Research Updates

• Stubborn MG Cases May Improve With IV Cyclophosphamide
• Designer Hormones Could Work Against SBMA
• Gene Chips Used in Studies of SBMA, Inflammatory Myopathies
• New Genetic Factor Implicated in FSH Muscular Dystrophy
• Culprit Gene Identified for Rare Muscular Dystrophy
• Help for Obtaining Daranide, Carnitor
• Vent Users Report Good Quality of Life
• Health Care Access an Obstacle for People With Disabilities
• Advances in Duchenne Muscular Dystrophy

Stubborn MG Cases May Improve With IV Cyclophosphamide

Daniel Drachman

Investigators have found that high-dose treatment with the immune-system suppressant cyclophosphamide (brand names Cytoxan and Neosar) can produce dramatic improvement in people with severe myasthenia gravis (MG) that resists conventional treatment. MG, in which the immune system mistakenly interferes with nerve-to-muscle signal transmission, is an autoimmune disease.

A recent study conducted in Argentina using the same drug in MG on a different schedule (see “Research Updates,” October-November 2002) also yielded positive results.

Although most patients with MG respond well to oral drugs to suppress the immune system and to increase signal transmission, some people either don’t respond adequately or can’t tolerate the treatment-associated side effects.

Neuromuscular disease specialist Daniel Drachman, who co-directs the MDA clinic at Johns Hopkins University in Baltimore, led a team that conducted a pilot study of high-dose, intravenous (by vein) cyclophosphamide in severe, treatment-resistant MG and presented results at a recent meeting of the American Academy of Neurology in Denver. The findings are scheduled to appear in the January issue of the journal Annals of Neurology.

The investigators report that three patients who received only one four-day course of intravenous cyclophosphamide tolerated it well, had marked improvement in their weakness and were able to reduce other immunosuppressive medications. The effects have lasted up to four years.

Drachman and colleagues note that this approach differs from immunosuppressive strategies that destroy the patient’s immune system and then require a bone marrow transplant to provide a new immune system.

“This treatment virtually eliminates the mature immune system but leaves the bone marrow’s stem cells intact,” Drachman said. “These stem cells then ‘reboot’ the immune system, resulting in long-lasting improvement.”

Physicians wishing to inquire about details of the cyclophosphamide regimen can contact Drachman atdandrac@aol.com.

Designer Hormones Could Work Against SBMA

Spinal-bulbar muscular atrophy (SBMA), also known as Kennedy’s

SBMA is caused by defects in the androgen receptor, a protein that controls gene activity inresponse to androgens. Entry into the nucleus is a necessary part of the androgen receptor’s function, but also appears to be a critical event in SBMA.

disease, occurs almost exclusively in men, causing motor neurons (muscle-controlling nerve cells) and muscles to degenerate in middle age. It’s caused by genetic mutations in the androgen receptor, a protein that allows cells to take up testosterone and other masculinizing hormones known as androgens.

Scientists have debated whether androgens or antiandrogens — drugs that block the receptor — might be useful for treating the disease (see “Kennedy’s Disease,” Quest, June-July 2002), but two new studies show that both kinds of drugs exacerbate SBMA in animals. However, they also offer insights into effects of the mutant androgen receptor, which could lead to the development of better drugs for SBMA.

The normal androgen receptor is located in the cell’s main compartment, where it waits for androgens to cross the cell’s outer membrane. Activation of the receptor by androgens sends it into the nucleus — the cell compartment that contains DNA — where it attaches to DNA to turn genes on or off. According to the new studies, both published in the Aug. 29 issue of Neuron, the receptor’s movement into the nucleus is critical in SBMA.

In one study, a team at Nagoya University Graduate School of Medicine in Japan generated mice with the androgen receptor mutations that cause SBMA. Just as in humans, the male mice eventually developed severe weakness but the female mice had only mild signs of the disease. Injections of testosterone gave the females full-blown SBMA, and in the males, castration largely prevented SBMA. The animals that developed SBMA had a buildup of the mutant receptor in their cell nuclei.

In the other study, scientists at the University of Tokyo genetically engineered fruit flies to produce mutant androgen receptors. Since fruit flies don’t have androgens, the mutant receptor alone didn’t cause disease, but feeding the flies testosterone or antiandrogens — which also send the receptor into the nucleus — caused their neurons to degenerate. Engineering the androgen receptor so that it couldn’t enter the nucleus blocked those effects.

