An MDA-supported clinical trial of intravenous gentamicin in children and adolescents with a form of Duchenne muscular dystrophy (DMD) caused by so-called "nonsense" mutations (also called "premature stop codon" mutations) found the drug was safe and that levels of the muscle protein dystrophin increased in some, but not all, participants who received the drug for six months.
The researchers saw biochemical indications that average muscle destruction may have slowed in the trial participants, but there were no significant changes in tests comparing muscle strength and function before and after treatment.
Experiments in mice and shorter trials conducted in humans showed that gentamicin can increase muscle levels of dystrophin. Lack of dystrophin is the underlying cause of DMD.
The researchers speculate that an unwanted immune response against the newly synthesized dystrophin protein may have thwarted the drug's effects, at least in some of the trial participants.
"Further work will be needed to clarify this point," said MDA grantee Jerry Mendell, who co-directs the MDA clinic at Nationwide Children's Hospital in Columbus, Ohio, and who coordinated the study team. "The immune response may be something we can overcome, now that we recognize it."
About DMD and BMD mechanisms
DMD is caused by any of a large number of mutations in the X-chromosome gene for the dystrophin protein that result in virtually no production of functional dystrophin and progressive degeneration of skeletal and cardiac muscles. The closely related disease, Becker muscular dystrophy (BMD), is caused by mutations in the dystrophin gene that allow for some functional dystrophin to be produced but not enough to sustain normal muscle function.
Approximately 15 percent of the mutations that lead to DMD or BMD are known as "nonsense," or "premature stop codon," mutations. These genetic flaws cause cells to cease synthesis of a protein before it is complete and functional, even if all the genetic instructions for making a complete protein are present.
About gentamicin and MD
Gentamicin is an aminoglycoside antibiotic, normally used for fighting infections. Several years ago, it was found that gentamicin is among the compounds that can cause "stop codon read-through," allowing cells to ignore (read through) a stop codon and synthesize a complete and functional protein.
In 1999, MDA-supported studies showed that mice missing dystrophin because of a stop codon began producing the protein after treatment with gentamicin. (See Antibiotic Shows Promise as Systemic Treatment for Muscular Dystrophy.) In these mice, the investigators saw some protection against contraction-induced muscle injury and a reduction of serum creatine kinase (CK), an enzyme that leaks out of damaged muscle cells. A reduction in serum CK is generally considered an indication that muscle destruction has slowed.
Investigators began to plan studies in humans, but they had to exercise extreme care, because long-term or high-dose gentamicin can cause kidney damage, hearing loss and impairment of balance. (A blood test before gentamicin treatment can reduce the incidence of side effects, Mendell said.)
Starting about 2000, investigators cautiously infused the drug in two small, two-week studies in people with DMD, BMD and limb-girdle muscular dystrophy (LGMD), announcing results in 2001. The results of both small studies were confusing: There was no evidence that dystrophin or the targeted LGMD protein increased, but both studies saw marked decreases in blood CK levels, indicating possible slowing of muscle damage. (See "Gentamicin Studies Yield Confusing Results," in Research Updates, Quest, June 2001.)
An Italian study of the drug in DMD patients, for which results were announced in 2003, found three out of four participants began producing dystrophin after receiving gentamicin for two six-day periods seven weeks apart.
These early results prompted Mendell and others to continue studying gentamicin as a possible therapy for DMD or BMD. They speculated that a longer course of treatment, accompanied by careful monitoring for drug toxicity, could result in significant dystrophin production.
About the new findings
Mendell, with colleagues at Nationwide and several other institutions in the United States and Canada, published the the new trial results online March 15, 2010, in Annals of Neurology.
The investigators divided the DMD patients into four groups: Group 1 consisted of 10 boys with DMD, all of whom had premature stop codon mutations in the dystrophin gene. These boys received two weeks of intravenous gentamicin. Group 2 contained eight boys with DMD, all of whom had mutations in the dystrophin gene that were not premature stop codons. They received the same treatment as group 1.
In group 3, there were 12 boys with DMD because of stop codons who received weekly infusions of gentamicin for six months. Group 4 contained four boys with DMD due to stop codons who received twice-weekly gentamicin infusions for six months.
In the two-week study, serum CK dropped by 50 percent in group 1, the group with the premature stop codon mutations, and did not change in group 2, the group with different mutations. This result indicated to the researchers that gentamicin was probably targeting the stop codon mutations specifically and that the drop in CK might indicate a slowing of muscle destruction in the stop codon patients.
In the six-month study, the investigators again saw a drop in CK levels in groups 3 and 4 of an average of about 50 percent.
The only toxic effect of gentamicin they observed was a transient diminution in hearing in one participant, who was removed from the trial and recovered his hearing after ceasing treatment. It was determined that he had mistakenly received more than the expected dose of intravenous gentamicin.
No tests of muscle strength or function showed any change that was considered statistically significant. However, average muscle strength scores, expected to decline by 0.2 units over six months, showed little or no decline in the treated boys. In addition, maximum voluntary muscle contraction scores showed slight improvement, and respiratory capacity increased slightly. These can be considered hints of possible effectiveness of gentamicin if it were given at a higher dose.
Perhaps the most intriguing results concerned dystrophin production. When results for groups 3 and 4 were combined, the overall average increase in dystrophin protein levels measured in pre- and post-treatment muscle biopsy samples was significant. However, there were marked individual differences among the 12 participants.
There was no increase at all in muscle dystrophin protein in six of the 12 boys in groups 3 and 4 after six months of weekly or twice-weekly infusions of gentamicin. Three of the 12, however, showed large dystrophin increases, while the remaining three showed smaller increases.
Complex analyses of the biopsy samples led researchers to conclude that some trial participants may have mounted an immune response against the parts of the newly synthesized dystrophin protein molecules that their muscles had not previously produced, while others apparently did not.
The researchers interpreted these results to mean that gentamicin can increase dystrophin protein production in some patients with DMD but that, in future studies, investigators must be aware of the potential for an immune response to interfere with increases in dystrophin levels.
They also concluded that, while the drug can be safely administered at the current doses, higher doses will likely be required to produce clinically meaningful results.
"All such studies must have close supervision to provide the proper safety environment for boys with DMD," Mendell said. "Only under these closely supervised conditions can it be determined if raising the dose will be effective and whether safety can be maintained with long-term treatment."
Meaning for people with DMD and BMD
The data from this latest gentamicin trial show that this stop codon read-through drug can, in some cases, increase dystrophin production, but still more dystrophin will be needed to improve muscle function.
Problems with further increases in gentamicin dosing include: 1) the drug may be toxic in high doses or when given for prolonged periods of time; 2) it has to be given intravenously, which is inconvenient and expensive; 3) only some dystrophin-deficient patients respond to it; and 4) researchers don't yet know what all the factors are that determine this response.
Recently, the oral drug ataluren (PTC124), an experimental stop codon read-through compound, failed to increase the distance people with DMD or BMD could walk in six minutes (see Ataluren Results Disappointing). Detailed analyses of those trial results are not yet available.
It's plausible (although not yet established) that an immune response may be responsible for the failure of gentamicin — and perhaps other stop codon read-through drugs — to sustain enhanced levels of dystrophin and therefore to improve function in DMD and BMD. If this proves to be the case, it may be possible to manipulate the immune system to overcome this barrier to treatment.