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Professor Stanley Froehner tells International Innovation how he came to realise the human impact of his work in repurposing drugs to treat Duchenne muscular dystrophy muscle loss in DMD, and the nitric oxide pathway seemed like a good place to start. Have you encountered any major challenges in the course of your research? How have these been overcome? When did you become interested in researching Duchenne muscular dystrophy (DMD)? In the late 1980s, we were attempting to identify novel proteins at the neuromuscular synapse, using an unbiased monoclonal antibody approach. When the DMD gene was identified in 1987 by Dr Lou Kunkel’s laboratory, we realised that we had antibodies to dystrophin and some of its associated proteins. This was very exciting. Research in our laboratory had already been supported by the Muscular Dystrophy Association (MDA) for several years, and suddenly we found ourselves working on the most devastating of all the muscular dystrophies. What caused you to turn your efforts toward identifying drugs for DMD treatment? In 2001, I was invited by Pat Furlong to attend the annual meeting of Parent Project Muscular Dystrophy (PPMD), to present a lay level talk about our research. Pat had lost two sons to DMD and started PPMD to help other Duchenne families deal with the devastating diagnosis and promote research into this disease. At the conference, attended by several hundred parents of DMD boys, I realised that I had never before met anyone with any connection to DMD. The parents were intensely interested in our research and had many questions. I left the meeting thinking about how we might use our basic science findings to find treatments. We continued our basic research but also began to consider drugs that might slow the relentless Maintaining adequate funding for the laboratory is a problem that all biomedical scientists are facing. We depend more now on private sources and foundations. Also, while collaborations with biotech and pharma are more common, the goals of academic medical research and the commercial world are not always compatible. With the shift in emphasis to translational research at the National Institutes of Health (NIH), applications that propose basic research are severely disadvantaged. This is unfortunate, because with so much not understood about the pathogenic mechanisms that cause DMD, important therapeutic targets may remain unidentified. As in many areas of biomedical research, collaboration among individuals with a broad range of expertise is absolutely necessary in the study of DMD. Our lab brings together individuals with expertise in biochemistry, molecular biology, cardiac and skeletal muscle physiology and animal husbandry, all with the single purpose of understanding the molecular pathogenesis of DMD. Of course, we collaborate widely with our colleagues here at the University of Washington and also at other research institutions, both nationally and internationally. (FDA), even for a different disease, has already been through toxicology and pharmacokinetic studies, an expensive and time-consuming process. Of course, if the drug has not been approved for use in children, then additional studies might be required. Nevertheless, the information available about a drug already in use can be an enormous help in meeting the requirements for a clinical trial and allow human studies to proceed much more rapidly. PROFESSOR STANLEY FROEHNER Teaching old drugs new tricks You are currently a member of the Scientific Advisory Committee for PPMD. What activities do you undertake within this role? As a member of the PPMD Committee, I participate in reviews of grant applications submitted to the foundation and serve on an oversight and advisory committee for a large research project funded by PPMD. Members of the Scientific Advisory Committee also advise PPMD on priorities as the foundation tries to balance the need for funding basic and translational research, clinical trials and high-risk, high-payoff proposals. How does the process of trialling an existing drug for new uses differ from testing a completely novel drug? An existing drug that is already approved for use by the US Food and Drug Administration WWW.INTERNATIONALINNOVATION.COM 79 PROFESSOR STANLEY FROEHNER Halting muscular degeneration Progress in the basic research of a rare muscular dystrophy has not yet translated into effective treatments, but novel insights from scientists at the University of Washington are providing new hope for patients DUCHENNE MUSCULAR DYSTROPHY (DMD) is a severe genetic disorder characterised by progressive muscle degeneration and consequent muscle weakness. As a recessive, X-linked condition, it almost exclusively impacts males – with approximately 200,000 thought to be