A systematic literature review and meta-analysis of the effectiveness of vitamin D supplementation for patients with Duchenne muscular dystrophy.

We conducted a systematic literature review and meta-analysis on the effectiveness of vitamin D supplementation in maintaining or restoring vitamin D levels in Duchenne muscular dystrophy. Due to a lack of randomised controlled trials, cross-sectional and retrospective and prospective cohort studies were taken as the best available evidence. Inclusion criteria included reporting mean serum vitamin D levels in a supplement-taking group. After screening 102 records; 13 were included in a narrative synthesis and eight of these in a meta-analysis. We show that current dosing regimens are preventing severe deficiency but are not effective at maintaining sufficient vitamin D levels within the Duchenne population. Despite high levels of daily vitamin D supplementation (>1000 International Units), at least 20 % of people with Duchenne remain vitamin D deficient. No significant association between dose and serum vitamin D levels was found (r2 = 0.3, p = 0.237). A meta-analysis of mean serum vitamin D levels across eight studies also revealed substantial variability in response to vitamin D supplementation and high heterogeneity (I2 = 99.59 %). These data could impact on an individual's risk and severity of osteoporosis and vertebral fractures.

Sulfur amino acid supplementation displays therapeutic potential in a C. elegans model of Duchenne muscular dystrophy.

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a common muscle disease that manifests with muscle weakness, wasting, and degeneration. An emerging theme in DMD pathophysiology is an intramuscular deficit in the gasotransmitter hydrogen sulfide (H2S). Here we show that the C. elegans DMD model displays reduced levels of H2S and expression of genes required for sulfur metabolism. These reductions can be offset by increasing bioavailability of sulfur containing amino acids (L-methionine, L-homocysteine, L-cysteine, L-glutathione, and L-taurine), augmenting healthspan primarily via improved calcium regulation, mitochondrial structure and delayed muscle cell death. Additionally, we show distinct differences in preservation mechanisms between sulfur amino acid vs H2S administration, despite similarities in required health-preserving pathways. Our results suggest that the H2S deficit in DMD is likely caused by altered sulfur metabolism and that modulation of this pathway may improve DMD muscle health via multiple evolutionarily conserved mechanisms.

Acute toxicity and regenerative dose finding of an extract of Miconia ferruginata DC. in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe disease with no cure caused by a genetic abnormality, promoting progressive muscle degeneration. Corticosteroids are used drugs in treatment associated with adverse effects. The extract of Miconia ferruginata (Melastomataceae) (MF) has demonstrated potent antioxidant and anti-inflammatory potential in vitro. This study used a DMD model (mdx) to determine the toxic dose of this plant and found a possible non-toxic dose with therapeutic effects. The mdx groups received an intraperitoneal injection of 0 (control group), 50, 100, 200, 300, and 2000 mg kg-1 of the aqueous leaf extract following a single-dose acute toxicity protocol and were observed for 14 days. The range of toxicity of the extract and LD50 were determined. Histopathological analysis, the quantification of fibrosis, and immunohistochemical analysis of the tissues were performed. The results demonstrated that 2000 mg kg-1 was highly toxic, inducing histopathological changes in the tissues evaluated, with 100% mortality in 48 hours. The other doses caused no behavioral changes or signs of toxicity. The MF extract led reduction in histopathological changes, fibrosis, and inflammation, a reduction in HSP70 and an increase in MCL-1 proteins. Doses of 50-200 mg kg-1 demonstrated regenerative tissue and anti-inflammatory potential.

Exploring the Therapeutic Potential of Ectoine in Duchenne Muscular Dystrophy: Comparison with Taurine, a Supplement with Known Beneficial Effects in the mdx Mouse.

