corticosteroids in DMD published in 2005.
Long-Term Enhancement of Skeletal Muscle Mass and Strength by Single Gene Administration of Myostatin Inhibitiors
OBJECTIVE: To determine the safety and efficacy of adeno-associated virus expressing follistatin (FS-344), for muscle enhancement in a mouse model for Duchenne Muscular Dystrophy and wild-type animals. BACKGROUND: Increasing the size and strength of muscles represents a promising therapeutic strategy for musculoskeletal disorders. Significant interest has focused on myostatin, a negative regulatory factor of muscle growth. Inhibition of myostatin significantly increases muscle mass. Several proteins including follistatin (FS), follistatin-related gene (FLRG) and growth and differentiation factor-associated serum protein 1 (GASP-1) inhibit myostatin. DESIGN/METHODS: 1x1011 particles of AAV1 vectors expressing Follistatin, FLRG, or GASP-1 were injected bilaterally into hindlimbs of mdx or wild-type mice and followed for 6-months or 2 years. RESULTS: Wild-type mice injected in the hindlimbs with of AAV1-FS, FLRG, or GASP-1 showed increased overall body mass, with 30% increase in hindlimb and forelimb grip strength compared to AAV1-GFP controls. Overall strength increase was greatest for animals receiving AAV1-FS followed by AAV1-FLRG and then AAV1-GASP-1. We next tested the potential of AAV1-FS to increase muscle mass and strength and delay muscle deterioration in the mdx mouse model. Mdx animals were injected at 3 weeks of age and followed for 6 months with AAV1-FS. Animals showed a 30-50% increase in skeletal muscle mass, enhanced muscle grip strength, and decreased serum creatine kinase levels compared to controls. Histology demonstrated myofiber hypertrophy locally and at remote sites to the injection. No adverse organ pathology was detected. Mdx animals treated with AAV1-Follistatin at 6.5 months of age also showed increased muscle strength, demonstrating the ability to improve strength in aged animals. CONCLUSIONS/RELEVANCE: Inhibition of myostatin by myostatin inhibitory proteins, in particular follistatin represents a promising therapeutic strategy warranting consideration for clinical trials in human muscle diseases.
Fidelity of Gamma-Glutamyl Transferase in Differentiating Skeletal Muscle from Liver Damage
OBJECTIVE: To validate that gamma-glutamyl transferase (GGT) is a flawless biomarker distinguishing liver and skeletal muscle damage. BACKGROUND: Serum creatine kinase (CK) is recognized to be a valuable biomarker for distinguishing myopathic and neurogenic disorders. Accompanying the leak of CK from skeletal muscle are other intracellular enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), pyruvate kinase, and lactic dehydrogenase. The initial interpretation of an elevated AST and ALT is a derivation from liver, frequently leading to liver biopsy. Many clinicians ignore the fact that muscle breakdown is an established cause for hypertransaminasemia. GGT is a membrane-bound enzyme produced in the liver, with little or none from skeletal muscle. We tested the validity of GGT as a marker to differentiate skeletal muscle from liver damage in an environment of unrestrained activity. DESIGN/METHODS: Duchenne muscular dystrophy subjects (n=28) with proven dystrophin mutations were enrolled (equally divided between ambulatory and non-ambulatory, and approximately one-half taking corticosteroids). Normal males (n=20) served as controls. Initial GGT and CK samples were taken between 8 and 9 am and redrawn 8 hours later. Subjects resumed normal play or left the clinic between blood draws. RESULTS: Not a single DMD patient, ambulatory or non-ambulatory, taking corticosteroids or not, showed a GGT outside the range of normal at any time point. CK levels at the same time points ranged from 14 to 200 times the normal subjects. CONCLUSIONS/RELEVANCE: Some have challenged the value of GGT as a distinguishing marker for liver disease in myopathic disorders. We found its value to be impeccable despite an environment greatly predisposed to leak intracellular enzymes into the serum. The importance of this first and only evidence-based study establishes an essential role: 1) for GGT in the management of patients required to take hepatotoxic drugs for inflammatory muscle diseases; and 2) for monitoring myopathic patients participating in clinical trials.
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. Supported by: Muscular Dystrophy Association, NIH (NCRR).