affected worldwide. DMD starts to show its effects during early childhood. The typical patient has difficulty standing upright from the prone position and struggles to climb stairs, together with a range of more subtle motor deficits. The individual’s motor functions rapidly deteriorate with disease progression and, by the age of only 10 years old, children with DMD are usually wheelchair-bound. Eventually the heart and breathing muscles are compromised, leading to a premature death from heart or respiratory failure. The best available treatments are palliative, or else aim to delay onset and slow progression, as there is currently no cure. The average life expectancy of DMD patients is only 25-30. Despite this dearth of effective treatment strategies, a fair amount is known about the genetic and molecular pathology of DMD. The gene responsible for DMD, dystrophin, was identified over 20 years ago. The dystrophin gene is 2.4 million bases long – the largest found in nature to date – and contains 79 exons that take over 16 hours to be transcribed and spliced. Dystrophin is part of a large complex of proteins linking the subsarcolemmal actin cytoskeleton with 80INTERNATIONAL INNOVATION extracellular membrane proteins. This structure is thought to be important for muscle health, since many dystrophies are the result of mutations in genes that encode members of this complex. One of the leading theories of DMD pathology is known as the ‘membrane fragility hypothesis’. This posits that dystrophin plays a primarily structural role, providing stability to the membrane and protecting it from damage that can be induced during the natural process of muscle contraction. It is thought that in the absence of dystrophin the membrane might be more easily damaged. More recent studies, however, have revealed that this theory is too simplistic and certainly not the full story. EXISTING STRATEGIES Several genetic treatments are being developed for use in DMD. These therapies target the faulty gene specifically, either by correcting the genetic mutation through gene therapy or using a technique known as exon skipping. Gene therapy involves the introduction – usually by viral vector – of a healthy copy of a diseaserelated gene into the cells of a patient, where it can restore healthy function. Despite its promise as an effective treatment for many genetic disorders, gene therapy for DMD is problematic because the dystrophin gene is too big for the development of conventional viral vectors. Methods to develop a smaller equivalent gene, called ‘micro-dystrophin therapy’, have also resulted in compromised dystrophin function and are therefore not fully effective. Exon skipping is another, similar strategy, which is achieved using an antisense oligonucleotide that binds to the defective region of the target gene, forcing the cell to skip this section of the precursor mRNA during translation. This treatment is also a compromise, since it does not provide a fully functional copy of the gene, but nonetheless it could be a large step towards effective treatment of DMD. “Gene therapy and exon skipping may improve the DMD pathology to be more like Becker muscular dystrophy – usually a less severe condition,” explains Professor Stanley Froehner, a DMD expert from the University of Washington. “This would be an enormous accomplishment”. Despite the future promise of genetic therapies, there is a need for alternatives that can be more readily delivered to patients and at a much lower cost. SHIFTING THE PARADIGM In an effort to find an alternative to the widely accepted, yet questionable, membrane fragility theory, Froehner and his team have pursued the idea that the dystrophin complex is intimately linked with a variety of muscle cell signalling mechanisms. He suggests that the dystrophin complex can act as a molecular scaffold, which facilitates the organisation of signalling proteins in the muscle membrane. Froehner’s team has been building substantial INTELLIGENCE REPURPOSING DRUGS FOR DUCHENNE MUSCULAR DYSTROPHY OBJECTIVES • To explore the dystrophin complex as a scaffold that organises signalling proteins on the muscle membrane • To repurpose existing US Food and Drug Administration (FDA)-approved drugs that target these signalling pathways for the treatment of Duchenne muscular dystrophy (DMD) Despite the future promise of genetic therapies, there is a need for alternatives that can be more readily delivered to patients evidence to back this up, having discovered a family of signal transducing adapter proteins called syntrophins, which associate with dystrophin and target several classes of signalling proteins to the sarcolemma (muscle fibre cell membrane). These signalling proteins include ion channels, enzymes, transporters and G protein-coupled receptors – key mediators of many pathways. The