Duchenne Muscular Dystrophy (DMD) is a debilitating muscle disorder that condemns patients to year-long dependency on glucocorticoids. Chronic glucocorticoid use elicits many unfavourable side-effects without offering satisfying clinical improvement, thus, the search for alternative treatments to alleviate muscle inflammation persists. Taurine, an osmolyte with anti-inflammatory effects, mitigated pathological features in the mdx mouse model for DMD but interfered with murine development. In this study, ectoine is evaluated as an alternative for taurine in vitro in CCL-136 cells and in vivo in the mdx mouse. Pre-treating CCL-136 cells with 0.1 mM taurine and 0.1 mM ectoine prior to exposure with 300 U/mL IFN-γ and 20 ng/mL IL-1ß partially attenuated cell death, whilst 100 mM taurine reduced MHC-I protein levels. In vivo, histopathological features of the tibialis anterior in mdx mice were mitigated by ectoine, but not by taurine. Osmolyte treatment significantly reduced mRNA levels of inflammatory disease biomarkers, respectively, CCL2 and SPP1 in ectoine-treated mdx mice, and CCL2, HSPA1A, TNF-α and IL-1ß in taurine-treated mdx mice. Functional performance was not improved by osmolyte treatment. Furthermore, ectoine-treated mdx mice exhibited reduced body weight. Our results confirmed beneficial effects of taurine in mdx mice and, for the first time, demonstrated similar and differential effects of ectoine.

Efficacy and Safety of Vamorolone vs Placebo and Prednisone Among Boys With Duchenne Muscular Dystrophy: A Randomized Clinical Trial.

Importance: Corticosteroidal anti-inflammatory drugs are widely prescribed but long-term use shows adverse effects that detract from patient quality of life. Objective: To determine if vamorolone, a structurally unique dissociative steroidal anti-inflammatory drug, is able to retain efficacy while reducing safety concerns with use in Duchenne muscular dystrophy (DMD). Design, Setting, and Participants: Randomized, double-blind, placebo- and prednisone-controlled 24-week clinical trial, conducted from June 29, 2018, to February 24, 2021, with 24 weeks of follow-up. This was a multicenter study (33 referral centers in 11 countries) and included boys 4 to younger than 7 years of age with genetically confirmed DMD not previously treated with corticosteroids. Interventions: The study included 4 groups: placebo; prednisone, 0.75 mg/kg per day; vamorolone, 2 mg/kg per day; and vamorolone, 6 mg/kg per day. Main Outcomes and Measures: Study outcomes monitored (1) efficacy, which included motor outcomes (primary: time to stand from supine velocity in the vamorolone, 6 mg/kg per day, group vs placebo; secondary: time to stand from supine velocity [vamorolone, 2 mg/kg per day], 6-minute walk distance, time to run/walk 10 m [vamorolone, 2 and 6 mg/kg per day]; exploratory: NorthStar Ambulatory Assessment, time to climb 4 stairs) and (2) safety, which included growth, bone biomarkers, and a corticotropin (ACTH)-challenge test. Results: Among the 133 boys with DMD enrolled in the study (mean [SD] age, 5.4 [0.9] years), 121 were randomly assigned to treatment groups, and 114 completed the 24-week treatment period. The trial met the primary end point for change from baseline to week 24 time to stand velocity for vamorolone, 6 mg/kg per day (least-squares mean [SE] velocity, 0.05 [0.01] m/s vs placebo -0.01 [0.01] m/s; 95% CI, 0.02-0.10; P = .002) and the first 4 sequential secondary end points: time to stand velocity, vamorolone, 2 mg/kg per day, vs placebo; 6-minute walk test, vamorolone, 6 mg/kg per day, vs placebo; 6-minute walk test, vamorolone, 2 mg/kg per day, vs placebo; and time to run/walk 10 m velocity, vamorolone, 6 mg/kg per day, vs placebo. Height percentile declined in prednisone-treated (not vamorolone-treated) participants (change from baseline [SD]: prednisone, -1.88 [8.81] percentile vs vamorolone, 6 mg/kg per day, +3.86 [6.16] percentile; P = .02). Bone turnover markers declined with prednisone but not with vamorolone. Boys with DMD at baseline showed low ACTH-stimulated cortisol and high incidence of adrenal insufficiency. All 3 treatment groups led to increased adrenal insufficiency. Conclusions and Relevance: In this pivotal randomized clinical trial, vamorolone was shown to be effective and safe in the treatment of boys with DMD over a 24-week treatment period. Vamorolone may be a safer alternative than prednisone in this disease, in which long-term corticosteroid use is the standard of care. Trial Registration: ClinicalTrials.gov Identifier: NCT03439670.

