An altered form of botulinum toxin provides long-lasting pain relief in mice without adverse effects and, in time, could replace opioid drugs as a safe and effective way of treating chronic pain, new research has found.
A team from University College London, the University of Sheffield and the Hospital for Sick Children, Toronto, deconstructed the botulinum molecule and reassembled it with an opioid called dermorphin to make Derm-BOT — a compound which successfully targets and silences pain signals from neurons in the spinal cords of mice.
“Injected into the spine, Derm-BOT relieves chronic pain—such as that caused by nerve damage—and avoids the adverse events of tolerance and addiction often associated with repeated opioid drug use. It doesn’t affect muscles like the botulinum toxin used to reduce wrinkles but it does block nerve pain for up to four months without affecting normal pain responses. It really could revolutionise how chronic pain is treated if we can translate it into clinic, removing the need for daily opioid intake,"
said co-corresponding author, Professor Steve Hunt, of UCL’s Cell & Developmental Biology department.
Key neurons in the spinal cord are targets for pain management as they directly ‘sense’ pain and send this information to the brain. Opioids like morphine and fentanyl are considered to be the gold standard for pain relief but there is little evidence that their long-term use is effective in treating chronic pain.
This is because the body builds up a tolerance to repeated drug use which over the long term. Paradoxically opioids can also increase the body’s sensitivity to pain.
[caption id=“attachment_96467” align=“aligncenter” width=“680”] Neurons expressing NK1R, which convey pain to the brain, took up the injected botulinum (bright green) without toxicity in the hindbrain of mice.
Credit: M. Maiarù et al., Science Translational Medicine (2018)[/caption]
Opioid medications can also activate brain reward regions, causing addiction. Over 2 million individuals in the US have ‘opioid use disorder’ with most starting with prescribed opioid painkillers and opioid overdose is now the second leading cause of death in the US.
In the UK, 5% of the population are on opioids with 80% reporting negative side effects.
Previous studies in rats and companion dogs show that precise injections of tiny amounts of toxic substances, such as substance P-saporin, into the spine kill neurons responsible for crippling, chronic pain. This approach relies on an analogue of ricin which is difficult to manufacture to clinical standards and clinicians are resistant to irreversibly kill nerve cells.
In contrast, Derm-BOT is safe to manufacture, is non-toxic and does not kill neurons.
“We needed to find the best pain targeting molecular parts to direct the botulinum silencing ‘warhead’ to the pain-controlling system in the spine. For this, we developed a molecular Lego system which allows us to link the botulinum ‘warhead’ to a navigation molecule, in this case, the strong opioid called dermorphin, allowing the creation of widely desired long-lasting pain killers without the side effects of opioids,"
said co-corresponding author Professor Bazbek Davletov from the Department of Biomedical Science at the University of Sheffield.
Dermorphin targets and binds to opioid receptors on the surface of neurons which allows the Derm-BOT compound to enter the cells where the botulinum ‘warhead’ then reversibly inhibits the release of neurotransmitter, silencing the cells essential for sending pain signals to the brain.
Botulinum toxin (BTX), also known as Botox, is a neurotoxic protein produced by the bacterium Clostridium botulinum and related species. It prevents the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junction and thus causes flaccid paralysis. Infection with the bacterium causes the disease botulism. The toxin is also used commercially in medicine, cosmetics and research.
Botulinum is the most acutely lethal toxin known. While botulinum toxin is generally considered safe in a clinical setting, there can be serious side effects from its use. Most commonly, botulinum toxin can be injected into the wrong muscle group or spread from the injection site, causing paralysis of unintended muscles.
Humans most commonly ingest the toxin from eating improperly-canned foods in which C. botulinum has grown.
[caption id=“attachment_96466” align=“aligncenter” width=“680”] Target molecules of botulinum neurotoxin (abbreviated BoNT) and tetanus neurotoxin (TeNT), toxins acting inside the axon terminal
Credit: Y tambe, CC BY-SA 3.0[/caption]
Botulinum toxin exerts its effect by cleaving key proteins required for nerve activation. First, the toxin binds specifically to nerves which use the neurotransmitter acetylcholine.
Once bound to the nerve terminal, the neuron takes up the toxin into a vesicle. As the vesicle moves farther into the cell, it acidifies, activating a portion of the toxin which triggers it to push across the vesicle membrane and into the cell cytoplasm.
Once inside the cytoplasm, the toxin cleaves SNARE proteins preventing the cell from releasing vesicles of neurotransmitter. This stops nerve signaling, leading to paralysis.
Over a five year period, 200 mice were used to simulate the early stages of human inflammatory and neuropathic pain and were treated with a single injection of either Derm-BOT, SP-BOT (a substance P-modified botulinum molecule) or morphine. The behaviour of the mice was monitored to track their pain-response and the locations and binding properties of the botulinum compounds were investigated.
“Both SP-BOT and Derm-BOT have a long-lasting effect in both inflammatory and neuropathic pain model, successfully silencing neurons without cell death. We were impressed to see that one tiny injection was enough to stop chronic pain caused by inflammation and nerve damage for at least a month. Furthermore, a single injection of Derm-BOT reduced mechanical hypersensitivity to the same extent as morphine. We hope to take our investigations forward with the aim of translating this into the clinic,"
concluded lead author, Dr. Maria Maiarù.
The work was supported by a grant from the Medical Research Council.
Maria Maiarù, Charlotte Leese, Michelangelo Certo, Irene Echeverria-Altuna, Antonina S. Mangione, Jason Arsenault, Bazbek Davletov, Stephen P. Hunt Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice Science Translational Medicine 18 Jul 2018: Vol. 10, Issue 450, eaar7384 DOI: 10.1126/scitranslmed.aar7384
Top Image: Spinal-parabrachial axons labelled with cSNAP25. Credit: M. Maiarù