A new drug offers hope for young boys with the progressive neuromuscular disease Duchenne muscular dystrophy (DMD) by potentially offering an alternative to high-dose glucocorticoids that have significant side effects.
Interim results from a 24-month clinical trial at Duke Health and other institutions suggest that the drug, vamorolone, may retain or improve the effects of current steroid treatments but reduces health risks associated with long-term steroid use.
Vamorolone is an anti-inflammatory steroid that differs from all 33 drugs in the corticosteroid class because of a distinct interaction with the body’s glucocorticoid receptors.
Duke’s participation in the study is part of a larger, multi-center global trial.
Published this month in the journal PLOS Medicine, the findings are significant because they offer a potential treatment option for young patients that may reduce the side effects that occur as a result of treatment with high-doses of such steroids as prednisone or deflazacort, while retaining the therapeutic benefit of this class of drugs.
Steroid therapy is currently the only treatment that has been shown to slow the effects of DMD, an irreversible, progressive muscle disease that gradually takes the strength of boys.
“This is potentially great news for these boys who are just beginning the steroid regimen that is our standard-of-care treatment,” says Edward C. Smith, M.D., a neurologist, co-director of the Duke Children’s Neuromuscular Program and a clinical investigator in the trial.
“One of our biggest concerns about high-dose steroid treatment in these patients is the effect on linear growth and bone development,” Smith said.
“So far, based on the interim results from this trial, we may be seeing a much less negative impact on bone health among patients using vamorolone.”
High-dose steroid use halts the development of growth plates in young patients and inhibits the lengthening of bones, Smith says.
Despite the side effects, steroid therapy has been shown to extend patients’ mobility and lives. Patients now frequently live into their 30s, but eventually experience heart and respiratory failure.
A severe type of muscular dystrophy, DMD is caused by a genetic inability to create dystrophin, a protein that protects skeletal and heart muscle from injury caused by normal contraction and relaxation.
The disorder is caused by an X chromosome mutation and affects mostly boys. There is no cure.
Smith says young patients typically present with some degree of delayed motor development and neurocognitive issues. Behavioral development may also be hampered.
“The boys tend to do relatively well until about ages four to six,” he says. “Then weakness becomes more pronounced and eventually impacts their ability to stand and then their ability to walk.”
Following completion of the six-month study, the 46 trial participants were given the option to transition to standard of care using prednisone or deflazacort or continue treatment with vamorolone through enrollment in a two-year long-term extension study. All participants opted to continue treatment with vamorolone.
“We saw statistically significant improvements in the outcome measures in this part of the overall trial in boys treated with the two highest doses of vamorolone for 18 months, with improvements in strength and function,” Smith said.
“These improvements appear to be similar to what is seen on steroid-treated boys, based on data from DMD natural history studies. Additionally, vamorolone appears it may have a much better side effect profile than traditional glucocorticoids, even at the highest doses tested.”
“Although this particular trial was not placebo-controlled, I am encouraged by the safety and efficacy data and look forward to results from the larger placebo-controlled trial (VBP15-004) that is currently underway at Duke and other sites,” Smith said.
Duchenne muscular dystrophy (DMD; OMIM: 310200) is a common X-Linked recessive degenerative neuromuscular disorder , which afflicts males with an incidence of 1 in 3300–5000 live births [2,3].
Patients with DMD begin to show symptoms between the ages of 2–5 years, these include calf muscle hypertrophy, frequent falls, walking on toes, waddling gait, difficulty in climbing stairs, Gower’s sign and progressive muscular degeneration. Patients become non-ambulatory and wheelchair dependent by the age of twelve and most often succumb to cardiopulmonary failure in their early 20s, although treatment with glucocorticoids extends mobility and lifespan .
Mutations in the DMD gene encoding the dystrophin protein are responsible for DMD as well as a milder form of the disease referred to as Becker Muscular dystrophy (BMD; OMIM: 300376). DMD is the largest gene in humans, being 2.4 Mb in size harboring 79 exons.
