Introduction
Duchenne muscular dystrophy (DMD) is a severe, genetic, degenerative muscle disease that affects young males. It the most common dystrophinopathy, with an incidence of 1 in 3,500-5,000 live male births [1] and a prevalence of 4.8 in 100,000 males worldwide [2]. DMD is an X-linked recessive disease caused by a mutation in chromosome Xp21 in the DMD gene [3].
The DMD gene is the third largest known human genes. It encompasses 79 exons and encodes dystrophin, a 3,685 amino acid protein [4]. The extremely large size of the gene results in a complex mutational spectrum, with over 7,000 mutations identified and a 30% spontaneous mutation rate [5]. The remaining 70% of cases are maternally inherited [6]. Two-thirds of all DMD mutations are caused by large deletions, involving one or more exons. The remaining mutations are caused by insertions, small deletions, point mutations or splicing mutations [7]. Out-of-frame mutations result in the most severe phenotype, due to a complete loss of the dystrophin protein [8, 9]. In-frame mutations allow for truncated, albeit, partially functional dystrophin protein to be synthesized, leading to more variable phenotypes including Becker muscular dystrophy or X-linked dilated cardiomyopathy [6]. Most deletions occur in a region spanning exons 45 to 53, as part of the major mutational “hotspot” [6].
Dystrophin is a cytoskeletal protein in the muscle membrane, and it is an integral part of the dystrophin associated glycoprotein complex [10]. Dystrophin is comprised of 4 separate functional domains including an actin-binding domain at the N-terminus, a rod domain with 25 triple-helical segments, a cysteine-rich dystroglycan binding site, and a C-terminal domain which is structurally homologous to the utrophin protein [11, 12]. It binds to dystroglycan via its cysteine-rich domain, to dystrobrevin and syntrophin via its C-terminal domain, and it is closely associated with other cytoskeletal proteins including F-actin via its N-terminus domain [13]. Through these binding sites, dystrophin provides structural stability to the skeletal muscle by connecting the sarcolemma and the basal lamina of the extracellular matrix to the inner cytoskeleton. Dystrophin also modulates vasomotor responses to physical activity via transmembrane signalling, which is essential to cell survival [14, 15]. Loss of dystrophin leads to a disruption of the dystrophin glycoprotein complex, which causes muscle membrane fragility. This leads to an influx of calcium into the sarcolemma, activation of proteases and pro-inflammatory cytokines, and mitochondrial dysfunction, all of which results in muscle degeneration [14]. As well, tissue ischemia occurs through the release of nitric oxide synthase which causes an increase in oxidative stress and reparative failure, ultimately leading to muscle fibrosis and necrosis with fatty tissue replacement [16].
The disruption of the dystrophin glycoprotein complex leads to the clinical symptoms of motor developmental delay, calf hypertrophy, joint contractures, and progressive muscle weakness in boys, accompanied by high levels of serum creatine kinase (CK) and dilated cardiomyopathy [17]. Ultimately, this leads to a shortened life span, with death typically occurring in the third or fourth decade, secondary to respiratory failure or severe cardiomyopathy [18]. Current standard of care treatments includes the use of corticosteroids to prolong ambulation and delay secondary complications such as cardiomyopathy and scoliosis [19]. Further cardioprotective measures and non-invasive positive pressure ventilation are also used as supportive strategies. Many emerging therapies from various therapeutics avenues have been pursued, all with the shared goal of targeting affected muscles in the body [20]. Gene therapies, including viral-mediated microdystrophin gene replacement, exon skipping to restore the reading frame, and nonsense suppression therapy to allow translation and production of a modified dystrophin proteins, are being investigated. Other therapies, including upregulation of compensatory proteins, reduction of the inflammatory cascade, prevention of muscle fibrosis, and augmentation of muscle regeneration are also being pursued. The purpose of this review is to highlight current and newly developed techniques and emerging therapies for DMD.
