ARTICLE IN BRIEF
In an experiment in rats and mice, botulinum neurotoxin A (BoNT, Botox) that was injected into whisker muscles of rats and mice ended up in remote areas of the CNS.
Italian investigators injected botulinum neurotoxin A (BoNT, Botox) into whisker muscles of rats and mice, as well as into various brain regions; they found that the toxin ended up in remote areas of the CNS.
While Matteo Caleo, PhD, of Italy's National Research Council and his colleagues said that this “novel pathway of BoNT-A trafficking in neurons” has “important implications for the clinical uses of this neurotoxin,” scientists and physicians who have worked with botulinum neurotoxins for decades say that the doses were much too high in the animals to extrapolate to humans and that the long-term clinical use of these medicines has yet to reveal similar problems.
“There is no evidence from long-term clinical use that BoNT injected into peripheral muscles, skin, or other tissues causes any central clinically detectable effects,” said Joseph Jankovic, MD, professor of neurology and director of the Parkinson's Disease Center and Movement Disorders Clinic at Baylor College of Medicine in Texas.
The study, reported in the April 2 Journal of Neuroscience, made national headlines because of the popularity of the toxins used to treat facial wrinkles and movement disorders.
STUDY METHODS, RESULTS
Dr. Jankovic said that the dose injected into the rat's whisker pad was about 150-fold higher than the dose typically used for facial dystonia. The Italian scientists also injected the neurotoxin into the hippocampus and the superior colliculus, the brain region that receives signals from the eye.
Three days after the injections, they found evidence of long-distance retrograde effects of the toxin. From the whisker muscle the toxin made its way to the brain stem; it crossed from one side of the brain to the other in the hippocampus and some toxin seemed to have moved from the superior colliculus to the eye.
They also reported a disruption of brain cell activity locally and in some of the distant regions identified that showed footprints of the toxin.
Dr. Jankovic, who conducted the first double blind, placebo-controlled study of botulinum neurotoxin A in the early 1980s, said that he has treated thousands of patients with BoNT and has never observed side effects related to adverse activity of BoNT in the brain or spinal cord. He added that “no such CNS effects have been described, even in patients injected therapeutically or inadvertently with very high doses of BoNT.”
Dr. Caleo said that they found “a fraction of the injected BoNT-A that does not stay confined to the injection site but migrates along axons by retrograde transport.” He explained that after transport, the active toxin was transferred to other synapses, resulting in long distance actions. “It was widely assumed that BoNT-A remains at the synaptic terminal and its effects are confined to the injection site. There was evidence of a cleaved protein (SNAP-25) that blocked hippocampal activity in the untreated hemisphere.” They used antibodies against the cleaved SNAP-25 to prove the retrograde spread of the toxin.
Dr. Jankovic and other scientists said that this antibody has not been well characterized and, therefore, the experiments have to be replicated.
In one experiment, investigators injected BoNT-A into rat facial muscles. “This resulted in the appearance of BoNT-A activity (cleavage of the BoNT-A substrate SNAP-25) into the brainstem region containing the motoneurons that innervate the infusion site,” Dr. Caleo said.
“In my view, it is premature at this stage to draw conclusions about the possible clinical implications of this finding. It will depend on the type of CNS synapses that are affected, and this requires more investigation,” he said. “One might imagine that these central actions potentiate the peripheral effect of BoNT-A,” and this may further silence motoneuron activity by blocking some excitatory input, he added. “It is also possible that they result in unwanted side effects. We definitely need to gain more information about this retrograde trafficking.”
Dr. Jankovic said that BoNT is safe when used appropriately and by trained physicians. First approved by the FDA in 1989 for blepharospasm, further studies on strabismus, facial spasm, cervical dystonia, hyperhidrosis, and cosmetic indications subsequently led to the federal green light. A list of reported side effects for each of these conditions is provided on the FDA Web site and on the labeling.
But Dr. Jankovic said that toxic spread into the brain would have resulted in cognitive, corticospinal, and other CNS problems that have not been documented in any of the millions of patients treated with BoNT worldwide and thousands of articles published on the clinical use.
CONCERNS ABOUT DOSAGE AT INJECTION SITE
Still, scientists agree that the concentration and the dosage at the injection site could influence the transport noted in the Italian study. “Side effects from botulinum neurotoxin could be prevented by blocking the toxin's transport into the tips of nerve cells,” said David Sherry, PhD, associate professor of cell biology and associate of the Center for Neuroscience at the University of Oklahoma Health Sciences Center. He called for more studies to characterize the mechanisms responsible for transporting the toxin across the brain.
The study was funded by the Telethon Foundation and the Investment Fund for Basic Research in Italy. •