Novel androgen-based drugs that attach to the receptor and keep it out of the nucleus could be effective treatments for SBMA, the two research groups conclude.

Gene Chips Used in Studies of SBMA, Inflammatory Myopathies

A team of scientists led by Kenneth Fischbeck, of the National Institutes of Health (NIH) in Bethesda, Md., is exploring how mutations in the androgen receptor gene lead to spinal and bulbar muscular atrophy (SBMA).

Kenneth Fischbeck

Scientists believe that those mutations alter the receptor’s ability to turn genes on and off. In an MDA-funded study, Fischbeck and his group examined those changes using gene chips, devices that can provide a snapshot of the activity of thousands of genes at once (see “Fast-Track Pharmacy,” Quest, August 2001).

By stimulating motor neuron-like cells with androgen, they found that the mutant receptor fails to regulate many of its normal target genes but does regulate other genes it would normally leave alone.

Some of these changes could represent the motor neurons’ attempts to repair themselves, and thus might offer clues to therapy, Fischbeck and his team suggest. Their study was published in the August 2002 issue of Human Molecular Genetics.

In another study, MDA grantees Louis Kunkel and Alan Beggs of Harvard-affiliated Children’s Hospital in Boston were part of a team that used gene chips to study dermatomyositis (DM), polymyositis (PM) and inclusion-body myositis (IBM).

All three diseases are considered inflammatory myopathies, but they differ in important ways. In DM, inflammatory (immune) cells attack the blood vessels surrounding muscles and in PM, the cells appear to attack muscle itself. Although IBM is associated with inflammation, it usually doesn’t respond to immunosuppressant drugs, leading scientists to question whether inflammation has a primary role in the disease.

DM, PM and IBM are usually diagnosed by a muscle biopsy, but even to a well-trained eye, the diseases can be hard to identify.

Kunkel, Beggs and their colleagues measured the activity of over 10,000 genes in muscle biopsies from 45 people with inflammatory myopathies, other neuromuscular diseases or no disease. Their results, published in the Oct. 22 issue of Neurology, show that patterns of gene activity can be used to distinguish the inflammatory myopathies from other diseases and, to some extent, from each other. They also show that many immune-related genes increase their activity in IBM, supporting an inflammatory component to the disease.

In a commentary, MDA grantee Charles Thornton of the University of Rochester (N.Y.) writes that gene chips have “great potential” for diagnosing inflammatory myopathies and studying the mechanisms behind them.

New Genetic Factor Implicated in FSH Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual genetic defect involving an increase in the activity of normally inactive genes — a phenomenon called transcriptional derepression. As if that’s not complicated enough, a study in the October issue of Nature Genetics points to additional genetic mechanisms behind FSHD.

In the late 1990s, scientists found that FSHD is linked to deletions (missing pieces) in a region of chromosome 4 called D4Z4. A recent study by MDA grantee Rossella Tupler showed that D4Z4 normally represses the transcription, or “turning on,” of nearby genes — so large deletions of D4Z4 lead to transcriptional derepression (see “Research Updates,” August-September 2002).

The new study, by MDA grantee Silvere van der Maarel and his colleagues at Leiden University in the Netherlands, centers on the fact that two normal genetic variations occur near D4Z4, known as 4qA and 4qB. Surprisingly, the researchers found that out of 80 FSHD patients, the D4Z4 deletion always occurred in association with the 4qA variant. In the patients’ unaffected parents and in 80 people without FSHD, 4qA and 4qB occurred in almost equal frequencies.

The researchers found additional evidence that D4Z4 deletions in a 4qA chromosome — but not a 4qB one — cause FSHD.

“In light of our findings, either unique characteristics of 4qA must cause or facilitate transcriptional derepression, or those of 4qB must protect against it,” they wrote. They’re now focusing on differences between 4qA and 4qB to gain insights into possible therapies.

Culprit Gene Identified for Rare Muscular Dystrophy

Scientists have found that genetic defects in the muscle protein titin (pronounced “titan”) are behind tibial muscular dystrophy (TMD), which causes weakness concentrated in the tibialis anterior muscle of the lower leg.

Titin, so named for being the largest protein in the body, provides elasticity to muscle fibers and helps maintain the contractile units that span the length of each fiber. Researchers have long suspected the titin gene as the culprit in TMD, but struggled to pinpoint the disease-causing mutations because of the gene’s large size.