Methods
Literature search: We conducted a literature search of PubMed from 1947 to February 2018 using the following search terms: Duchenne muscular dystrophy, Duchenne muscular dystrophy in combination with; guidelines, gene therapies, exon skipping, drisapersen, eteplirsen, nonsense suppression, myoblasts, cardiomyopathy, glucocorticosteroids, anti-inflammatory treatments, anti-fibrotic treatments, muscle regeneration, and membrane stabilization. Non-English language publications were excluded from this review.
Clinical manifestations and diagnosis
Clinical manifestations
DMD typically presents in boys between age 3 and 5 years with initial symptoms of gross motor delays, abnormal gait, troubles with rising from the floor and frequent falling, in the context of an elevated serum CK. The characteristic weakness pattern initially involves the proximal lower limbs and neck flexor muscles, followed by the upper limbs and distal muscles. Examination findings include a waddling gait, calf muscle hypertrophy, and a positive Gower’s sign. Most boys with DMD will maintain strength and motor skills until approximately 6 years of age. After this age, progressive muscle deterioration occurs, and affected boys will be wheelchair bound by 11-12 years of age if left untreated [21]. In addition, boys affected by DMD may have variable degrees of cognitive impairment, speech delays, and learning disabilities [17, 22]. The average intelligence quotient in the patients with DMD is 85, approximately one standard deviation below the population norms [23]. This intellectual disability is static and does not correlate with the degree of muscle weakness [23].
Cardiac manifestations include dilated cardiomyopathy and arrhythmias. Progressive cardiomyopathy is one of the leading causes of death in DMD [24, 25]. Clinically apparent cardiomyopathy is first evident after 10 years of age, with one-third of patients affected by 14 years, and 100% of DMD patients affected over 18 years of age [19]. Pre-clinical involvement has been detected in 25% of patients under age 6 year with persistent tachycardia [26]. Due to their physical inactivity, most DMD patients are relatively asymptomatic. As such, the 2010 DMD guidelines recommend baseline electrocardiogram (ECG) and echocardiogram (ECHO) at diagnosis, followed by repeat testing every 2 years, and then annually after 10 years of age [19].
Other secondary complications of DMD include chronic respiratory insufficiency, scoliosis, and joint contractures. Chronic respiratory insufficiency develops due to progressive restrictive lung disease. Obstructive sleep apnea (OSA) is a prominent cause of sleep-disordered breathing (SDB) in the first decade [27], and OSA without hypercapnia is usually the first manifestation of respiratory insufficiency in DMD. In the second decade, hypoventilation occurs, which further progresses to SDB with nocturnal hypercapnia, and then finally develops into diurnal hypercapnia [28]. Without corticosteroid treatment, almost all boys will develop scoliosis [29]. Scoliosis compromises the respiratory vital capacity, and progresses further when ambulation is lost [30]. Joint contractures are common at the hips, knees, ankle joints and iliotibial bands, due to fibrosis and muscle weakness [29].
Diagnosis
The diagnosis of DMD is made based on the clinical features on history and examination with confirmation by additional investigations including muscle biopsy and/or genetic testing [17]. Serum CK is usually markedly elevated, ranging from 20,000 – 40,000U/L, and can be accompanied by elevated serum alanine and aspartate transaminase levels. Confirmatory genetics testing includes multiplex polymerase chain reaction focusing on the most commonly deleted regions or assays that interrogate all 79 exons, such as multiplex ligation-dependent probe amplification (MLPA) or comparative genomic hybridization (CGH) microarray [31, 32]. If a disease-causing deletion or duplication is not identified, then complete DMD gene sequencing is required. Muscle biopsies can also be obtained for dystrophin immunostaining and extraction of ribosomal nucleic acid (RNA) to produce complementary DNA, in order to confirm a dystrophin mutation. Typical pathologic features found on muscle biopsies include ongoing muscle degeneration and regeneration and absent or markedly reduced dystrophin staining [33, 21]. Identifying a specific mutation is essential for accurate diagnosis, individualized treatment, and prognosis for the affected males, and also to provide appropriate genetic counselling for their families [34].
