K. Roger Aoki

Activation of TRPV1 Mediates Calcitonin Gene-Related Peptide Release, Which Excites Trigeminal Sensory Neurons and Is Attenuated by a Retargeted Botulinum Toxin with Anti-Nociceptive Potential

Excessive release of inflammatory/pain mediators from peripheral sensory afferents renders nerve endings hyper-responsive, causing central sensitization and chronic pain. Herein, the basal release of proinflammatory calcitonin gene-related peptide (CGRP) was shown to increase the excitability of trigeminal sensory neurons in brainstem slices via CGRP1 receptors because the effect was negated by an antagonist, CGRP8–37. This excitatory action could be prevented by cleaving synaptosomal-associated protein of Mr 25,000 (SNAP-25) with botulinum neurotoxin (BoNT) type A, a potent inhibitor of exocytosis. Strikingly, BoNT/A proved unable to abolish the CGRP1 receptor-mediated effect of capsaicin, a nociceptive TRPV1 stimulant, or its elevation of CGRP release from trigeminal ganglionic neurons (TGNs) in culture. Although the latter was also not susceptible to BoNT/E, apparently attributable to a paucity of its acceptors (glycosylated synaptic vesicle protein 2 A/B), this was overcome by using a recombinant chimera (EA) of BoNT/A and BoNT/E. It bound effectively to the C isoform of SV2 abundantly expressed in TGNs and cleaved SNAP-25, indicating that its /A binding domain (HC) mediated uptake of the active /E protease. The efficacy of /EA is attributable to removal of 26 C-terminal residues from SNAP-25, precluding formation of SDS-resistant SNARE complexes. In contrast, exocytosis could be evoked after deleting nine of the SNAP-25 residues with /A but only on prolonged elevation of [Ca2+]i with capsaicin. This successful targeting of /EA to nociceptive neurons and inhibition of CGRP release in vitro and in situ highlight its potential as a new therapy for sensory dysmodulation and chronic pain.

Preclinical update on BOTOX® (botulinum toxin type A)-purified neurotoxin complex relative to other botulinurn neurotoxin preparations

Ongoing investigations evaluated clinically relevant properties of BOTOX® (botulinum toxin type A) relative to other botulinum neurotoxin preparations based on the same (type A) or different (types B, C, E and F) serotypes. The mouse Digit Abduction Scoring (DAS) assay was used to compare muscle weakening efficacy, the antigenic potential of two BOTOX® preparations was measured in rabbits, and the presence of antibodies to serotypes A and B was analysed in humans. BOTOX® and new BOTOX® produced similar degrees of dose‐related muscle weakness in mice. Both preparations of BOTOX® were approximately four times more potent than Dysport®. Preparations of serotypes B, C, and F also demonstrated lower potency than BOTOX®, with serotype F also having a shorter duration of action. Neutralising antibodies were found in rabbits 3 months post‐treatment with BOTOX®, but were undetected 8 months post‐treatment with new BOTOX®. A high incidence of antibodies to type B was observed in individuals with no known exposure to type B toxin: highest in groups with the highest incidence of type A antibodies. The safety margin for BOTOX®, calculated using DAS median effective dose (ED,) and the minimum dose producing a 10% reduction in body weight, was more than twice that of Dysport®. In conclusion, each botulinum toxin serotype tested exhibited a different muscle weakening efficacy; BOTOX® consistently exhibited the greatest eficacy. Importantly, BOTOX® and Dysport® exhibited markedly different efficacy and safety profiles, and should not be considered interchangeable. Antibody distribution in humans suggests that there may be immunological cross‐reactivity between serotypes A and B.

Botulinum toxin type A normalizes alterations in urothelial ATP and NO release induced by chronic spinal cord injury

The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.

Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

Intramuscular injection of 125I-botulinum neurotoxin-complex versus 125I-botulinum-free neurotoxin: time course of tissue distribution.

The diffusion from the site of intramuscular injection of 900 kDa botulinum neurotoxin-hemagglutinin complex (BoNT/A-complex) and 150 kDa free-botulinum neurotoxin (free-BoNT/A) was compared. Radioiodinated compounds were injected into the gastrocnemius muscle of rats (70Units (U) 125I-BoNT/A-complex, 67 or 344 U free-125I-BoNT/A, or free-125I-iodide) and the eyelids of rabbits (24 U 125I-BoNT/A-complex or 108 U free-125I-BoNT/A), and measured in various tissues at different time points. There were no detectable systemic effects or generalized botulinum neurotoxin toxicity in either rats or rabbits, indicating that most of the toxin, whether as 125I-BoNT/A-complex or free-125I-BoNT/A, remained at the injection site. In rats, 125I-BoNT/A-complex and free-125I-BoNT/A diffused in a pattern that was grossly similar. Almost no radioactivity was recovered from the brain. Radioactivity recovered from distant tissues (thyroid, skin, and contralateral muscle) was primarily attributable to either low molecular weight 125I-containing peptides or 125I-iodide. After injection into rabbit eyelids, neither 125I-BoNT/A-complex nor free-125I-BoNT/A spread to distant structures, including the eye. The results indicate that most of the neurotoxin does not diffuse from the injection site, whether in free or complexed form, and this may reduce the potential for systemic effects.