An MDA-funded team led by Bjarne Udd at Vasa Central Hospital in Finland identified titin mutations in 13 unrelated families with TMD. Figuring out why most muscles are spared from the effects of titin mutations might hold insights to therapy, they write in the September issue of the American Journal of Human Genetics.

Help for Obtaining Daranide, Carnitor

Dichlorphenamide (brand name Daranide), formerly manufactured by Merck & Co., is used by some patients with periodic paralysis. Merck has discontinued production and sale of this medication, but it can still be obtained through compounding pharmacies, special retail outlets that can make up drugs that aren’t prepackaged.

To locate a compounding pharmacy near you, check your telephone book or contact the International Academy of Compounding Pharmacists (IACP), in Sugar Land, Texas, at (800) 927-4227 or iacpinfo@iacprx.org. The IACP’s directory of compounding pharmacies is available on its Web site, www.iacprx.org.

Carnitine is a natural substance that helps the body use fatty acids to produce energy. Carnitine and other natural compounds help transport fatty acids from the main compartments of cells into the mitochondria, the energy-producing parts of cells.

People with carnitine deficiency and some other metabolic diseases of muscle may need supplemental carnitine. The Carnitor brand of carnitine, manufactured by Sigma-Tau Pharmaceuticals, may be recommended.

The National Organization for Rare Disorders (NORD), located in Danbury, Conn., has an assistance program for those who need Carnitor but are unable to pay for it. Contact NORD at www.rarediseases.org (click on Programs and Services, then Medication Assistance Programs), (800) 999-NORD or mmccourt@rarediseases.org.

Vent Users Report Good Quality of Life

“Breathtaking Metamorphosis” by Erin Brady Worsham reflects a good quality of life for a ventilator user.

A Canadian study sponsored by the Gazette International Networking Institute (GINI) has found that people who depend on mechanical ventilation report they have a good quality of life but that improvements in equipment, services and education are needed to improve it. The study of 26 invasive (tracheostomy) and noninvasive (without tracheostomy) home ventilator users surveyed people living in Edmonton, Alberta, and Toronto, Ontario.

Although the study participants noted that the general public and health care professionals tend to view mechanical ventilation as an intrusive burden, they themselves regarded it as assistive technology not unlike a wheelchair. They saw themselves as generally healthy.

The participants said they needed more flexibility in equipment choices; improved funding and coordination of services; better access to buildings, travel and recreation; better designed equipment to reduce noise and size; more education on mechanical ventilation for the public and for health care professionals; and better and more timely provision of social services. (Canada has a national health insurance program.)

Several participants noted that their need for airway suctioning interfered with independence because they lacked adequate support persons.

The 115-page report is available at www.post-polio.org or from International Ventilator Users Network (IVUN), 4207 Lindell Blvd., Suite 110, St. Louis, MO 63108; gini_intl@msn.com.

Health Care Access an Obstacle for People With Disabilities

A study sponsored by the National Institute on Disability and Rehabilitation Research (NIDRR), a U.S. government agency, has found that at least some people with disabilities report limited access to needed health-related services.

The investigators, who published their findings on the Internet in October, interviewed 30 people with either cerebral palsy, multiple sclerosis or a spinal cord injury, asking them about barriers to their care and the consequences of delayed care or services.

Everyone surveyed reported some problems with care access. The main ones included difficulty with transportation to and from appointments; inaccessible medical offices and equipment; lack of knowledge by professionals about disabilities; delays in the referral process and scheduling of needed services; and limited coverage of equipment and therapy, with costs incurred by deductibles and “co-pays” adding up.

Among the consequences of these access problems were a decline in the primary physical condition of the participant and an increase in secondary complications of that condition; reduced self-esteem, depression and stress; loss of income and missed time from work; and interference with social life and ability to live independently.

The investigators concluded that the following are required to improve the situation:

• increased understanding by health care providers and plans of barriers
to care and the consequences of
delayed or denied services

• more autonomy for people with disabilities regarding their health care

• increased knowledge and skills on the part of health care providers and plans with respect to disability (“disability literacy”)

• increased access to maintenance therapies and durable medical equipment

The full report is available at www.ilru.org/ku-ilru/online/calendar.html; click on the Oct. 2 presentation and then on “text transcript of the Web cast.” You can also obtain it by calling the National Rehabilitation Information Center at (800) 346-2742.