Genotype Analysis Predicts Dilated Cardiomyopathy in Becker Muscular Dystrophy
OBJECTIVE: To identify dystrophin mutations predictive of dilated cardiomyopathy in patients with Becker muscular dystrophy (BMD) or X-linked dilated cardiomyopathy (XLDCM). BACKGROUND: BMD is caused by dystrophin gene mutations resulting in skeletal and cardiac muscle degeneration. When cardiac manifestations are out of proportion to skeletal muscle symptoms, the disorder is diagnosed as XLDCM. Dystrophin mutations predictive of XLDCM have been suggested, but representative examples from the literature are insufficient in number to establish with certainty. Defining the clinical presentations of specific mutations is crucial for risk classification, early detection and treatment to prolong survival. DESIGN/METHODS: BMD and XLDCM patients with dystrophin deletion/missense mutations and with cardiac histories were collected from databases at Nationwide Childrens Hospital, The Ohio State University, the Utah Dystrophinopathy Project, the Leiden Open Variation Database, and published case reports. Phenotype-genotype correlations were established based on age, skeletal muscle and cardiac manifestations, and echocardiograms. Statistical analysis was performed using Mann-Whitney U tests. RESULTS: Data from 126 patients were analyzed. Deletions in the N-terminus domain of dystrophin resulted in the earliest mean age of cardiac involvement (23 years) compared to mutations affecting the rod domain (41 years) or the hinge 3 region (39 years). Mutations affecting the spectrin repeats in the rod domain which preserve hinge 3 lead to an earlier onset (31 years) of cardiomyopathy than mutations which remove hinge 3 (47 years) (p<0.05). Mutations affecting the spectrin repeats preserving hinge 3 were subdivided based on whether they disrupted (out-of-phase) or preserved (in-phase) the spectrin repeats structure. Out-of-phase mutations predisposed to early onset (25 years) cardiomyopathy compared to in-phase mutations (37 years) (p<0.05). CONCLUSIONS/RELEVANCE: This genotype-phenotype analysis included the largest number of BMD patients to date. The results define dystrophin mutations predisposing to cardiomyopathy and enable clinicians to provide early intervention for at-risk patients that could improve the disease natural history.
Immediate Release Oral Pentoxifylline Is Poorly Tolerated in Duchenne Muscular Dystrophy Boys
OBJECTIVE: The objective of this open label pilot study of oral, immediate release pentoxifylline was to assess the tolerability, safety and efficacy on muscle strength and function in Duchenne Muscular Dystrophy (DMD). BACKGROUND: Severe to complete dystrophin deficiency produces the DMD phenotype. Untreated DMD boys demonstrate progressive, eventually fatal, muscle weakness. In relevant animal models, pentoxifylline slows disease progression. DESIGN/METHODS: This was an open label, prospective study with a 3 month lead in period followed by 12 months of treatment with a 20mg/kg/day dose of pentoxifylline (maximal dose 598 mg/day). Study subjects were 4-9 year old steroid-naive DMD boys. The primary efficacy measure was a total quantitative muscle testing (QMT) score. Secondary and exploratory endpoint measures were manual muscle strength (MMT), arm and leg QMT, timed function tests, muscle MRI, serum TNF- and TGF- levels and adverse event profiles. RESULTS: Of twenty patients screened, 17 enrolled and 9 completed the protocol. Age range was 4.3-8.5 years (mean 6.0, median 5.8). QMT and MMT measures did not show a significant change during the 3 month lead-in or 12 month treatment period. Five of 8 patient withdrawals were due to intolerable adverse events. Eleven of 17 patients experienced nausea and vomiting. Two patients experienced moderate (grade 3) to severe (grade 4) leucopenia. CONCLUSIONS/RELEVANCE: The immediate release oral formulation of pentoxifylline is not well tolerated in children with DMD. The 12% incidence of leucopenia in this study was higher than the reported incidence rate in adults (<1%). Leucopenia improved post drug withdrawal. The 65% incidence of vomiting was also higher than reported in adults (4.5%). The lack of deterioration in strength and function over 12 months in steroid naive DMD children (mean age 6 years) suggests a possible beneficial effect on disease progression and warrants further study with a different formulation of pentoxifylline.