Therapeutic interventions
General approaches
The management of boys with DMD requires a multidisciplinary team with focuses on both symptomatic and rehabilitative management. With this approach the mean age of death from DMD has improved from 14.4 years in the 1960’s to 25.3 years in the 1990’s [24, 35].
Comprehensive guidelines published in 2010 and further updated in 2018 have helped standardized the global care of boys and young men with DMD. Corticosteroids are the only current pharmacologic therapy that is effective as a disease-modifying treatment for all patients with DMD. Initial short-term randomized controlled trials demonstrated improved muscle strength and respiratory function. Within 10 days of starting corticosteroids, muscle strength began to improve, with maximal effects seen at 3 months and maintained up to 18 months [36, 37]. Longer term studies demonstrated that corticosteroids prolonged ambulation by a mean of 3 years, reduced cardiopulmonary functional decline, decreased the risk of scoliosis, and improved life expectancy [36, 38]. Current practice guidelines endorse the use of corticosteroid treatment in all DMD patients [17, 39]. Daily oral prednisone (0.75mg/kg/day) or deflazacort (0.9mg/kg/day) are the recommended doses, with intermittent scheduling dosing options available including 10 days on/10 days off, or high dose weekends only [39, 40]. There is little evidence to suggest the optimal timing to begin treatment with corticosteroids or the duration of treatment. The current consensus is to start corticosteroid treatment in the early ambulatory stage or definitively before motor decline [17]. Continued treatment after loss of ambulation has demonstrated benefit in reducing the progression of scoliosis and delaying the decline in cardiopulmonary function [17, 39, 41, 42].
Patients with DMD receiving corticosteroid treatment require close monitoring and surveillance for side effects. Weight gain and a cushingoid appearance are the most common side effects, along with short stature, obesity, and cataracts [43, 44]. Deflazacort has a lower risk of weight gain and is an alternate option to prednisone [36]. Vertebral and long bone fractures occur at a higher rate compared to corticosteroid-naïve boys, with at least one-third of boys developing vertebral compression factures [17, 45]. The fractures are secondary to long-term corticosteroid use, but also related to progressive muscle weakness, impaired vitamin D and calcium absorption, and immobilization. As part of a bone health management plan, regular exercise, a calcium enriched diet, vitamin D supplementation, and a baseline lateral spine X rays with intermittent follow radiographs and bone densities with a dual energy X-ray absorptiometry (DEXA) scan are recommended [19, 46]. Intravenous bisphosphonates are indicated in children with asymptomatic moderate and severe vertebral fractures, and symptomatic vertebral compression fractures [46].
Cardiopulmonary dysfunction also requires dedicated treatment with early use of cardioprotective measures, including angiotensin-converting enzyme (ACE) inhibitors or beta-blockers as they may delay the progression of cardiomyopathy [19, 47]. Eplerenone, an aldosterone antagonist, in addition to ACE inhibitors may attenuate the decline in left ventricular systolic function in boys with DMD with preserved ejection fractions [48].
Treatments have also been standardized for respiratory insufficiency associated with DMD. Respiratory evaluations should occur annually, starting when the patient is 5-6 years, and then biannually in non-ambulatory boys to monitor for early signs of respiratory insufficiency [19, 46]. Overnight pulse oximetry or sleep studies with capnography to detect SDB should be considered, including in ambulatory boys, especially if experiencing weight gain due to glucocorticoid therapy. Sign and symptoms of sleep hypoventilation and sleep disordered breathing include fatigue, dyspnea, morning or continuous headaches, nocturnal awakenings, difficulty concentrating, hypersomnolence, and frequent nightmares, and are an indication for the use of nocturnal non-invasive intermittent positive pressure ventilation (NIPPV) with a back-up rate as a safe and effective treatment [46]. However, some patients remain asymptomatic and thus NIPPV should be initiated if the patient’s forced vital capacity (FVC) is less than 50% predicted or when the absolute value of maximum inspiratory pressure is less than 60 cm H2O [46]. NIPPV increases sleep quality, decreases daytime sleepiness, enhances gas exchange, improves quality of life, delays the onset of daytime hypercapnia, and improves life expectancy [24, 49, 50, 51, 52]. Further progression to daytime ventilation occurs when despite nocturnal NIPPV, symptoms or signs of hypoventilation and hypercarbia persist or when any of the following are true: end-tidal or transcutaneous partial pressure of carbon dioxide in the blood (petCO2 or ptcCO2, respectively) are more than 45 mm H;, arterial, venous, or capillary blood pCO2 is more than 45mm Hg; or the baseline SpO2 is less than 95% on room air [46]. The decision to ventilate patients with DMD via tracheostomy or invasive ventilation is a controversial questions and requires advanced care directives that are discussed with the patients and their families prior to initiating [46, 53]. As a prophylactic measure, both the 23-valent pneumococcal polysaccharide and the annual trivalent inactivated influenza vaccines should be offered in addition to routine childhood immunizations. Acute respiratory infections should be treated with antibiotics, chest physiotherapy, and non-invasive positive pressure ventilation if required [19, 54, 46].