Advances in Duchenne Muscular Dystrophy Progress, Challenges in Gene Therapy for DMD New experiments on mice with Duchenne muscular dystrophy (DMD) show that gene therapy can reverse some symptoms of the disease even in very old mice. Balanced against another recent study, the results offer hope — but raise technical questions — regarding the potential of gene therapy for DMD. In most previous studies, researchers have delivered the gene for dystrophin (the protein missing in DMD) to mice 2 months old or younger. In the new study, a team led by Jeffrey Chamberlain at the University of Washington in Seattle delivered the gene to mice that were a year old — the equivalent of middle age in humans. Each mouse received a single intramuscular injection of a virus carrying the gene. A month later, dystrophin was present in 25 percent to 30 percent of the injected muscle. In DMD, repeated muscle contraction leads to damage and a decline in force production, but in the treated muscles, this contraction-induced injury was corrected by 40 percent. The results, published in the Oct. 1 issue of the Proceedings of the National Academy of Sciences, contrast with those from a recent study by MDA grantee Paula Clemens, who gave similar injections to 8-week-old mice. Clemens found that the injection provoked an immune response, the dystrophin levels declined after two months and the injected muscles continued to degenerate (see “Research Updates,” October-November 2002). Why the differences? For one thing, Chamberlain’s group monitored the mice for a shorter period of time. But they’ve begun to see persistent dystrophin at later time points, he said. He also noted that in his experiments, the mice did have an immune reaction to gene therapy, but it was “a fairly mild one.” Much gene therapy research is focused on developing vectors (gene delivery vehicles) that can slip past the immune system. In their experiments, Chamberlain and Clemens used slightly different versions of an adenoviral vector, a cold-causing virus that’s been “gutted” — stripped of its own genes, so that it’s less likely to trigger the immune system. Their disparate results highlight the need for further investigation into how the vector used, the dose delivered and the time of treatment affect the outcome, Chamberlain said. He predicts that clinical trials of gene therapy for DMD could begin in two years. Calpain Inhibitors, Albuterol Look Promising Researchers have found that a genetic blockade of proteins called calpains can slow muscle wasting in mice with DMD, suggesting drugs that inhibit calpains might be similarly beneficial to boys with the disease. One such drug, albuterol, is already under testing in an MDA-funded trial. Muscle cells contain three types of calpains, calcium-activated proteins that chew up other proteins. Some studies have found that when dystrophic muscle breaks down, calcium leaks into the cells, leading to a surge of calpain activity that might destroy essential proteins. But it wasn’t clear whether the increased calpain activity was a contributing factor in the muscle breakdown or a consequence of it. So, Melissa Spencer of the University of California at Los Angeles and Ronald Mellgren of the Medical College of Ohio in Toledo probed the connection between calpains and DMD by giving mice with the disease extra copies of the gene for calpastatin, a natural inhibitor of calpains 1 and 2. In 4-week-old mice, overproduction of calpastatin dampened the activity of calpains 1 and 2, and led to a corresponding decrease in muscle cell death and inflammation. Calpastatin didn’t repair the leakiness of the muscle cell membrane, showing that it can’t fully compensate for the primary defect in DMD (loss of the membrane protein dystrophin). Still, “these data suggest that inhibition of calpain is a potentially promising treatment for DMD,” Spencer and Mellgren write in the Oct. 15 issue of Human Molecular Genetics. The results bode well for the albuterol trial, which is headed by Spencer. The drug, commonly used to treat asthma, has been shown to increase muscle mass and strength in people without neuromuscular disease, and to increase calpastatin levels in muscle. Systemic IGF1 Strengthens Respiratory Muscles Systemic delivery of the growth-promoting protein IGF1 improves the strength of respiratory muscles in mice with Duchenne MD, according to an MDA-funded study. A previous study showed that genetically engineering the mice to produce extra IGF1 largely prevented the muscle weakness and wasting associated with DMD (see “Research Updates,” April-May 2002). Although the experiment was an encouraging prelude to gene therapy, clinical testing of IGF1-based gene therapy against DMD could take several years. In the new study, Gordon Lynch and colleagues at the University of Melbourne in Australia delivered IGF1 to the mice by a method with a strong clinical basis. The researchers surgically fitted each mouse with a subcutaneous (under the skin) pump that sent IGF1 into its bloodstream. Lynch and his team implanted the pumps around the time that signs of muscle wasting appear in the mice, and after eight weeks, they examined the treatment’s effect on the diaphragm (a muscle that controls breathing). Compared to those from untreated mice, diaphragms from the treated mice had improved contractile force and increased resistance to fatigue. Those results were published