Double-Blind Randomized Controlled Trial of SNT-MC17/Idebenone in Duchenne Muscular Dystrophy
OBJECTIVE: This 12-month study has evaluated the efficacy and tolerability of treatment with SNT-MC17/idebenone (450 mg/day) compared to placebo in children with Duchenne muscular dystrophy (DMD). BACKGROUND: Idebenone supports mitochondrial respiratory chain function and reduces oxidative stress, pathways that are involved in DMD pathogenesis. Previous long-term blinded controlled preclinical studies in the homologous mdx mouse have indicated that idebenone is cardioprotective and improves exercise performance in murine dystrophin-deficiency. DESIGN/METHODS: 21 DMD patients (8-16 y) with cardiac dysfunction were enrolled in the double-blind randomized placebo-controlled trial. Comedication with glucocorticoids was allowed at stable dosage; use of ACE-inhibitors was excluded. Thirteen patients received SNT-MC17 as a daily dose of 450 mg for 52 weeks, 8 patients were randomized to the placebo group. RESULTS: All subjects completed the study; the frequency and type of adverse events was comparable in the active and placebo treatment groups indicating good safety and tolerability of SNT-MC17. The primary endpoint was the change in peak systolic radial strain of the left ventricular inferolateral wall, the region of the heart which in DMD is affected early and most severely. For the observed cases, patients on SNT-MC17 improved by 17.313.2% from baseline (22.99.1%) to week 52, while patients on placebo improved by 7.512.0% from baseline (33.79.7%; p= 0.066 comparison SNT-MC17 vs placebo). Secondary outcome measures included respiratory function tests. Whereas SNT-MC17 treatment was associated with improvement in peak flow (30.854.4 L/min) and peak flow % predicted (2.813.8%) during the 52 week study period, patients on placebo deteriorated in peak flow (- 13.851 L/min; p= 0.039) and peak flow % predicted (- 8.513.8%; p= 0.042). CONCLUSIONS/RELEVANCE: This the first indication of clinical efficacy with SNT-MC17/idebenone on functional cardiac and respiratory parameters in DMD. The results provide the basis for the planning of further clinical development studies with SNT-MC17/idebenone in DMD.
Implementation of a Newborn Screening Program for Duchenne Muscular Dystrophy (DMD)
OBJECTIVE: Introduce a comprehensive newborn screening paradigm for Duchenne muscular dystrophy (DMD). BACKGROUND: The incidence of DMD is presumed to be 1:3500 newborn males, however, this has not been confirmed in an outbred population. Only small cohorts of newborns have been screened based on creatine kinase (CK) testing. The most useful, exacting and baby friendly paradigm includes testing both CK and DNA on the dried blood spots (DBSs) taken within the first 24 to 48 hours of life. DESIGN/METHODS: DBSs obtained within the newborn period were used to test for CK combined with a rapid direct method of sequence analysis of the dystrophin gene referred to as SCAIP sequencing. RESULTS: The first goal of this study was to establish a range of CK from 40,000 anonymous newborn male DBSs (250 + 111). Mutational analysis was tested on DBSs taken from 8 boys with known DMD mutations. Blood was obtained from the finger simulating heel stick samples obtained in the newborn period. Samples were analyzed blindly using modifications of SCAIP and matched successfully with known mutations. Once having established a CK testing range based on >3SD above the mean and a DNA confirmational method on DBSs, the second goal was achieved with implementing a newborn screening program in 6 birthing hospitals in central Ohio and Cincinnati areas. To date we have enrolled over 6000 newborn males. CONCLUSIONS/RELEVANCE: A successful paradigm for screening newborns for DMD has been established that will provide a definitive diagnosis of DMD in the newborn period. Survey of over 500 participants reveals a high degree of enthusiasm (>99%) for participation in this program. The long-term benefits include: 1) prevention of an unsuspecting second child with DMD; 2) establishing a registry for early intervention for clinical trials; and 3) determining the incidence of DMD in newborn males in an outbred population.
A Gene Therapy Approach To Treat Cardiomyopathy in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)
OBJECTIVE: At least 95% of DMD patients develop a cardiomyopathy, critical to their long-term prognosis. Most evolving strategies for replacing the missing dystrophin protein in skeletal muscle are not applicable to the heart. Our goal is to use adeno-associated virus (AAV) to replace the mutant gene. BACKGROUND: Dystrophinopathy victims historically died from respiratory complications while cardiac failure appeared catastrophically related to the stress of respiratory infections. Contemporary methods now protect the respiratory system related to improved equipment, antibiotics and vaccines; consequently, the progressive cardiomyopathy of DMD mandates attention. We have previously shown AAV serotype 8 to efficiently transduce skeletal muscle when delivered through the vasculature, and therefore chose to test its efficiency in transducing cardiac muscle of mdx mice, a model with similar lesions to DMD patients. DESIGN/METHODS: Using AAV8 carrying green fluorescent protein (GFP) as a marker gene we treated mdx mice at the neonatal stage (P2) [2 x 1011 vector genomes (vg)] by intraperitoneal injection, or at 8 weeks-old by intravenous tail vein injections (8 x 1011 vg). Hearts were harvested and stained for GFP four weeks post delivery. In addition, we developed a testing paradigm to serially measure in vivo cardiovascular function in mdx using echocardiography and electrocardiography (ECG). RESULTS: Gene delivery to mdx mice resulted in 73% gene expression in mdx mice treated with AAV8.GFP treated as adults and 65% expression in neonates. In addition, using our previously described methods, we observed a 52% reduction in left ventricular fractional shortening % (FS%) in the mdx mice as compared to the wild-type controls (52.05-2.77 vs. 27.513.06; Ctrl vs. mdx; p<0.05). CONCLUSIONS/RELEVANCE: Together these data support treatment of the cardiomyopathy of neonatal or adult mdx mice using rAAV8 to deliver a therapeutic dystrophin transgene while concurrently monitoring cardiac performance using serial, non-invasive outcome measures similar to those used in the clinic.
