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Xeomin ® is a commercial formulation of botulinum neurotoxin type A (BoNT/A) clinically authorized for treating neurological disorders, such as blepharospasm, cervical dystonia, limb spasticity, and sialorrhea. We have previously demonstrated that spinal injection of laboratory purified 150 kDa BoNT/A in paraplegic mice, after undergoing traumatic spinal cord injury (SCI), was able to reduce excitotoxic phenomena, glial scar, inflammation, and the development of neuropathic pain and facilitate regeneration and motor recovery. In the present study, as proof of concept in view of a possible clinical application, we studied the efficacy of Xeomin ® in the same preclinical SCI model in which we highlighted the positive effects of lab-purified BoNT/A. Data comparison shows that Xeomin ® induces similar pharmacological and therapeutic effects, albeit with less efficacy, to lab-purified BoNT/A. This difference, which can be improved by adjusting the dose, can be attributable to the different formulation and pharmacodynamics. Although the mechanism by which Xeomin ® and laboratory purified BoNT/A induce functional improvement in paraplegic mice is still far from being understood, these results open a possible new scenario in treatment of SCI and are a stimulus for further research., Competing Interests: The funders had no role in the design of the study; collection, analyses, or interpretation of data; writing of the manuscript; or decision to publish the results. F.P., S.L., S.M., and V.V. are the inventors of the patent Nr. WO2016170501A1 “A new therapeutic use of the botulinum neurotoxin serotype A”.

Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin is able to block the release of pro-inflammatory molecules for months after its administration. Here we demonstrate the extraordinary capacity of BoNT/A to neutralize the complete paralysis and pain insensitivity induced in a murine model of severe spinal cord injury (SCI). We show that the toxin, spinally administered within one hour from spinal trauma, exerts a long-lasting proteolytic action, up to 60 days after its administration, and induces a complete recovery of muscle and motor function. BoNT/A modulates SCI-induced neuroglia hyperreactivity, facilitating axonal restoration, and preventing secondary cells death and damage. Moreover, we demonstrate that BoNT/A affects SCI-induced neuropathic pain after moderate spinal contusion, confirming its anti-nociceptive action in this kind of pain, as well. Our results provide the intriguing and real possibility to identify in BoNT/A a therapeutic tool in counteracting SCI-induced detrimental effects. Because of the well-documented BoNT/A pharmacology, safety, and toxicity, these findings strongly encourage clinical translation.

Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background : The serotype A (BoNT/A) has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B) are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods : As model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control) or BoNT/B (5 or 7.5 pg/mouse) into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse). Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state., Competing Interests: The authors declare no conflict of interest.

The injection of safe doses of botulinum neurotoxin A (BoNT/A) have been reported to be useful for the treatment of neuropathic pain, but it is still unknown how functional recovery is induced after peripheral nerve injury. We evaluated the effects of intranerve application of BoNT/A, on regeneration and sensorimotor functional recovery in partial and complete peripheral nerve injuries in the mouse. After sciatic nerve crush (SNC) and intranerve delivery of BoNT/A (15pg), axonal regeneration was measured by nerve pinch test at different days. Regeneration of myelinated and unmyelinated fibers was assessed by immunohistochemical double labeling for NF200/GAP43 and CGRP/GAP43. S100 was used as Schwann cells marker. Medial footpad skin reinnervation was assessed by PGP staining. Motor functions were assessed by means of nerve conduction tests. In other mice groups, nerve conduction tests were performed also after chronic constriction injury (CCI) of the sciatic nerve and intraplantar injection of BoNT/A (15pg). In SNC mice, BoNT/A increased the rate of axonal regeneration. The advantage of regrowing myelinated axons after BoNT/A injection was evidenced by longer NF200+ nerve profiles and confirmed by nerve histology. We observed also a higher expression of S100 in the distal portion of BoNT/A-injected regenerated nerves. In CCI mice, BoNT/A induced an increase in reinnervation of gastrocnemius and plantar muscles. These results show that a low dose of BoNT/A, insufficient to produce muscular dysfunction, conversely speeds up sensorimotor recovery by stimulating myelinated axonal regeneration, and points out its application as a multipotent treatment for peripheral neuropathies., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Botulinum neurotoxin type A (BoNT/A) and minocycline are potent drugs used in clinical therapies. The primary molecular mechanism of BoNT/A is the cleavage of SNARE proteins, which prevents cells from releasing neurotransmitters from vesicles, while the effects of minocycline are related to the inhibition of p38 activation. Both BoNT/A and minocycline exhibit analgesic effects, however, their direct impact on glial cells is not fully known. Therefore, the aim of the present study was to determine the effects of those drugs on microglial and astroglial activity after lipopolysaccharide (LPS) stimulation and their potential synergistic action. Our results show that BoNT/A and minocycline influenced primary microglial cells by inhibiting intracellular signaling pathways, such as p38, ERK1/2, NF-κB, and the release of pro-inflammatory factors, including IL-1β, IL-18, IL-6, and NOS2. We have revealed that, in contrast to minocycline, BoNT/A treatment did not decrease LPS-induced release of pro-inflammatory factors in the astroglia. In addition, BoNT/A decreased SNAP-23 in both types of glial cells and also SNAP-25 expressed only in astrocytes. Moreover, BoNT/A increased TLR2 and its adaptor protein MyD88, but not TLR4 exclusively in microglial cells. Furthermore, we have shown the impact of BoNT/A on microglial and astroglial cells, with a particular emphasis on its molecular target, TLR2. In contrast, minocycline did not affect any of those factors. We have revealed that despite of different molecular targets, minocycline, and BoNT/A reduced the release of microglia-derived pro-inflammatory factors. In conclusion, we have shown that BoNT/A and minocycline are effective drugs for the management of neuroinflammation by dampening the activation of microglial cells, with minocycline also affecting astroglial activity.