Routine health surveillance and growth monitoring are important in DMD patients. Optimal nutritional status should be targeted, as defined by weight or body mass index between the 10th and 85th percentiles for age, based on national growth charts [19].
Novel therapeutic avenues
Genetic therapies
Dystrophin gene replacement using virus vectors
The large size of the dystrophin gene has made previous attempt to develop a gene therapy difficult. More recent attempts have addressed this problem by deleting selective regions of the DMD gene and creating mini- or micro-dystrophins proteins. A suitable vector such as the adeno-associated virus (AAV) is used to introduce smaller but functional copy of the dystrophin gene to muscle fibres. The benefits of AAV vectors include prolonged expression of the gene, inability to independently replicate without the presence of a helper virus, and the ability to infect both dividing and non-dividing cells [55]. AAV vectors carrying mini/microdystrophin injected into dystrophic canine models of DMD have resulted in improvements in muscle histology [56]. However, in a study of 6 boys with DMD with frameshift mutations that were injected with AAV associated mini-dystrophin gene, transgene expression was not successfully attained; this was attributed to an unexpected primed T-cell-mediated immune response to the mini-dystrophin protein [57]. Further studies looking at recombinant AAV (National clinical trial (NCT) 03368742) and more efficient vector delivery systems to replace defective genes in DMD are currently in progress [58].
Exon Skipping
A promising therapeutic strategy for DMD is exon skipping [59]. By using synthetic antisense oligonucleotide (AON) sequences to “skip” the specified exon(s) in certain dystrophin gene mutations, this strategy aims to change DMD into a milder Becker muscular dystrophy phenotype [60]. Synthesized AONs are transferred into the cell using a series of diverse chemical backbones including peptide nucleic acids, 2’O-methyl-phosphorothioate oligoribonucleotide , and phosphorodiamidate morpholino oligomer [61]. During pre-messenger RNA (pre-mRNA) splicing of the dystrophin gene, specific exons are skipped, causing a restoration of the reading frame leading to partial production of an internally truncated protein. Approximately 83% of DMD patients with deletions can potentially be targeted and improved by exon skipping [62, 63].
Drisapersen, also known as PRO051, a 2’O-methyl-phosphorothioate oligoribonucleotide, skips exon 51 in dystrophin pre-mRNA. In an early Phase 1 clinical trial, 4 boys with DMD treated with a single intramuscular injection into the tibialis anterior showed a partial (17-35%) restoration of dystrophin [64]. In a follow up Phase II study, 12 boys with DMD received weekly dose of 6mg/kg subcutaneous injections of drisapersen. An increase of dystrophin expression, as well as an improvement in the 6-minute walk test (6MWT) was seen after a 3 month open-label extension treatment [65]. In a larger Phase III clinical trial involving 186 boys with DMD treated with 9mg/kg dose, there was no significant improvement in the 6MWT compared to placebo. However, a cohort of 80 boys in the Phase III trial who had a baseline 6MWT of 300-400 meters and the ability to rise off the floor showed a statistically significant improvement in the 6MWT, suggesting that a subgroup of less impaired DMD patients was able to benefit from drisapersen [66]. A Phase II study in younger boys (mean age 7.3 years) treated with weekly 9mg/kg dose of drisapersen continuously for 25 weeks demonstrated an improvement in the 6MWT and increased expression of dystrophin, however, this was not sustained at week 49 [67]. As well, formal muscle strength testing did not differ between participants on continuous or intermittent drisapersen regimens and placebo [67]. Drisapersen was generally well tolerated; side effects including injection site reactions, transient proteinuria, and fever [66, 67, 68] were noted. An open-label extension study (NCT02636686) was underway but stopped due to the company’s decision to stop pursuing exon skipping therapies.