The full length transcript gives rise to a 427kD protein (3685 amino acids) that is composed of four domains: an amino-terminal actin binding domain (ABD), a central rod domain with spectrin-like repeats, a cysteine-rich domain, and a unique carboxy-terminal domain. DMD mRNA is expressed mainly in the muscles, heart and brain [5,6]
Within the muscle fiber, dystrophin protein associates with the dystrophin associated protein complex (DAPC), a multi-protein complex harboring α- and β-dystrobrevins, dystroglycans, sarcoglycans, sarcospan, syntrophins, and laminins. DAPC links the intracellular actin cytoskeleton to the extracellular matrix, providing structural stability during muscular activity.
Loss of the dystrophin protein as a result of mutations in the DMD gene leads to loss of membrane integrity. Chronic bouts of myofiber degeneration and regeneration then lead to aberrant activation of the NF-κB (nuclear factor kappa B) inflammatory pathway resulting in progressive membrane damage, ultimately leading to muscular degeneration .
Thus far no definitive curative treatment options are available for patients with DMD (Rev in ). The use of glucocorticoid anti-inflammatory drugs such as prednisone or deflazacort to alleviate inflammation, represent the current standard of care. While corticosteroids have been associated with partial improvement in muscle strength, cardiopulmonary function and ambulation, serious side effects, including increased bone fragility, stunting of growth, weight gain, adrenal suppression and delayed puberty are the biggest drawbacks .
More recently, a first in class dissociative steroidal anti-inflammatory drug, vamorolone, showed an improved safety profile compared to prednisone. Additionally, vamorolone demonstrated anti-inflammatory activity in a phase IIa trial, presumably through stabilizing the myofiber membrane leading to improved muscle strength .
This promising drug however awaits further testing in patients and final FDA approval.
The most encouraging therapeutic options available to date thus far involve an exon-skipping approach and a stop codon read through approach. The former enables DMD patients to produce dystrophins such as found in Becker patients, albeit at significantly lower levels, leading to a slowdown in disease progression, while the latter attempts to reestablish the functional integrity of the protein [11,12]. Both these approaches require very precise identification of the mutational status of the DMD gene in DMD/BMD patients.
In the absence of curative treatment options, the early diagnosis of patients with suspected DMD is vital to implementing effective disease management strategies, keeping the quality of life of the patient in mind. In this regard, genetic counseling for patients and their families must be emphasized.
In providing a humane and compassionate platform through which disease ontology, disease progression and its inevitable outcome is disseminated to patients and their families, the importance of genetic counseling should not be underestimated. Genetic counseling also stresses the importance of carrier testing in female relatives of DMD patients , and serves to help families understand and manage the impact of having one or more DMD patients in the family.
In this report we present the first comprehensive genetic analysis of a large cohort of 961clinically suspected, 99% unrelated DMD patients at our center (MDCRC) in Coimbatore in the southern Indian state of Tamil Nadu. We have effectively implemented a region-appropriate, molecular diagnostic algorithm, which sequentially utilizes multiplex PCR, multiplex ligation dependent probe amplification (MLPA) [14,15] and next generation sequencing (NGS) to detect genetic variants in the 79 exons of the DMD gene.
As expected, the majority of the patients had deletion (66%) and duplication (7.5%) mutations in the DMD gene. A striking pattern was observed in DMD patients defying the “reading frame rule,” where patients with in-frame DMD gene mutations demonstrated an enrichment of deletion and duplication mutations in the proximal hotspot region of the DMD gene.
Additionally, we found patients with 114 novel DMD gene deletion, duplication and point mutations, and describe a family with three affected males and two carrier females harboring a rare non-contiguous mutation. NGS analysis further identified patients with small mutations and provided unequivocal clarity in distinguishing DMD versus non-DMD muscular dystrophies.
Our mutational analyses identified a subset of patients who would likely benefit from recently approved FDA and EMA drugs. Genetic analyses also demonstrated that carrier testing, especially in mothers with an older DMD child, coupled with genetic counseling could provide informed family planning options to prevent the propagation of this deadly disease.