alteration or loss of these signalling pathways is likely to have many downstream pathological effects and may therefore play a significant role in the pathology of DMD. If this is true, then it represents an extremely exciting finding for the field. Signalling pathways provide an accessible route for the development of novel treatments. Froehner is now shifting his focus towards the development of treatments for DMD. Drugs that restore these pathways could be rapidly implemented clinically, especially if such treatments arise through the repurposing of existing drugs – something that should be straightforward and inexpensive in the case of generic signalling pathways. “Although not likely to be cures, these drugs could improve quality of life and lengthen survival, thus extending the time for genetic treatments,” Froehner explains. OTHER AVENUES This line of research and drug development has led Froehner to the study of nitric oxide (NO) and its production by the enzyme neuronal nitric oxide synthase (nNOSµ). In healthy muscle, nNOSµ is localised to the muscle membrane through dystrophin and alpha-syntrophin dependent processes, but this is not the case in DMD muscle – a shortcoming that corresponds with reduced production of NO at the muscle cell membrane. NO is involved in many signalling pathways, one of which leads to an increase in the production of cyclic guanosine monophosphate (cGMP) and consequent protein phosphorylation. What is interesting about this pathway is that there are already clinically effective drugs that target it, specifically sildenafil and tadalafil (also known as Viagra and Cialis), both of which inhibit the phosphodiesterase (PDE)-mediated degradation of cGMP. These drugs are used for a variety of ailments, from erectile dysfunction to artery hypertension, and they represent a promising therapeutic strategy that can be readily applied to DMD. PRECLINICAL TRIALS The Washington team has been conducting some preclinical trials of these drugs and found that they improved both respiratory and cardiac function. The two drugs are currently being tested for efficacy in human patients: “So far, two limited trials with sildenafil have not demonstrated skeletal muscle improvement,” reveals Froehner. “However, a clinical trial of Cialis, a more potent PDE inhibitor with higher specificity, is currently recruiting DMD participants aged seven to 14 years.” Reactive oxygen species (ROS)-mediated oxidative stress has also caught Froehner’s attention, since ROS generation is known to occur in DMD skeletal muscle. The team found that a pathological upregulation of NOX2 in DMD leads to excessive ROS production in skeletal muscle of the mdx mouse. Froehner hopes that drugs which reduce oxidative stress, scavenge ROS or inhibit their production through NOX2 may provide the basis for effective treatments in the future. These lines of inquiry are beginning to show promise in terms of DMD treatment, and represent an important paradigm shift in basic DMD research. However, these novel and repurposed treatments may not be enough to tackle all DMD symptoms: “Combinatorial approaches that target distinct pathways altered in DMD offer a very promising approach that deserves more attention from both researchers and clinicians,” explains Froehner. “The limitations of genetic therapies may be improved by co-administration with drugs that target specific signalling pathways.” This may one day provide realistic hope of effective treatment for those afflicted by this otherwise devastating condition. KEY COLLABORATORS Marvin Adams, PhD; Nicholas Whitehead, PhD; Min Jeong Kim, PhD; Kenneth Bible, DVM, University of Washington, Seattle, USA • Kathryn R Wagner, MD, PhD, Kennedy Krieger Institute/Johns Hopkins, USA FUNDING National Institutes of Health (NIH) Raymond and Beverly Sackler Foundation Parent Project Muscular Dystrophy CONTACT Professor Stanley Froehner Raymond and Beverly Sackler Professor and Chair Department of Physiology and Biophysics University of Washington 1705 NE Pacific Street HSB Campus Box 357290 Seattle Washington 98195 USA T +1 206 543 0950 E [email protected] http://depts.washington.edu/dmdlab/ http://linkd.in/1yRK1Vp STANLEY C FROEHNER is the Raymond and Beverly Sackler Professor and Chair of the Department of Physiology and Biophysics at the University of Washington, Seattle. Previous to this he was Professor and Chair of Cell and Molecular Physiology at the University of North Carolina at Chapel Hill and Professor of Biochemistry at Dartmouth Medical School (from 1978-92). He has a PhD in Biochemistry and Neurophysiology from the California Institute of Technology. Froehner has served as a visiting professor in Australia and France and was recognised with an NIH Javits Neuroscience Investigator Award. WWW.INTERNATIONALINNOVATION.COM 81