2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Effectiveness of Neridronate in the Management of Bone Loss in Patients with Duchenne Muscular Dystrophy: Results from a Pilot Study.

INTRODUCTION: Bone loss is a major issue in patients affected by Duchenne muscular dystrophy (DMD), a rare musculoskeletal disorder, particularly in those treated with glucocorticoids (GCs). We aimed to assess the effectiveness of neridronate in terms of bone mineral density (BMD) changes in this population. METHODS: We retrospectively reviewed the records of patients affected by DMD receiving GCs referred to our outpatient from 2015 to 2020. All patients were treated with an intramuscular (IM) injection of neridronate (25 mg every month). Bone density was measured at the lumbar spine (LS; L1-L4 tract) using dual-energy x-ray absorptiometry (DXA) (GE Lunar), no more than 4 weeks before (T0) and after 1 year from neridronate treatment (T1). RESULTS: Eight boys with DMD were included with a mean age at diagnosis of 4.75 ± 2.81 years. Six of them were non-ambulant and two of them had previous low-trauma fractures (a distal femur fracture and a vertebral compression fracture, respectively). All patients were receiving deflazacort [median duration of therapy 11.5 years (interquartile range 2-25)]. At the DXA evaluation (T0), the mean L1-L4 BMD value was 0.716 ± 0.164 g/cm2. Six patients (75%) showed an L1-L4 Z-score height-adjusted of less than - 2. The mean age of neridronate initiation was 18.87 ± 6.81 years. All patients were supplemented with calcium carbonate and vitamin D at baseline. After 12 months of treatment (T1), the mean L1-L4 BMD value was 0.685 ± 0.190 g/cm2. Seven patients (87.5%) showed an L1-L4 Z-score of less than - 2. Changes in LS BMD and Z-score were not significant between T0 and T1 in our cohort (p = 0.674 and p = 0.208, respectively) as well as among non-ambulant patients with DMD without previous fragility fractures. CONCLUSIONS: In this study, we reported for the first time that neridronate may slow bone loss in GC-treated patients with DMD at 1-year follow-up.

Reduced bone mineral density in adolescents with Duchenne Muscular Dystrophy (DMD) and scoliosis.

Duchenne muscular dystrophy is a progressive disease usually associated with loss of ambulation and progressive scoliosis. Immobilisation and glucocorticoid treatment are predisposing factors for reduced bone mineral density (BMD). Analysis of quantitative computed tomography revealed low BMD in thoracic and lumbar vertebrae in comparison to age- and sex-matched healthy controls. INTRODUCTION: Evaluation of vertebral bone mineral density (BMD) in Duchenne Muscular Dystrophy (DMD) adolescents with untreated advanced scoliosis and comparison with the BMD values of healthy age-matched controls, based on quantitative computer tomography. METHODS: Thirty-seven DMD adolescents (age 15.6 ± 2.5 years) with spinal deformity were evaluated clinically and radiologically prior to definite spinal fusion and compared to 31 male and age-matched healthy individuals (age 15.7 ± 2.3 years). Data related to previous medical treatment, physiotherapy and ambulatory status was also analysed. Scoliotic curves were measured on plain sitting radiographs of the spine. The BMD Z-scores of the thoracic and lumbar vertebrae were calculated with QCTpro® (Mindways Software Inc., USA), based on data sets of preoperative, phantom pre-calibrated spinal computed tomography scans. RESULTS: A statistically significant lower BMD could be found in DMD adolescents, when compared to healthy controls, showing an average value for the lumbar spine of 80.5 ± 30.5 mg/cm3. Z-scores deteriorated from the upper thoracic towards the lower lumbar vertebrae. All but the uppermost thoracic vertebrae had reduced BMD values, with the thoracolumbar and lumbar region demonstrating the lowest BMD. No significant correlation was observed between BMD and the severity of the scoliotic curve, previous glucocorticoid treatment, cardiovascular impairment, vitamin D supplementation, non-invasive ventilation or physiotherapy. CONCLUSION: DMD adolescents with scoliosis have strongly reduced BMD Z-scores, especially in the lumbar spine in comparison to healthy controls. These findings support the implementation of a standardised screening and treatment protocol. Level of evidence/clinical relevance: therapeutic level III.