We present here the first large comprehensive genetic study on DMD patients from India. Previous studies from India aiming to diagnose DMD at the genetic level have thus far used mPCR and MLPA methods [30–33].
In one study, the DMD gene mutation detection rate was reported to be 62% leaving 36% of clinically suspected DMD patients with no detected mutations , underscoring the need for a comprehensive diagnostic approach. More recently a small study conducted in India focused on NGS as a single platform replacing the need for mPCR and MLPA in diagnosing patients with DMD .
However, larger numbers of patients need to be considered before the NGS platform alone can be confirmed as a single comprehensive platform for the diagnosis of DMD.
In light of this, we implemented a simple, minimally invasive algorithm which includes NGS, particularly suited for developing and low income countries, and used this approach to diagnose DMD or BMD in a cohort of 961 clinically suspected DMD patients. In the process, we have made several significant observations with important repercussions for DMD patients.
Given that an accurate diagnosis of DMD is an essential first step to implementing an effective disease management strategy , we first used multiplex PCR as an inexpensive and easy method to initially identify common hot spot deletion mutations in 30 of the 79 exons in the DMD gene.
Patients lacking such mutations, and those in which the borders of deletion mutations were unclear, were then tested by MLPA, which is designed to identify large deletions and duplications in the DMD gene. While the majority of patients (75%) tested positive for DMD gene mutations by these two methods, 25% of patients remained undiagnosed and hence were tested by the relatively more expensive NGS method, yielding an additional 10% of patients with “small” mutations in the DMD gene.
NGS analysis revealed that C>T transition followed by G>A transitions were the most common point mutations in our cohort, and invariably resulted in termination mutations. While all C>T transitions resulted in termination mutations, all termination mutations did not harbor C>T transitions.
This observation has been previously reported in a study where a C>T transition in a hotspot CpG dinucleotide island region in the DMD gene (C.8713C>T/p.R2905X) was observed . The reason for the large numbers of C>T transitions in DMD associated point mutations remains unclear.
The majority of the remaining 15% of patients who lacked DMD gene mutations were found to harbor mutations in genes associated with other muscular dystrophies (OMDs), particularly Limb Girdle Muscular Dystrophies (LGMDs), or other unrelated disorders such as Charcot Marie Tooth and Nemaline myopathy.
This set of patients would have most likely been misdiagnosed as having DMD, a disease with considerably higher morbidity and mortality compared to other MDs, without the NGS component of our diagnostic algorithm. NGS is fast replacing other methods of genetic diagnosis, particularly since the price tag associated with it has plummeted over the past decade, making it relatively more accessible to patients, especially in developed countries.
Our algorithm is recommended particularly in communities, such as in rural Tamil Nadu, India, where the cost of diagnostic tests could decide whether a patient undergoes the test or not. Thus, using the NGS option as a last step in our algorithm has made DMD diagnosis both accurate and affordable for most patients.
The inclusion of NGS has also circumvented the need for painful muscle biopsies , which were previously required on a routine basis in the event the diagnostic results from mPCR or MLPA tests were found to be negative [32,38].
Ninety eight percent of the 961 clinically suspected DMD patients in our cohort received a definite genetic diagnosis, lending credence to the significance of our diagnostic algorithm. Sixteen patients from our cohort of 961 (2%) were however, found to be devoid of mutations in genes interrogated in our NGS gene panel, despite having some DMD-like clinical manifestations. Further genetic clarity on these patients will likely emerge upon whole exome or whole genome sequencing of their blood or muscle biopsy samples.
The identification of 50 DMD patients with in-frame deletions or duplications in the DMD gene of which 30 (60%) had mutations originating in the proximal hotspot region of the DMD gene was surprising, because a recent study reporting 24 in-frame DMD patients showed a preference for mutations in the distal hotspot region of the DMD gene .