Eteplirsen (AVI-4658) is a phosphoramidate morpholino oligomer designed specifically for exon 51 skipping. This will target approximately 13% of boys with a dystrophin gene mutation [69]. Results from earlier phase II/III studies of eteplirsen have led to its accelerated approval by the United States Food and Drug Administration (FDA) in 2016 [70]. In a double-blind placebo-controlled trial, 12 boys with DMD (age 7-12 years) were treated with either 30mg/kg or 50mg/kg of eteplirsen weekly for 24 weeks, followed by an open-label extension treatment. Encouraging results were found in a subset of patients with significant improvement in walking ability and stabilization in clinical function. Histological evidence of de novo dystrophin production and restoration of the dystrophin glycoprotein complex were found on participants’ muscle biopsies [71]. An open-label, dose escalating study of eteplirsen is currently recruiting patients (NCT03218995); as well, there are other ongoing (NCT02420379, NCT01540409, and NCT02286947) and confirmatory studies (PROMOVI, NCT02255552). Further studies on exons 45 (NCT02530905, NCT02310906, NCT02667483), 51 (NCT03375255), and 53 (NCT01385917, NCT02500381, NCT02740972) skipping are also underway [72].
Limitations of early exon skipping strategies include low efficacy in cardiac muscles, poor cellular uptake, and fast clearance from the circulation system; as such, frequent administrations are required to reach therapeutic benefits [73]. This has prompted further investigations into alternative exon skipping strategies. In order to enhance potency and delivery, these strategies utilize cell penetrating peptides that conjugate to uncharged AONs [74]. Highly active peptides such as phosphorodiamidate oligomer internalizing peptides can induce high levels of dystrophin protein expression after a single low dose intravenous infusion, especially in cardiac muscles of the mdx mouse [75]. As member of a new class of AONs, tricylo-DNA is more effective in restoring dystrophin expression in skeletal muscles, heart, and brain after exon 23 skipping in mdx mice [76]. However, these new exon skipping strategies require further assessment in regard to toxic effects before being tested in humans.
Stop Codon Read-through Agents
Point mutations, which account for approximately 10%-15% of DMD cases, cause premature stop codons within the coding sequence [7, 8, 77]. Premature stop codons are nucleotide triplets within mRNA that signal termination of protein translation by causing the ribosomal subunits to dissociate and release a shortened amino acid chain. This results in the loss of a functional dystrophin protein. Nonsense suppression therapy induces read-through of these premature stop codons via binding to the ribosomes and preventing recognition of the stop signals in the DMD cell lines, allowing for the generation of full-length modified dystrophin protein [78, 79, 80]. Nonsense mutations suppression agents such as gentamicin, an aminoglycoside, induced higher expression of dystrophin in sarcolemma and increased resistance to contraction-induced damage in mdx mice [80]. Human trials, however, have had conflicting results in regard to increased dystrophin production and clinical benefit. While an increase in dystrophin expression and reduced serum CK was found in a 6-month study after treatment with gentamicin, there was no significant improvement in maximum isometric concentration or timed function tests [80, 81]. As well, the undesirable side effects of oto- and nephrotoxicity have limited the human applications of gentamicin [82]. Currently, another aminoglycoside, Arbekacin, is being studied in a phase II trial (NCT01918384).