Paeonia lactiflora extract improves the muscle function of mdx mice, an animal model of Duchenne muscular dystrophy, via downregulating the high mobility group box 1 protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. is an ethnopharmacological medicine with a long history of human use for treating various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Duchenne muscular dystrophy (DMD) is an X-linked degenerative muscle disease affecting 1 in 3500 males and is characterized by severe muscle inflammation and a progressive decline in muscle function. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (PL) on the muscle function in the muscular dystrophy X-linked (mdx) mouse, the most commonly used animal model of DMD. MATERIALS AND METHODS: Male mdx mice and wild-type controls aged 5 weeks were orally treated with PL for 4 weeks. The corticosteroid prednisolone was used as a comparator drug. Muscle strength and motor coordination were assessed via the grip-strength and rotarod tests, respectively. Muscle damage was evaluated via histological examination and assessment of plasma creatine-kinase activity. Proteomic analyses were conducted to identify the muscle proteins whose levels were significantly affected by PL (ProteomeXchange identifier: PXD028886). Muscle and plasma levels of these proteins, and their corresponding mRNAs were measured using western blotting and ELISA, and quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: The muscle strength and motor coordination of mdx mice were significantly increased by the oral treatment of PL. PL significantly reduced the histological muscle damage and plasma creatine-kinase activity. Proteomic analyses of the muscle showed that PL significantly downregulated the high mobility group box 1 (HMGB1) protein and Toll-like receptor (TLR) 4, thus suppressing the HMGB1-TLR4-NF-κB signaling, in the muscle of mdx mice. Consequently, the muscle levels of proinflammatory cytokines/chemokines, which play crucial roles in inflammation, were downregulated. CONCLUSION: PL improves the muscle function and reduces the muscle damage in mdx mice via suppressing the HMGB1-TLR4-NF-κB signaling and downregulating proinflammatory cytokines/chemokines.

Pro-inflammatory monocytes are increased in Duchenne muscular dystrophy and suppressed with omega-3 fatty acids: A double-blind, randomized, placebo-controlled pilot study.