Pentoxifylline Treatment Fails To Rescue Muscle Strength and Function Deterioration in Prednisone-Treated Duchenne Muscular Dystrophy
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. DESIGN/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- and TNF-. 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. CONCLUSIONS/RELEVANCE: 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.
Imatinib Attenuated Skeletal Muscle Dystrophy in mdx Mice
OBJECTIVE: To test anti-inflammatory and anti-fibrotic effects of imatinib on mdx mice, a mouse model of Duchenne muscular dystrophy (DMD). BACKGROUND: DMD is the most common genetic muscle disease. DMD is lethal and currently untreatable. Muscle necrosis, inflammation, and fibrosis are prominent pathological features, and pharmacotherapy to ameliorate these pathological changes may represent an important therapeutic approach for DMD. Imatinib is an anti-neoplastic therapy that selectively inhibits the tyrosine kinase activity of c-abl, c-kit, and PDGF receptors (PDGFRs). It is also a promising anti-inflammatory and anti-fibrotic therapy as demonstrated by its substantial inhibition of inflammation and fibrosis of liver, kidney, lung, or skin in various animal models of disease. DESIGN/METHODS: We treated mdx mice with intraperitoneal injection of imatinib at the peak of limb muscle inflammation and at the onset of diaphragm fibrosis; controls received PBS vehicle or were left untreated. Muscle necrosis and inflammation were quantified by measuring serum CK activity and percentage of CD45 immunoreactive area, respectively. Muscle fibrosis was evaluated by collagen III immunostaining. Muscle function was assessed by measuring hindlimb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was studied by Western blot. RESULTS: Compared with control mdx mice, imatinib-treated mdx mice showed striking pathological and functional benefit. Serum CK level, diaphragm and quadriceps inflammation areas, and diaphragm fibrosis were markedly reduced. Hindlimb grip strength was significantly improved. Reduced clinical disease was accompanied by suppression of TNF-alpha and IL-1beta mRNA expression, and inhibition of c-abl and PDGFR phosphorylation. CONCLUSIONS/RELEVANCE: 1) Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis. 2) Imatinib likely exerts its therapeutic effects on mdx mice by inhibiting c-abl and PDGFR signaling and downstream inflammatory cytokine and fibrotic gene expression.
How Frequently Are Corticosteroids Used in Boys with Dystrophinopathy in the USA: Data from the MDSTARnet
OBJECTIVE: To review the use of corticosteroids for the treatment of Duchenne Muscular Dystrophy (DMD) in clinical practice from 1991 to 2005 in a large population- based cohort in the USA. BACKGROUND: Corticosteroids are the only pharmacological treatment that has been shown to have positive effect on muscle strength and function in DMD in three randomized controlled trials and a 3 year follow up open study. These studies were published in 1991. DESIGN/METHODS: The Muscular Dystrophy SurveillanceTracking and Research Network (MD STARnet) is a population-based surveillance system funded by the Centers for Disease Control to identify all cases of Duchenne /Becker Muscular Dystrophy (DBMD) born since 1982 and/or residing within Arizona, Colorado, Iowa and Western New York State. A pooled database containing clinical information abstracted from medical records on 433 individuals was analyzed. RESULTS: The proportion of boys receiving corticosteroids increased from 27% in 1991 to 44% in 2005. The proportion of boys receiving corticosteroids in 2005 by state is: NY: 57 % (clinic A= 75%; clinic B= 5 %), CO: 29%, AZ: 38%, IA: 50 %. Mean age when corticosteroids are started is 7.4 years (median= 7; range 3-18). Mean duration of treatment is 57 months. Most common reasons for discontinuing corticosteroids are: weight gain, behavioral side effects and becoming wheelchair bound. Most common reasons for changing corticosteroids dose are: weight gain, behavioral side effects and parents request. CONCLUSIONS/RELEVANCE: The proportion of DMD boys taking corticosteroids has increased in the past 15 years but is overall still less than 50%. Large variability in practice remains between States and between Neuromuscular Clinics. We plan a repeat analysis in 2 years to assess the impact of the AAN Practice Parameter on the use of corticosteroids in DMD published in 2005.