Ataluren, also known as Translarna, is an oral small molecule read-through agent designed to overcome premature nonsense mutations [83]. In a phase IIa dose-finding open label study that compared pre-treatment and post-treatment muscle histology, ataluren was found to increase dystrophin expression by 11% [84]. In a subsequent double-blind placebo-controlled phase IIb study, 174 boys with DMD were treated with ataluren for 48 weeks. A clinically significant improvement in the mean 6MWT distance of greater than 30m was found among participants treated with low-dose ataluren at 40mg/kg/day compared to placebo. This improvement was not seen in the boys receiving high-dose ataluren at 80mg/kg/day. These results were attributed to ataluren’s bell-shaped dose response curve [85]. The European Medicines Agency has given ataluren conditional approval since August 2014 [86]. The confirmatory trial (NCT02090959) is currently active with results pending. Additional clinical trials of ataluren are ongoing (NCT02819557 and NCT01247207).
Utrophin Modulators
Utrophin is an autosomal analog of dystrophin, with 80% similarity between the proteins [87]. Utrophin is upregulated when there is muscle injury; it is increased in DMD, but not to the extent of being able to compensate for the dystrophic symptoms [88, 89].
Ezutromid (SMT C1100), a 2-aryl benzoaxole utrophin modulator, increases utrophin in mdx mice, and improves muscle strength and decreases muscle fatigue post-exercise. In a phase 1 trial, increased levels of utrophin was found in both skeletal and cardiac muscles after treatment with ezutromid at a dose of 100mg/kg 3 times daily for 11 days [89]. A phase II trial is currently ongoing (NCT02858362) with results from completed clinical trials (NCT0256808 and NCT02383511) pending. Another utrophin modulator, SMT022357 is an oral second-generation utrophin modulator that is in development [60].
Genome Editing Technology
RNA-guided clustered regularly interspaced short palindromic repeats-CAS9 (CRISPR/Cas9) system allow for efficient DMD genome editing in in vitro and in vivo studies [90, 91]. Using AAV vectors, CRISPR/Cas9 has shown functional recovery in mdx mice, as well as reversal of dystrophic changes in both skeletal muscle fibres and cardiomyocytes [69, 92, 93]. Human clinical trials have not yet been performed.
Symptomatic Agents
Anti-oxidants and Anti-inflammatory drugs
Idebenone is a benzoquinone with antioxidant properties that has the potential to delay the onset of respiratory failure in DMD [94]. The phase IIa DELPHI study compared DMD patients (ranging from 8-16 years) treated with 450mg of idebenone daily compared to placebo. There was a significant improvement in the mean and percent predicted peak expiratory flow (PEF) in the idebenone treated group. A post-hoc analysis found that glucocorticosteroid-naïve patients had a greater increase in their PEF than glucocorticosteroid-treated patients, suggesting that idebenone had a greater effect in steroid-naïve patients [95]. The DELOS study, a 52-week trial, examined the effect of idebenone in glucocorticosteroid-naïve patients and found that the PEF remained stable in the idebenone group while the placebo group had a decrease in PEF [96]. Currently, a phase III trial (SIDEROS) is recruiting patients (NCT02814019).
Vamorolone (VBP15) is a 9,11 glucocorticosteroid analogue that may offer benefits similar to corticosteroid without the adverse effects. In mdx mice, vamorolone increased muscle strength without causing immunosuppression or hormone changes [97]. Currently, phase II trials are investigating the safety and efficacy of vamorolone in young boys with DMD (NCT02760264, NCT03038399, and NCT02760277).
In DMD, IB kinase/nuclear factor-kappa B (NF-B ) signaling is persistently elevated in muscle fibres and immune cells. A newer agent, CAT-1004 (Edasalonexent), inhibits activated NF-B. In a Phase 1 safety study in adults, after 2 weeks of edasalaonexent treatment, the NF-B pathway was significant decreased and safely, tolerated with mild side effects including mild diarrhea and headache [98]. Further studies (NCT02439216) are underway. Previously tried potential anti-inflammatory therapies included N-acetylcysteine [99], green tea extract [100], melatonin [101], and pentoxifylline [102] had inclusive results. As well, flavocoxid, a blend of plant-derived flavonoids with anti-inflammatory activity (NCT01335295) is currently being investigated.
