Already a well-established treatment for different autonomic and movement disorders, the use of botulinum toxin type A (BoNT/A) in pain conditions is now continuously expanding. Currently, the only approved use of BoNT/A in relation to pain is the treatment of chronic migraines. However, controlled clinical studies show promising results in neuropathic and other chronic pain disorders. In comparison with other conventional and non-conventional analgesic drugs, the greatest advantages of BoNT/A use are its sustained effect after a single application and its safety. Its efficacy in certain therapy-resistant pain conditions is of special importance. Novel results in recent years has led to a better understanding of its actions, although further experimental and clinical research is warranted. Here, we summarize the effects contributing to these advantageous properties of BoNT/A in pain therapy, specific actions along the nociceptive pathway, consequences of its central activities, the molecular mechanisms of actions in neurons, and general pharmacokinetic parameters.

The antinociceptive action of botulinum toxin type A (BoNT/A) has been demonstrated in behavioral animal studies and clinical settings. It was shown that this effect is associated with toxin activity in CNS, however, the mechanism is not fully understood. Substance P (SP) is one of the dominant neurotransmitters in primary afferent neurons transmitting pain and itch. Thus, here we examined association of SP-mediated transmission and BoNT/A antinociceptive action by employing gene knockouts. Antinociceptive activity of intraplantarly (i.pl.) injected BoNT/A was examined in mice lacking the gene encoding for SP/neurokinin A (tac1-/-) or SP-preferred receptor neurokinin 1 (tac1r-/-), compared to control C57Bl/6J wild type animals. BoNT/A action was assessed in inflammatory pain induced by formalin and CFA, and neuropathic pain induced by partial sciatic nerve ligation. BoNT/A activity in CNS was examined by c-Fos and BoNT/A-cleaved SNAP-25 immunohistochemistry. In wild type mice, acute (formalin-evoked) and chronic pain (neuropathic and inflammatory) was reduced by peripherally injected BoNT/A. In tac1-/- and tac1r-/- knockout mice, BoNT/A exerted no analgesic effect. In control animals BoNT/A reduced the formalin-evoked c-Fos expression in lumbar dorsal horn, while in knockout mice the c-Fos expression was not reduced. After peripheral toxin injection, cleaved SNAP-25 occurred in lumbar dorsal horn in all animal genotypes. BoNT/A antinociceptive activity is absent in animals lacking the SP and neurokinin 1 receptor encoding genes, in spite of presence of toxin's enzymatic activity in central sensory regions. Thus, we conclude that the integrity of SP-ergic system is necessary for the antinociceptive activity of BoNT/A.

Background and Purpose: Botulinum toxin type A (BoNT/A) injections into hyperactive muscles provide effective treatment for spasticity and dystonias, presumably due to its local effects on extrafusal and intrafusal motor fibres. A recent discovery of toxin's retrograde axonal transport to CNS might suggest additional action sites. However, in comparison to cholinergic peripheral terminals, functional consequences of BoNT/A direct central action on abnormally increased muscle tone are presently unknown. To address this question, the central effects of BoNT/A were assessed in experimental local spastic paralysis.Experimental Approach: Local spastic paralysis was induced by injection of tetanus toxin (1.5 ng) into rat gastrocnemius. Subsequently, BoNT/A (5 U·kg⁻¹) was applied i.m. into the spastic muscle or intraneurally (i.n.) into the sciatic nerve to mimic the action of axonally transported toxin. Functional role of BoNT/A transcytosis in spinal cord was evaluated by lumbar i.t. application of BoNT/A‐neutralizing antitoxin. BoNT/A effects were studied by behavioural motor assessment and cleaved synaptosomal‐associated protein 25 (SNAP‐25) immunohistochemistry.Key Results: Tetanus toxin evoked muscular spasm (sustained rigid hind paw extension and resistance to passive ankle flexion). Subsequent injections of BoNT/A, i.m. or i.n, reduced tetanus toxin‐evoked spastic paralysis. Beneficial effects of i.n. BoNT/A and occurrence of cleaved SNAP‐25 in ventral horn were prevented by i.t. antitoxin.Conclusions and Implications: Axonally transported BoNT/A relieves muscle hypertonia induced by tetanus toxin, following the trans‐synaptic movement of BoNT/A in the CNS. These results suggest that such direct, centrally mediated reduction of abnormal muscle tone might contribute to the effectiveness of BoNT/A in spasticity and hyperkinetic movement disorders.

Botulinum neurotoxin type A1 (BoNT-A) reduces the peripheral peptide and cytokine upregulation in rats with antigen-evoked persistent immunogenic hypersensitivity (PIH) of the temporomandibular joint (TMJ). Herein, we examined the effects of two preparations of BoNT-A, abobotulinumtoxinA (aboBoNT-A; Dysport) and onabotulinumtoxinA (onaBoNT-A; Botox), on spontaneous and evoked nociceptive behaviors, as well as on central neuronal and astroglial activation. The antigen-evoked PIH was induced in rats via repeated systemic and unilateral intra-articular (i.a.) injections of methylated bovine serum albumin (mBSA). Rats were subsequently injected with unilateral i.a. aboBoNT-A (14 U/kg), onaBoNT-A (7 U/kg), or the vehicle (saline). After i.a. treatments, spontaneous and mechanically evoked nocifensive behaviors were assessed before and after the low-dose i.a. formalin (0.5%) challenge. The central effects of BoNT-A were assessed by an immunohistochemical analysis of cleaved synaptosomal-associated protein 25 (cSNAP-25) presence, c-Fos, GFAP, and CGRP expression in the trigeminal nucleus caudalis (TNC). Both BoNT-A preparations similarly reduced the formalin-induced spontaneous pain-related behaviors and mechanical allodynia of the hypernociceptive rats. Likewise, their effects were associated with the central occurrence of cSNAP-25 and reduction of c-Fos and GFAP upregulation in the TNC. BoNT-A antinociceptive activity on the PIH is associated with the toxin axonal transport to trigeminal sensory areas and reduction of neuronal and glial activation in central nociceptive regions.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.