Losartan, an FDA-approved treatment for hypertension in children, delays fibrosis and improves strength in a mouse model of Duchenne muscular dystrophy (DMD), according to a Jan. 21 study published in advance of print in Nature Medicine. These results pave the way for a quick start to a trial of losartan in human DMD, even while questions remain in the minds of some researchers about results in the mouse.
CLUES FROM MARFAN SYNDROME
The study grew out of work with losartan in Marfan syndrome, a common disorder caused by loss of fibrillin-1, an extracellular muscle matrix protein that, along with its role in forming the elastic fibers found in connective tissue, suppresses activation and signaling of the cytokine transforming growth factor-beta (TGF-beta). TGF-beta signaling reduces myocyte differentiation and increases fibrosis in response to injury.
While best known clinically for its effect on bone growth and cardiac complications, “The vast majority of people with Marfan syndrome have some skeletal muscle abnormality, including weak or small muscles,” according to Harry C. Dietz, MD, professor of medicine and genetics at Johns Hopkins School of Medicine. “With better care, these patients are living longer, and many develop a clinical myopathy later in life,” including failure of muscle regeneration and increased fibrosis, which is also typical of muscle in DMD. “A major similarity of both is failure of muscle regeneration,” he added.
In the current study, Dr. Dietz showed that blocking TGF-beta activity with an antibody could reverse the myopathic changes in fibrillin-negative mice. The same rescue was effected by treatment with losartan, whose known anti-TGF-beta activity is believed to be independent of its antihypertensive effect. Losartan restored normal muscle architecture in both acute and chronic injury models. Clinical trials of losartan in Marfan syndrome are now underway.
TGF-BETA IN DUCHENNE DYSTROPHY
“When we delved more deeply into Marfan syndrome, we saw there were normal numbers of satellite cells — [cells responsible for muscle regeneration] — but they had lost the ability to respond to proliferation signals,” Dr. Dietz said. “This told us something about suppression of muscle regeneration, and we asked whether this might be relevant to Duchenne dystrophy. Young children with Duchenne dystrophy have active muscle regeneration despite the ongoing muscle breakdown, and only after exhaustion of regeneration capacity do they develop serious clinical myopathy.” This led his group to ask whether TGF-beta might be involved in DMD muscle pathology, and whether losartan might offer a way to ameliorate the disease.
TGF-beta is increased in muscle in the standard mouse model of DMD, the mdx mouse, in which dystrophin is absent, serum creatine kinase levels are high, and inheritance is X-linked, as found in the human disease. Dr. Dietz's group first showed that mdx muscle displays the signs of TGF-beta signaling, including activation of the same downstream molecules they had seen in the Marfan syndrome mouse. Next they shut down TGF-beta signaling, again with either antibodies or losartan. Both treatments improved regeneration in the mdx mouse, accompanied by lack of activation of the downstream molecules that carry TGF-beta's signal to the nucleus.
After six to nine months of losartan treatment, beginning when the mice were six weeks old, fibrosis in different muscle groups had slowed significantly compared to untreated animals. In the diaphragm, the most affected muscle in the mouse, fibrosis occupied 18 percent of the cross-sectional area in treated mice, compared with 32 percent in untreated mice, a significant difference (p< 0.03).
The improvement was not due to restoration of the dystrophin complex, the molecular defect in DMD. Rather, Dr. Dietz said, it was a function of improved regeneration. This may have clinical relevance, since in DMD, “functional decline correlates with loss of regenerative capacity and progressive fibrotic changes.”
QUESTIONS ABOUT MUSCLE MASS
To demonstrate a functional benefit, the researchers assessed both muscle mass and muscle strength. It is here that some outside experts have raised questions about the results. These results showed that the mass of a representative muscle from one-year-old untreated mdx mice was significantly less than the non-mdx control, and that treatment normalized the muscle weight. Similarly, untreated mdx muscle was weaker than control muscle, but was normalized by treatment.