Long-Term Enhancement of Skeletal Muscle Mass and Strength by Single Gene Administration of Myostatin Inhibitiors
OBJECTIVE: To determine the safety and efficacy of adeno-associated virus expressing follistatin (FS-344), for muscle enhancement in a mouse model for Duchenne Muscular Dystrophy and wild-type animals. BACKGROUND: Increasing the size and strength of muscles represents a promising therapeutic strategy for musculoskeletal disorders. Significant interest has focused on myostatin, a negative regulatory factor of muscle growth. Inhibition of myostatin significantly increases muscle mass. Several proteins including follistatin (FS), follistatin-related gene (FLRG) and growth and differentiation factor-associated serum protein 1 (GASP-1) inhibit myostatin. DESIGN/METHODS: 1x1011 particles of AAV1 vectors expressing Follistatin, FLRG, or GASP-1 were injected bilaterally into hindlimbs of mdx or wild-type mice and followed for 6-months or 2 years. RESULTS: Wild-type mice injected in the hindlimbs with of AAV1-FS, FLRG, or GASP-1 showed increased overall body mass, with 30% increase in hindlimb and forelimb grip strength compared to AAV1-GFP controls. Overall strength increase was greatest for animals receiving AAV1-FS followed by AAV1-FLRG and then AAV1-GASP-1. We next tested the potential of AAV1-FS to increase muscle mass and strength and delay muscle deterioration in the mdx mouse model. Mdx animals were injected at 3 weeks of age and followed for 6 months with AAV1-FS. Animals showed a 30-50% increase in skeletal muscle mass, enhanced muscle grip strength, and decreased serum creatine kinase levels compared to controls. Histology demonstrated myofiber hypertrophy locally and at remote sites to the injection. No adverse organ pathology was detected. Mdx animals treated with AAV1-Follistatin at 6.5 months of age also showed increased muscle strength, demonstrating the ability to improve strength in aged animals. CONCLUSIONS/RELEVANCE: Inhibition of myostatin by myostatin inhibitory proteins, in particular follistatin represents a promising therapeutic strategy warranting consideration for clinical trials in human muscle diseases.
Fidelity of Gamma-Glutamyl Transferase in Differentiating Skeletal Muscle from Liver Damage
OBJECTIVE: To validate that gamma-glutamyl transferase (GGT) is a flawless biomarker distinguishing liver and skeletal muscle damage. BACKGROUND: Serum creatine kinase (CK) is recognized to be a valuable biomarker for distinguishing myopathic and neurogenic disorders. Accompanying the leak of CK from skeletal muscle are other intracellular enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), pyruvate kinase, and lactic dehydrogenase. The initial interpretation of an elevated AST and ALT is a derivation from liver, frequently leading to liver biopsy. Many clinicians ignore the fact that muscle breakdown is an established cause for hypertransaminasemia. GGT is a membrane-bound enzyme produced in the liver, with little or none from skeletal muscle. We tested the validity of GGT as a marker to differentiate skeletal muscle from liver damage in an environment of unrestrained activity. DESIGN/METHODS: Duchenne muscular dystrophy subjects (n=28) with proven dystrophin mutations were enrolled (equally divided between ambulatory and non-ambulatory, and approximately one-half taking corticosteroids). Normal males (n=20) served as controls. Initial GGT and CK samples were taken between 8 and 9 am and redrawn 8 hours later. Subjects resumed normal play or left the clinic between blood draws. RESULTS: Not a single DMD patient, ambulatory or non-ambulatory, taking corticosteroids or not, showed a GGT outside the range of normal at any time point. CK levels at the same time points ranged from 14 to 200 times the normal subjects. CONCLUSIONS/RELEVANCE: Some have challenged the value of GGT as a distinguishing marker for liver disease in myopathic disorders. We found its value to be impeccable despite an environment greatly predisposed to leak intracellular enzymes into the serum. The importance of this first and only evidence-based study establishes an essential role: 1) for GGT in the management of patients required to take hepatotoxic drugs for inflammatory muscle diseases; and 2) for monitoring myopathic patients participating in clinical trials.