Botulinum toxin, a neurotoxin made by the bacterium Clostridium botulinum, inhibits neuronal signaling by blocking neurotransmitter release, leading to paralysis. Researchers from University College London (UCL), UK; the University of Sheffield, UK; and the Hospital for Sick Children, Toronto, Canada, have now used a “protein stapling” technique to develop two modified botulinum molecules that relieve pain, but without the toxicity characteristic of the unaltered toxin.
The group shows that a single injection of the new botulinum constructs ease pain in mouse models of neuropathic and inflammatory pain by targeting substance P (SP)-expressing neurons, or mu opioid receptor-expressing cells, in the spinal cord. The botulinum molecules did not kill the neurons, offering a significant advantage over other attempts to alleviate pain using SP attached to saporin, a toxin that does result in cell death. If the results translate to people, the new approach could provide an alternative to opioids and other analgesics in patients with chronic pain.
Tony Yaksh, University of California, San Diego, US, who has investigated botulinum’s potential to target nociceptive processing but was not involved with the new study, calls the work a “truly exciting advance.”
“The spinal delivery of this modified toxin allows for the selective regulation of spinal processing initiated by cells expressing receptors commonly found on small afferents, and on second-order neurons, that carry nociceptive information to higher brain centers,” he says. “This ability to produce a long-lasting but reversible block of transmission in the pain pathway after a single treatment could be a major step in reducing the persistent need for medications like opiates, which can have quite adverse side effects.”
The research was published July 18, 2018, in Science Translational Medicine.
Protein stapling: a form of LEGO assembly
Even a small amount of botulinum toxin can result in paralysis, and even death, as it disrupts communication between neurons and muscle cells. Several studies have suggested it might also dampen nociceptive signaling, but botulinum’s toxicity made it difficult for researchers to fully investigate its potential as an analgesic.
Bazbek Davletov, co-senior author at the University of Sheffield, originally became interested in botulinum toxin as a molecular tool to understand nerve-muscle signaling at the neuromuscular junction. The toxin contains a light chain zinc endopeptidase and a heavy chain polypeptide. The heavy chain allows the toxin to bind neuronal receptors, and then helps translocate the light chain across the endosomal membrane. The light chain can then disrupt neuronal communication by cleaving SNAP25, a protein that governs synaptic transmission. (It is possible to monitor botulinum activity by immunochemistry for this cleaved SNAP25 [cSNAP25]).
“Botulinum toxin is a very dangerous substance—one of the most toxic toxins known to man,” he says. “But it could have some utility in alleviating pain if you can create a functional molecule that is not paralytic.”
Previously, using a technique called “protein stapling,” Davletov and co-senior author Stephen Hunt, UCL, reengineered the toxin so that they could attach the light chain/translocation domain to specific proteins. For one of these molecules, called SP-BOT, the researchers attached substance P (SP) to the translocation domain, allowing them to target spinal neurons expressing the neurokinin-1 receptor (NK1R), to which substance P binds. For the second modified molecule, called DERM-BOT, the researchers conjugated botulinum toxin to dermorphin (DERM), a potent mu-opioid receptor (MOR) agonist, allowing for targeting of MOR-expressing cells.
The investigators had found that, in vitro, these novel botulinum substances could cleave SNAP25 and silence neuronal cells (Arsenault et al., 2013). The results were striking enough to inspire them to test the molecules in animal models of pain.
“It’s a molecular LEGO system, in a sense. And we saw, in cultures of neurons, that it was quite effective in silencing neuronal activity,” says Davletov. “It offers real utility at the molecular level when you can take this ‘mix and match’ approach to making new botulinum molecules to target specific cells. With so few options to treat chronic pain, we thought this method could offer a new solution.”
A long-term reduction in pain
For the new study, to see whether SP-BOT and DERM-BOT could relieve pain in behaving animals, Davletov, Hunt, first author Maria Maiarù, also of UCL, and colleagues tested the modified botulinum molecules in mouse models of neuropathic and inflammatory pain.
In the first set of experiments, the researchers injected SP-BOT or saline into the spine of both wild-type and NK1R knockout mice that had also received complete Freund’s adjuvant (CFA) in the ankle or hind paw. They also injected SP-BOT into mice with spared nerve injury (SNI), a model of neuropathic pain.
Using von Frey filaments to assess mechanical hypersensitivity, and the rotarod apparatus to examine locomotor activity, the researchers discovered that the wild-type mice started to experience substantial pain relief from SP-BOT about three days after the injection—and, what’s more, this lasted for weeks. In contrast, the NK1R knockout- mice showed no such relief, suggesting that the analgesic effects of SP-BOT were mediated by NK1R receptors. The animals also showed no signs of motor impairment.
The researchers next used double-fluorescent immunochemistry in tissue from CFA-treated mice to determine the activity pattern of SP-BOT. Ninety-six hours after injection of the botulinum construct, they saw botulinum activity in cell bodies, and axonal and dendritic branches, of NK1R-positive cells in the spinal cord, using antibodies against cSNAP25 and NK1R.
They then looked at the parabrachial nucleus of the hindbrain, a region to which the NK1R spinal neurons project, and observed cSNAP25 there, which they attributed to axonal transport of cSNAP25 and/or botulinum protease. Furthermore, the investigators saw decreased expression of c-Fos, an indicator of neuronal activity, in the parabrachial nucleus.
Notably, Yaksh says, the investigators observed no cell death in the dorsal horn of the SP-BOT-treated mice.
“The issue in the past with saporin-substance P is that once it is taken up by the NK1R cells, it blocks ribosomal function, so these cells die. And while that will produce irreversible changes in pain processing, that cell loss has been a concern,” he says.
A second round of experiments showed similar findings after injection of DERM-BOT. Here, too, DERM-BOT alleviated pain in models of inflammatory and neuropathic pain without causing cell death. And, upon comparing DERM-BOT to morphine in the SNI model, the group found it had just as strong of an analgesic effect as the classic opioid. Immunohistochemistry showed that DERM-BOT specifically silenced the activity of MOR-expressing cells in the spinal cord.
“These new botulinum molecules bind to spinal neurons without toxicity,” says Davletov. “We removed the toxicity by targeting the botulinum molecules specifically to those neurons instead of muscles. And we saw that we could block chronic pain and continue to block it for months at a time.”
Moving forward
Davletov and colleagues plan to follow up their study by testing SP-BOT and DERM-BOT in companion dogs suffering from age-related chronic pain. Yaksh is also interested to see whether the modified botulinum toxin molecules will have similar analgesic effects in other animals. If so, this could offer the possibility that chronic pain patients could use lower doses of opioids, or not need them.
“Even if this is something that just reduces the opioid requirement, it would be quite significant,” Yaksh says. And, “it could be a very significant therapeutic alternative for patients who suffer from chronic pain without all of the issues of tolerance and addiction.”
Davletov agrees. “Remodeling botulinum neurotoxin gives us hope for new and more effective pain treatments,” he says. “With rigorous studies in the future, I’m hopeful that we will see this translate to patients and become something that will help clinicians control not only chronic pain but also other chronic neurological conditions.”
Kayt Sukel is a freelance writer based outside Houston, Texas.
Image: Crystal structure of botulinum neurotoxin type A. Credit: Protein Data Bank (PDB) 3BTA. Lacy et al., 1998. Nat Struct Mol Biol. 5: 898-902.