in the December issue of the American Journal of Pathology. IGF1, short for insulin-like growth factor 1, is believed to stimulate the activity of muscle-forming cells called satellite cells. Although delivery of the protein wouldn’t correct the underlying genetic defect in DMD, it might stimulate enough muscle repair to slow the disease. New Dosing Schedules May Minimize Prednisone Risks Taking the corticosteroid drug prednisone in high doses twice weekly instead of lower doses every day may allow boys with DMD to tolerate the drug better while keeping its benefits with respect to muscle strength preservation. Prednisone and its close chemical cousin prednisolone have been found to slow the decline of strength in boys with DMD and may even allow for temporary strength gains, but such gains come at the cost of many serious side effects. These include significant weight gain, slowing of growth, red stripelike marks on the skin, bone-density losses and psychological side effects, such as irritability. Now, Anne Connolly in the Department of Neurology and Pediatrics at Washington University in St. Louis and colleagues report that giving prednisone at a dose of 5 milligrams per kilogram (a kilogram is 2.2 pounds) every Friday and Saturday appears to be as effective as giving 0.75 milligrams every day, but with far fewer side effects. The findings are in the December issue of the journal Neuromuscular Disorders. Connolly has MDA support to study the role of the immune system in animal models of DMD and congenital muscular dystrophies. Prednisone may act in part through immunologic mechanisms. The twice-a-week prednisone schedule improved strength over six to 12 months in the majority of the 20 boys who received this treatment. The boys grew normally, and their rate of obesity didn’t differ from that of untreated boys with DMD. However, the prednisone didn’t slow the development of contractures (frozen joints), and it appeared to cause irritability in about a third of the boys on the two days following the high doses. Across the Atlantic, neuromuscular disease specialist Victor Dubowitz at Hammersmith Hospital in London has been studying the use of prednisolone in DMD, using two other schedules. The Dubowitz group has tried a prednisolone schedule of 0.75 milligrams per kilogram taken either for the first 10 days of each month, or taken for 10 days alternating with 10 days off the drug. In an October supplement to Neuromuscular Disorders, Dubowitz and colleagues report on 32 older boys with DMD and four boys under age 5 with the disease who were treated on one of these new schedules. In the older boys, studied for at least 18 months, there was improvement in muscle strength over six months with slow decline after that. Weight gain and other corticosteroid side effects were considerably less than in boys treated with continuous medication. In young boys on the 10 days on and 10 days off schedule, there were no side effects related to the prednisolone. Growth and weight remained normal for age, and gains in muscle function were considerably more striking than in the older boys. The investigators emphasize that larger and longer studies must be conducted before definite conclusions about the risks and benefits of corticosteroids in DMD can be drawn. Both note that the mechanism by which corticosteroids act is still uncertain. New Study Supports Respiratory Training in DMD A French study published in the August issue of Neuromuscular Disorders lends still more support to a growing body of knowledge that suggests special respiratory training exercises, performed at home, can improve breathing endurance in boys with Duchenne MD. Building on previous studies, particularly one conducted in Austria a few years ago (see “Research Updates,” 2000, no. 5), the current study found that such exercises can increase inspiratory muscle endurance after six weeks. Inspiratory muscle strength, however, didn’t increase. Inspiratory muscles are those used to inhale air. The investigators, at the St. Eloi University Hospital Center in Montpellier, France, studied 16 boys with DMD, all of whom were using wheelchairs and had moderate respiratory impairment. Half the group were given special respiratory training that mildly exercised their inspiratory muscles by providing resistance to inhalation, and the other half were given “placebo” training, meaning training with almost no resistance. At the start of the study, both groups were able to keep up the prescribed breathing pattern for an average of about 5 minutes. After six weeks of training, the resistance group could keep up the pattern for an average of 7 minutes, 30 seconds, while there was no increase in the endurance in the placebo training group. The researchers concluded that “this simple method of home training may be used as an effective adjunct to other therapies in the future.”

MORE MDA RESEARCH NEWS

For up-to-the-minute news on MDA research developments, visit MDA’s Web site at www.mda.org. Click on “Research” for information on recent research developments and active clinical trials, and links to major medical/research sites. Look at the Web site’s “What’s New” section for news bulletins about breaking research announcements.

For research news about amyotrophic lateral sclerosis, see The MDA/ALS Newsletter or go to www.www.als-mda.org.

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