Myostatin Inhibitors
Muscle regeneration may be increased with the inhibition of myostatin. Histone deacetylase inhibitors decrease myostatin levels and may be a potential treatment for boys with DMD [103]. In mdx mice, muscle regeneration was increased and muscle fibrosis was reduced with the use of givinostat, a histone deacetylase inhibitor [104]. In a 12-month phase II study of givinostat in patients with DMD, there was a significant increase in muscle fibre area fraction and a significant decrease in total fibrosis, necrosis, and adipose-tissue replacement [103]. A phase III study will be evaluating the efficacy of givinostat in ambulatory DMD patients (NCT02851797), with other studies (NCT03373968 and NCT01761292) underway. Other myostatin inhibitors, including follistatin (NCT01519349), PF-06252616 (NCT02310763 and NCT02907619), and BMS-986089 (NCT02515669) are ongoing.
Granulocyte Colony-Stimulating Factor
The use of granulocyte colony-stimulating factor (GCSF) increases myocytes in mdx mice. GCSF is a cytokine that induces the bone marrow to release stem cells, especially after muscle injury. These stem cells then differentiate into myocytes that can develop into skeletal muscle [105]. A phase I clinical trial of filgastrim, a GCSF analog, is recruiting patients (NCT02814110).
Phosphodiesterase-5-Inhibitors
Muscle ischemia occurs due to a reduction in neuronal nitric oxide synthase, leading to increased oxidative stress and ongoing muscle injury [13]. The use of phosphodiesterases inhibitors to increase blood flow to the muscle has been pursued. Tadalafil, a phosphodiesterase-5 (PDE-5) inhibitor, has shown increased muscle performance in mdx mice [106]. However, clinical trials (NCT01865084 and NCT01070511) in patients with DMD was terminated early due to lack of efficacy.
Preventing muscle damage
As part of a 16-week study, treatment with metformin and L-arginine demonstrated an increase in nitrotyrpsine, a marker of NO production, which improves mitochondrial function and decreases the accumulation of reactive oxygen species, thereby decreasing muscle damage [107]. A phase III clinical study regarding metformin and L-arginine is completed, with results pending (NCT01995032). Creatine with and without glutamine has been investigated as another way to improve mitochondrial function. A 4-month course of creatine monohydrate demonstrated improvement in handgrip strength and increased fat-free muscle mass [108]. Conversely, a 6-month course of creatine monohydrate failed to show an increase in muscle strength [109]. Finally, in mdx mice, heparin therapy for 4 weeks increased levels of utrophin, leading to functional benefits [110].
Anti-fibrotic drugs
Repeated degeneration and regeneration leads to muscle fibrosis in DMD patients. TGF- is elevated in muscular dystrophies which stimulates the formation of fibrosis by blocking activation of satellite cells [111]. Losartan, an angiotensin-II type 1 receptor blocker that reduces the expression of TGF- has been tested in murine models of DMD [111, 1112]. FG-3019, a monoclonal antibody to connective tissue growth factor (CTGF), has also been investigated. CTGF contributes to the development of muscle fibrosis. In mdx mice, less fibrosis and skeletal damage was seen after 2 months treatment with FG-3019 [113]. A phase II trial of FG-3019 is recruiting (NCT02606136). Other anti-fibrotic agents such as targeted microRNAs [114] and HT-100 (halofuginone) [115] and are being investigated, however, the halofuginone trial is currently on hold due to severe adverse events. Another agents such as, Rimeporide, a sodium/proton type 1 exchanger inhibitor is being investigated (NCT 02710591).
Conclusion
DMD is a complex, heterogeneous, degenerative genetic disease that limits the lives of many young men. Given the complexity of the underlying pathobiology, multiple interventions have been investigated to target the different diseases processes. Novel therapies aim to target both the underlying genetics as well as the secondary complications with hopes of slowing the disease progression. Further confirmatory data from human trials are urgently needed to provide more definitive treatment options for DMD.
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