Similar analysis on larger numbers of such patients will help resolve this apparent inconsistency. Our finding that in-frame mutations in DMD patients are associated with a severe phenotype could be explained by the enrichment of mutations in this cohort in the proximal hotspot region, which more than likely results in the abrogation of DMD mRNA expression and loss of the DMD protein.
According to the TREAT-NMD DMD database, approximately 10–15% of DMD patients have a nonsense mutation in the DMD gene , leading to protein truncation and subsequent degradation.
The finding that aminoglycoside antibiotics such as Gentamycin can interact with ribosomes and allow for stop codon read through, resulting in the formation of a full length protein , led to the development of Ataluren (Translarna©) (PTC Therapeutics, USA).
This drug had previously been shown to restore levels of the dystrophin protein in cell lines and in a mouse model of DMD (mdx mice).
However, in the phase 2b randomized, double-blind, placebo controlled clinical trial (PTC124-GD-007-DMD), a small but measurale beneficial effect of ataluren was accepted to be clinically relevant, and provided a much needed ray of hope for DMD patients.
Based on this, conditional approval of Atalauren for use in Europe was granted in 2014 by the European Commission . We identified 35 patients in our cohort of 101 NGS tested patients harboring a stop codon, who might potentially benefit from Ataluren treatment.
Another mutation specific therapeutic option involving exon skipping involves the use of antisense oligomers (AOs) designed to target specific exons in the DMD gene. Success using this approach has led to FDA approval of Exondys51 and Golodirsen (Sarepta, USA), antisense drugs specific for exon 51 and exon 53 skipping respectively, which applies to the largest group of patients .
Similar AOs are also being developed for exon 45. A survey of our DMD cohort also found the largest number of patients (117) amenable to exon 51 skipping. These patients could potentially benefit from exon skipping therapy, which is currently undergoing further refinement in AO chemistry to improve uptake by muscle cells .
More recently, the therapeutic rationale for skipping Exons 45–55 has been proposed by Toshifumi Yokota  and is based on the well documented observation that patients with in-frame deletion mutations in the DMD gene involving exons 45–55 deletions result in mild symptoms or may even be asymptomatic [26,43].
It is presumed that mRNA stability following exon 45–55 deletion likely contributes to this phenomenon. Our data are consistent with this observation, as 12/14 patients diagnosed with DMD having in- frame deletion mutations involving exons 45–55 were ambulant, with two in particular, who despite having onset of symptoms at ages 5 and 10 remained ambulant at ages 27 and 22 respectively.
Our data also show that 14 BMD patients with deleted exons 45–55 had mild symptoms with an average age of onset at 15.2 years and age of diagnosis at 27.6 years. All 14 BMD patients remained ambulant.
We have further identified DMD gene mutations in 138 /316 consenting carrier mothers of DMD probands, demonstrating that 43.7% of DMD cases analyzed in our cohort had familial disease while the rest were sporadic. This percentage is significantly lower than the theoretically derived value of two thirds of carrier mothers being predicted to be carrier positive [28,29]. This discrepancy requires further insight and study in larger cohorts of patients, specifically in the large DMD databases.
Our study has demonstrated that the power of mutational analysis coupled with genetic counseling can provide DMD carrier mothers with the opportunity to make informed family planning decisions to prevent the birth of subsequent DMD positive offspring.
This is of vital importance particularly in rural India where impoverished mothers, often alone, are left to shoulder the burden of care for, in some cases, multiple DMD afflicted sons. In lieu of currently available effective treatment options for patients with DMD, and with the multi-exon skipping approach likely to face regulatory challenges and unlikely to be developed clinically in the near future , informed family planning decisions hold great promise to prevent the propagation of DMD, an invariably deadly disease.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304910/
More information: Edward C. Smith et al, Efficacy and safety of vamorolone in Duchenne muscular dystrophy: An 18-month interim analysis of a non-randomized open-label extension study, PLOS Medicine (2020). DOI: 10.1371/journal.pmed.1003222