BACKGROUND: Omega-3 long chain polyunsaturated fatty acids (LCPUFA) reduce circulating cytokines produced by monocytes. Nevertheless, whether the omega-3 LCPUFA regulate the monocytes and their cytokines in Duchenne muscular dystrophy (DMD) is unknown. The aim of this study was to evaluate whether circulating pro-inflammatory monocytes are increased and whether omega-3 LCPUFA selectively suppress these monocytes and their cytokines in patients with DMD. METHODS: This was a double-blind, randomized, placebo-controlled pilot study carried out in patients with DMD supplemented with omega-3 LCPUFA (n = 6) or sunflower oils (placebo, n = 6) for 6 months. Monocytes and their cytokines were measured at baseline and after 1, 2, 3, and 6 months of supplementation. RESULTS: The anti-inflammatory monocytes (median, [95% CI]) are increased at month 3 (-0.46 [-13.5-9.5] vs. 8.4 [5.5-12.5], p = 0.05) in the omega-3 LCPUFA group compared with the placebo group. The pro-inflammatory monocytes (-5.7 [-63.8-114.1] vs. -51.9 [-91.2 to -25.4], p = 0.026 and -16.4 [-50.8-50.6] vs. -57.9 [-86.9 to -18.5], p = 0.045 at months 3 and 6, respectively) and their cytokine interleukin 6 (-11.9 [-93.5-148.9] vs. -64.7 [-77.8 to -42.6], p = 0.019 at month 6) decreased in the omega-3 LCPUFA group compared with the placebo group. Pro-inflammatory monocytes decreased and anti-inflammatory monocytes were augmented (p < 0.05) during the 6 months of supplementation with omega-3 LCPUFA. CONCLUSIONS: This pilot study suggests that supplementation with omega-3 LCPUFA could have a selective reductive effect on pro-inflammatory monocytes and their cytokines in patients with DMD. These findings also support the performance of studies in a significant population to explore the role of omega-3 LCPUFA on monocyte populations and their cytokines in patients with DMD. This research was registered at clinicaltrials.gov (NCT018264229).

Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

ß-Glucans as Dietary Supplement to Improve Locomotion and Mitochondrial Respiration in a Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration. A lack of dystrophin in DMD leads to inflammatory response, autophagic dysregulation, and oxidative stress in skeletal muscle fibers that play a key role in the progression of the pathology. ß-glucans can modulate immune function by modifying the phagocytic activity of immunocompetent cells, notably macrophages. Mitochondrial function is also involved in an important mechanism of the innate and adaptive immune responses, owing to high need for energy of immune cells. In the present study, the effects of 1,3-1,6 ß-glucans on five-day-old non-dystrophic and dystrophic (sapje) zebrafish larvae were investigated. The effects of the sonication of ß-glucans and the dechorionation of embryos were also evaluated. The results showed that the incidence of dystrophic phenotypes was reduced when dystrophic embryos were exposed to 2 and 4 mg L-1 of 1,3-1,6 ß-glucans. Moreover, when the dystrophic larvae underwent 8 mg L-1 treatment, an improvement of the locomotor performances and mitochondrial respiration were observed. In conclusion, the observed results demonstrated that 1,3-1,6 ß-glucans improve locomotor performances and mitochondrial function in dystrophic zebrafish. Therefore, for ameliorating their life quality, 1,3-1,6 ß-glucans look like a promising diet supplement for DMD patients, even though further investigations are required.

Bone density and bone health alteration in boys with Duchenne Muscular Dystrophy: a prospective observational study.

OBJECTIVES: Boys with Duchenne Muscular Dystrophy (DMD) are at increased risk for compromised bone health, manifesting as low-impact trauma long bone fractures and vertebral compression fractures. METHODS: In a prospective observational study, we studied bone health parameters in North Indian boys with DMD. We consecutively enrolled ambulatory boys with DMD on glucocorticoid therapy. Bone health was evaluated with X-ray spine, Dual-energy X-ray absorptiometry (DXA), serum calcium, vitamin D3 (25[OH]D), 1,25-dihyroxyvitamin D3 (1,25[OH]2D3), serum osteocalcin, osteopontin, and N terminal telopeptide of type 1 collagen (Ntx) levels. RESULTS: A total of 76 boys with DMD were enrolled. The median age was 8.5 (interquartile range [IQR] 7.04-10.77) years. Among these, seven (9.2%) boys had long bone fractures, and four (5.3%) had vertebral compression fractures. Fifty-four (71%) boys underwent DXA scan, and among these 31 (57%) had low bone mineral density (BMD, ≤-2 z-score) at the lumbar spine. The mean BMD z-score at the lumbar spine was -2.3 (95% confidence interval [CI] = -1.8, -2.8), and at the femoral neck was -2.5 (95% CI = -2, -2.9). 25(OH)D levels were deficient in 68 (89.5%, n=76) boys, and 1,25(OH)2D3 levels were deficient in all. Mean serum osteocalcin levels were 0.68 ± 0.38 ng/mL (n=54), serum osteopontin levels were 8.6 ± 4.6 pg/mL (n=54) and serum Ntx levels were 891 ± 476 nmol/L (n=54). Boys with low BMD received glucocorticoids for longer duration, in comparison to those with normal BMD (median, IQR [16.9 (6-34) months vs. 7.8 (4.8-13.4) months]; p=0.04). CONCLUSIONS: Bone health is compromised in North Indian boys with DMD. BMD at the lumbar spine is reduced in more than half of boys with DMD and nearly all had vitamin D deficiency on regular vitamin D supplements. Longer duration of glucocorticoid therapy is a risk factor for low BMD in our cohort.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