Updates on the antinociceptive mechanism hypothesis of botulinum toxin A

Botulinum toxin A has been traditionally viewed as a motor nerve specific treatment. However, clinical uses for botulinum toxin A have continued to expand, with increased use in conditions implicating sensory pain nerve dysfunction. Chronic pain is associated with excess pain fiber activity. When the site of this excess activity resides in the peripheral portion of the pain pathway, a condition of peripheral sensitization can establish. During this state, excess pain signaling reaches the central nervous system, which can then lead to a condition of central sensitization, manifesting as the symptoms associated with chronic pain (i.e. burning, electric pain, lowered pain threshold to normal stimuli, etc). Experimentally, botulinum toxin type A has been shown to reduce neuropeptides and neurotransmitter release from treated cells or nerve endings and to attenuate nociception in both neuropathic and non-neuropathic pain models. This review summarizes the literature to update the hypothesis for the mechanism by which botulinum toxin type A can modulate chronic pain.

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

Botulinum neurotoxin — from laboratory to bedside

Botulinum neurotoxins (BoNTs) have been used clinically since 1980, with an ever increasing range of clinical applications. This has coincided with a period of tremendous advance in the scientific understanding of neurotoxin structure and function, including the description of their endopeptidase activity in 1992. These developments have provided an increased understanding of the mechanisms underpinning the clinical use of the neurotoxins. The expanding clinical use of neurotoxin has created challenges for both the clinicians and manufacturers of BoNT preparations to ensure continuing efficacy and safety margins for the new clinical settings. The increased understanding of the mechanism of neurotoxin action has provided improved technologies to support their clinical use, including biochemical and pharmacological based assays of toxin function. These developments and opportunities, emphasise the need to maintain an active dialogue between clinicians and basic scientists to ensure that advances in the laboratory are translated into clinical benefit and that clinical developments are supported by the scientific research activity. This article is based upon presentations given in a workshop at the 5th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxin in Denver in June, 2005 seeking to address issues relating to the laboratory/clinic interface.

Identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a Protein Receptor for Botulinum Neurotoxin Serotype A (BoNT/A)

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Complete DNA sequences of the botulinum neurotoxin complex of Clostridium botulinum type A-Hall (Allergan) strain ☆

BOTOX is manufactured with the purified native 900-kDa type A neurotoxin complex from Clostridium botulinum type A-Hall (Allergan) strain. This complex is composed of the botulinum neurotoxin (BoNT) and several toxin associated proteins known as the hemagglutinins (HAs) and the non-toxic non-hemagglutinin protein (NTNH). We describe here the complete gene sequences of the BoNT complex of type A-Hall (Allergan) strain. Using a polymerase chain reaction-based approach, we sequenced six open reading frames (ORFs) encoding BoNT (1296 amino acids), the toxin-associated proteins: HA70, 625 aa; HA17, 147 aa; HA34, 291 aa; NTNH, 1193 aa; and the regulatory component botR/OrfX, 178 aa. Comparative alignments of the amino acid sequence of BoNT/A shows a 98-100% sequence identity among different strains of the type A, except for the Kyoto-F strain (90%), whereas the sequence identity between BoNT/A and other toxin serotypes is only 30.4-39.1%. Similar to the neurotoxin, the toxin-associated proteins and botR from type A-Hall strain also share more than 95% identity to the homologous proteins found in type A-NCTC2916 strain. Among all the toxin associated proteins, NTNHs and HA70s are the most conserved with 65-87% identity across different serotypes. On the other hand, HA34s, present only in serotypes A-D, show greater diversity than all other toxin-associated proteins; HA34/A has 90% identity to HA34/B and only approximately 35% identity to HA34/C and HA34/D. Relatively higher sequence identity ( approximately 60%) is seen in HA17 and botR of Hall A when compared to their counterparts in serotypes C or D. Of all proteins within the toxin complex, NTNH and HA70 have the highest degree of conservation across serotypes and this may underscore a critical role for these proteins in the formation of the complexes. Physiologically, different duration of action in different serotypes may be due to different modifications of toxins by neuronal enzymes, which lead to different compartmentalization of different toxins. Computer-assisted motif analysis reveals that toxins contain several potential sites for phosphorylation by casein kinase II, protein kinase C, tyrosine kinases, glycogen synthase kinase 3, cGMP dependent protein kinase (PKG) that are well conserved. The reported sequence information for type A-Hall strain will potentially facilitate elucidation of the toxin interactions with the nontoxin proteins in the complex.