Botulinum neurotoxin serotype A (BoNT/A) shows antinociceptive properties, and its clinical applications in pain therapy are continuously increasing. BoNT/A specifically cleaves SNAP-25, which results in the formation of a non-functional SNARE complex, thereby potently inhibiting the release of neurotransmitters and neuropeptides, including those involved in nociception. The aim of the present study was to determine the effects of BoNT/A (300pg/paw) on pain-related behavior and the levels of glial markers and interleukins in the spinal cord and dorsal root ganglia (DRG) after chronic constriction injury (CCI) to the sciatic nerve in rats. Glial activity was also examined after repeated intraperitoneal injection of minocycline combined with a single BoNT/A injection. Our results show that a single intraplantar BoNT/A injection did not influence motor function but strongly diminished pain-related behaviors in naïve and CCI-exposed rats. Additionally, microglial inhibition using minocycline enhanced the analgesic effects of BoNT/A. Western blotting results suggested that CCI induces the upregulation of the pronociceptive proteins IL-18, IL-6 and IL-1β in the ipsilateral lumbar spinal cord and DRG, but no changes in the levels of the antinociceptive proteins IL-18BP, IL-1RA and IL-10 were observed. Interestingly, BoNT/A injection suppressed the CCI-induced upregulation of IL-18 and IL-1β in the spinal cord and/or DRG and increased the levels of IL-10 and IL-1RA in the DRG. In summary, our results suggest that BoNT/A significantly attenuates pain-related behavior and microglial activation and restores the neuroimmune balance in a CCI model by decreasing the levels of pronociceptive factors (IL-1β and IL-18) and increasing the levels of antinociceptive factors (IL-10 and IL-1RA) in the spinal cord and DRG., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Botulinum neurotoxin A (BoNT/A) is a toxin produced by the naturally-occurring Clostridium botulinum that causes botulism. The potential of BoNT/A as a useful medical intervention was discovered by scientists developing a vaccine to protect against botulism. They found that, when injected into a muscle, BoNT/A causes a flaccid paralysis. Following this discovery, BoNT/A has been used for many years in the treatment of conditions of pathological muscle hyperactivity, like dystonias and spasticities. In parallel, the toxin has become a "glamour" drug due to its power to ward off facial wrinkles, particularly frontal, due to the activity of the mimic muscles. After the discovery that the drug also appeared to have a preventive effect on headache, scientists spent many efforts to study the potentially-therapeutic action of BoNT/A against pain. BoNT/A is effective at reducing pain in a number of disease states, including cervical dystonia, neuropathic pain, lower back pain, spasticity, myofascial pain and bladder pain. In 2010, regulatory approval for the treatment of chronic migraine with BoNT/A was given, notwithstanding the fact that the mechanism of action is still not completely elucidated. In the present review, we summarize experimental evidence that may help to clarify the mechanisms of action of BoNT/A in relation to the alleviation of headache pain, with particular emphasis on preclinical studies, both in animals and humans. Moreover, we summarize the latest clinical trials that show evidence on headache conditions that may obtain benefits from therapy with BoNT/A.