Duchenne muscular dystrophy hiPSC-derived myoblast drug screen identifies compounds that ameliorate disease in mdx mice.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. In the present study, when human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patient hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD-relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD Pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-ß signaling. Preclinical tests in mdx mice showed that treatment with these 2 hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study revealed that fenofibrate could inhibit mitochondrion-induced apoptosis in DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified 2 promising small molecules with in vivo efficacy.

Identification of therapeutics that target eEF1A2 and upregulate utrophin A translation in dystrophic muscles.

Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISA-based high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.

Janus effect of glucocorticoids on differentiation of muscle fibro/adipogenic progenitors.

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1,120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in sub-confluent FAPs. However, both drugs have a pro-adipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.

Teaching an Old Molecule New Tricks: Drug Repositioning for Duchenne Muscular Dystrophy.

: Duchenne muscular dystrophy (DMD) is one of the most severe forms of inherited muscular dystrophies. The disease is caused by the lack of dystrophin, a structurally essential protein; hence, a definitive cure would necessarily have to pass through some form of gene and/or cell therapy. Cell- and genetic-based therapeutics for DMD have been explored since the 1990s and recently, two of the latter have been approved for clinical use, but their efficacy is still very low. In parallel, there have been great ongoing efforts aimed at targeting the downstream pathogenic effects of dystrophin deficiency using classical pharmacological approaches, with synthetic or biological molecules. However, as it is always the case with rare diseases, R&D costs for new drugs can represent a major hurdle for researchers and patients alike. This problem can be greatly alleviated by experimenting the use of molecules that had originally been developed for different conditions, a process known as drug repurposing or drug repositioning. In this review, we will describe the state of the art of such an approach for DMD, both in the context of clinical trials and pre-clinical studies.

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 µM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive muscle wasting and weakness and premature death. Glucocorticoids (e.g. prednisolone) remain the only drugs with a favorable impact on DMD patients, but not without side effects. We have demonstrated that glycine preserves muscle in various wasting models. Since glycine effectively suppresses the activity of pro-inflammatory macrophages, we investigated the potential of glycine treatment to ameliorate the dystrophic pathology. Dystrophic mdx and dystrophin-utrophin null (dko) mice were treated with glycine or L-alanine (amino acid control) for up to 15 weeks and voluntary running distance (a quality of life marker and strong correlate of lifespan in dko mice) and muscle morphology were assessed. Glycine increased voluntary running distance in mdx mice by 90% (P < 0.05) after 2 weeks and by 60% (P < 0.01) in dko mice co-treated with prednisolone over an 8 week treatment period. Glycine treatment attenuated fibrotic deposition in the diaphragm by 28% (P < 0.05) after 10 weeks in mdx mice and by 22% (P < 0.02) after 14 weeks in dko mice. Glycine treatment augmented the prednisolone-induced reduction in fibrosis in diaphragm muscles of dko mice (23%, P < 0.05) after 8 weeks. Our findings provide strong evidence that glycine supplementation may be a safe, simple and effective adjuvant for improving the efficacy of prednisolone treatment and improving the quality of life for DMD patients.