Two long-time DMD researchers, Terry Partridge, PhD, and Lee Sweeney, PhD, both of whom have extensive experience with the mdx mouse, expressed concern over the muscle masses presented in the supplemental data that accompanied the article. Dr. Partridge is a researcher at the Center for Genetic Medicine at Children's National Medical Center in Washington, DC. Dr. Sweeney is professor and chair of physiology at the University of Pennsylvania School of Medicine.
In this study, the weight of explanted, untreated mdx muscle was only 80 percent of the control muscle weight, even after correcting for differences in total mass between the mice. Yet, according to Dr. Sweeney, “mdx muscle is normally slightly larger than control. There is a compensatory hypertrophy in younger animals,” including mice of the age in this study.
“In this study, it's the other way around. Also, the mass of different muscles was much larger than we've ever seen in mdx muscles.” Dr. Partridge concurred: “mdx mice do not lack muscle; they have bigger muscles than normal. It's not so efficient, and so the strength is usually about the same.”
First author Ronald Cohn, MD, assistant professor of pediatrics and neurology at the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University, responded: “It is a universally accepted finding that mdx mice show a hypertrophic response of skeletal muscle within the first few months of life. In contrast, observations of a sustained hypertrophy of mdx muscles beyond six months of age are controversial; some groups have found decreases in muscle mass beyond one year of age, while others have not.”
Dr. Partridge also noted that lack of muscle regeneration is not a characteristic of the mdx mouse. “We've looked at this for years, and it's not something you see in mice at this age.”
Dr. Cohn replied, “It is true that in vitro analyses of satellite cells from mdx mice have not shown evidence of decreased proliferation or differentiation. However, these experiments are performed under non-physiological conditions. Assessment of muscle regeneration in older mdx mice has shown significant impairment of the skeletal muscle repair process.”
Ultimately, the controversy about these issues will be settled when other labs either replicate these findings, or find they cannot. In his own work with losartan, Dr. Sweeney's group has seen a beneficial effect on the diaphragm, but no effect on regeneration or muscle strength.
CLINICAL TRIALS PLANNED
Despite these concerns, Dr. Sweeney thinks losartan is worth investigating further. “This study does present good evidence that losartan slowed fibrosis in the diaphragm,” and fibrosis does eventually block regeneration. “If losartan's effect on diaphragm fibrosis carries over into DMD, that could be beneficial.”
Because losartan already has FDA approval, clinical trials are unlikely to wait for complete replication. Indeed, the Muscular Dystrophy Association is already beginning to plan a trial, according to Sharon Hesterlee, PhD, vice president for translational research.
“We haven't worked out the details yet, but it shouldn't be a major undertaking to plan such a trial,” she said. Losartan, marketed as Cozaar, (Merck), is FDA-approved for treatment of hypertension in children as young as age six, and it has been extensively studied in younger children with chronic renal disease.
One important decision for such a trial will be the enrollment ages for boys with DMD. “It would be best to get them as young as possible,” Dr. Hesterlee said, “but we don't have the natural history data for very young boys. The best data we have are for those between ages eight and fourteen, where we see lots of functional loss.”
As for the questions raised about the mdx results, Dr. Hesterlee said. “It would be nice to see another study from another lab.”
The length of a trial is also a critical question, and one that hinges on the primary outcome. According to Dr. Sweeney, a clinical trial based on delaying fibrosis alone might have to be much longer than one that expected to see improved strength. “That's why this issue is so important,” he said.
Dr. Dietz has been in consultations about a DMD trial, even as the trial in children with Marfan syndrome is proceeding. “We'll be watching that trial closely to see if there are unanticipated side effects,” he said. He said he knows many patients who have found a physician willing to prescribe losartan, and are starting the drug outside of the trial.
“But we really don't know what the side effects are going to be in this population. The most responsible approach is in a large trial, and we should have answers fairly quickly.” Everyone concerned with Duchenne muscular dystrophy will eagerly await those answers, he added.
ARTICLE IN BRIEF
Losartan restored normal muscle architecture in both acute and chronic injury mouse models of Duchenne muscular dystrophy.