The use of botulinum neurotoxin type A (BoNT/A) against pain, with emphasis for its possible use in alleviating chronic pain, still represents an outstanding challenge for experimental research. In this study, we examined the effects of BoNT/A on morphine-induced tolerance during chronic morphine treatment in neuropathic CD1 mice subjected to sciatic nerve lesion according to the Chronic Constriction Injury (CCI) model of neuropathic pain. We measured the effects of BoNT/A on CCI-induced allodynia and hyperalgesia and on the expression of glial fibrillary acidic protein (GFAP, marker of astrocytes), complement receptor 3/cluster of differentiation 11b (CD11b, marker of microglia), and neuronal nuclei (NeuN) at the spinal cord level. We also analyzed the colocalized expression of GFAP, CD11b and NeuN with phosphorylated p-38 mitogen-activated protein kinase and with μ-opioid receptor (MOR). A single intraplantar injection of BoNT/A (15 pg/paw) into the injured hindpaw, the day before the beginning of chronic morphine treatment (9 days of twice daily injections of 40 mg/kg morphine), was able to counteract allodynia and enhancement of astrocytes expression/activation induced by CCI. In addition, BoNT/A increased the analgesic effect of morphine and countered morphine-induced tolerance during chronic morphine treatment. These effects were accompanied, in neurons, by re-expression of MORs that had been reduced by repeated morphine administration. The combinatory effects of BoNT/A and morphine could have relevant therapeutic implications for sufferers of chronic pain who could benefit of pain relief reducing tolerance due to repeated treatment with opiates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Over the recent years compelling evidence has accumulated indicating that botulinum neurotoxin serotype A (BoNT/A) results in analgesic effects on neuropathic as well as inflammatory pain, both in humans and in animal models. In the present study, the pharmacological interaction of BoNT/A with morphine in fighting inflammatory pain was investigated in mice using the formalin test. Moreover, the effects of BoNT/A on the tolerance-induced by chronic administration of morphine were tested and the behavioral effects were correlated with immunofluorescence staining of glial fibrillary acidic protein, the specific marker of astrocytes, at the spinal cord level. An ineffective dose of BoNT/A (2 pg/paw) combined with an ineffective dose of morphine (1 mg/kg) exerted a significant analgesic action both during the early and the late phases of formalin test. A single intraplantar injection of BoNT/A (15 pg/paw; i.pl.), administered the day before the beginning of chronic morphine treatment (7 days of s.c. injections of 20 mg/kg), was able to counteract the occurrence of tolerance to morphine. Moreover, BoNT/A reduces the enhancement of the expression of astrocytes induced by inflammatory formalin pain. Side effects of opiates, including the development of tolerance during repeated use, may limit their therapeutic use, the possibility of using BoNT/A for lowering the effective dose of morphine and preventing the development of opioid tolerance would have relevant implications in terms of potential therapeutic perspectives., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100β and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain is typified by injuries to the peripheral and central nervous system and derives from such causes as cancer, diabetes, multiple sclerosis, post-herpetic neuralgia, physical trauma or surgery, and many others. Patients suffering neuropathic pain do not respond to conventional treatment with non-steroidal anti-inflammatory drugs and show a reduced sensitivity to opiates often associated with serious side effects. Recently, it has been demonstrated that botulinum neurotoxin serotype-A (BoNT/A) is able to induce analgesia in inflammatory pain conditions. The goal of this research was to test if BoNT/A was able to relieve also neuropathic pain symptoms. By using chronic constriction injury of the sciatic nerve, a mouse model of neuropathic pain, we observed that peripheral administration of BoNT/A strongly reduced the mechanical allodynia associated with this neuropathy. Remarkably, a single non-toxic dose of BoNT/A was sufficient to induce anti-allodynic effects, which lasted for at least 3 weeks. This result is particularly relevant since neuropathic pain is poorly treated by current drug therapies. This communication enlarges our knowledge on potentially new medical uses of BoNT/A in efforts to ameliorate human health conditions, with very important implications in the development of new pharmacotherapeutic approaches against neuropathic pain.

Strains of Clostridium botulinum produce seven antigenically distinct botulinum neurotoxins (BoNTs) designated as serotypes A-G. All serotypes interfere with neural transmission by blocking the release of acetylcholine in cholinergic neurons. They cleave specific sites on proteins of the SNARE [soluble n-ethylmaleimide-sensitive factor (NSF) attachment protein receptor] complex, which play a key role in neuroexocytosis. This study assessed the behavioural effects due to central administration of BoNTs in mice. CD1 mice were injected intracerebroventricularly (icv) with sub-lethal doses of BoNT/A or /B and their behavioural responses in conditioning of active avoidance, object recognition test and pharmacologically induced locomotor activity were tested. Compared to control mice, BoNT-treated mice showed: (1) a reduced capacity to discriminate a novel object within a familiar environment; (2) an enhanced stimulant effect by scopolamine and a depressant effect by oxotremorine on locomotor activity. In contrast, central injection of BoNTs did not alter active avoidance acquisition. These results suggest an in vivo functional alteration due to the action of BoNTs directly administered into the central nervous system. The present data demonstrate that BoNTs may represent an analytical tool for studying the functional role of cholinergic neurons.

Botulinum neurotoxins (BoNTs) act specifically on cholinergic nerve terminals, where they cause a sustained block of acetylcholine release, and therefore they are powerful tools to study the role of cholinergic neurons in neuronal processes. Peripheral effects of BoNTs are widely documented while central effects have not been studied. Here, we report for the first time on the central toxicity of BoNT serotypes A and B following their direct intracerebroventricular (icv) injection in CD1 mice. The LD50 values were found to be in the range 0.5-1.0 x 10(-6)mg/kg. We recorded the following signs preceding animal death: piloerection and weight decrease appear first, followed by temperature decrease, eyelid closure, loss of sensorimotor reflexes, dehydration, dyspnea. Mice died of heart or respiratory failure. The surviving mice recovered completely within 4-6 days and regained the initial healthy conditions. At sub-lethal doses, the same clinical signs appear in a lighter form and with a longer time course.