A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism.

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism.

Symptomatic treatment of botulism with a clinically approved small molecule.

Botulinum neurotoxins (BoNTs) are potent neuroparalytic toxins that cause mortality through respiratory paralysis. The approved medical countermeasure for BoNT poisoning is infusion of antitoxin immunoglobulins. However, antitoxins have poor therapeutic efficacy in symptomatic patients; thus, there is an urgent need for treatments that reduce the need for artificial ventilation. We report that the US Food and Drug Administration-approved potassium channel blocker 3,4-diaminopyridine (3,4-DAP) reverses respiratory depression and neuromuscular weakness in murine models of acute and chronic botulism. In ex vivo studies, 3,4-DAP restored end-plate potentials and twitch contractions of diaphragms isolated from mice at terminal stages of BoNT serotype A (BoNT/A) botulism. In vivo, human-equivalent doses of 3,4-DAP reversed signs of severe respiratory depression and restored mobility in BoNT/A-intoxicated mice at terminal stages of respiratory collapse. Multiple-dosing administration of 3,4-DAP improved respiration and extended survival at up to 5 LD50 BoNT/A. Finally, 3,4-DAP reduced gastrocnemius muscle paralysis and reversed respiratory depression in sublethal models of serotype A-, B-, and E-induced botulism. These findings make a compelling argument for repurposing 3,4-DAP to symptomatically treat symptoms of muscle paralysis caused by botulism, independent of serotype. Furthermore, they suggest that 3,4-DAP is effective for a range of botulism symptoms at clinically relevant time points.

Toxina botulínica para tratamento de síndrome do Choro Assimétrico: relato de caso / Botulinum toxin in the treatment of asymmetric crying face syndrome: a case report

Síndrome do choro assimétrico é uma condição congênita secundária à hipoplasia ou ausência do músculo depressor do ângulo da boca. Trata-se de uma condição não tão incomum que pode cursar com assimetria facial ao chorar e sorrir, além de poder estar associadas a outras malformações congênitas. Crianças com essa deformidade podem sofrer dificuldades psicossociais e introversão. O arsenal terapêutico dessa condição já foi estudado e discutido na literatura com ênfase em abordagens cirúrgicas e invasivas. Relatamos aqui um caso de uma criança de 9 anos com essa síndrome, tratada, de forma menos invasiva, com toxina botulínica, com um bom resultado e satisfação.

Asymmetric crying face syndrome is a congenital condition secondary to hypoplasia or absence of the depressor muscle at the mouth angle. It is a common condition that presents with facial asymmetry while crying and smiling and may be associated with other congenital malformations. Children with this deformity may experience psychosocial difficulties and introversion. The therapeutic arsenal of this condition has already been studied and discussed in the literature with an emphasis on surgical and invasive approaches. We report here a case of a 9-year-old child with this syndrome, treated less invasively with botulinum toxin, with good result and satisfaction.

Dysfunctional voiding: the importance of non-invasive urodynamics in diagnosis and treatment.

In Dysfunctional voiding, failure of the external sphincter-pelvic floor complex to relax during micturition results in bladder outflow obstruction with a spectrum of presentation from more benign lower urinary tract dysfunction including recurrent urinary tract infections, to significant upper tract pathology and end-stage renal failure. There is no underlying neurological or anatomical cause and the condition is postulated to be a largely learnt behavior. Diagnosis relies on non-invasive urodynamics and in particular uroflowmetry, plus or minus EMG, which is also used in biofeedback, the mainstay of treatment. The etiology, presentation, diagnosis, and treatment with particular emphasis on non-invasive urodynamics are covered.

Epitope targeting of tertiary protein structure enables target-guided synthesis of a potent in-cell inhibitor of botulinum neurotoxin.

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target-guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate-mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope-targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active-site-adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model.

A novel use for botulinum toxin A in the management of ileostomy and urostomy leaks.

PURPOSE: To assess the efficacy of Botulinum toxin A (BoNT-A) in the treatment of patients with hypercontractile stomas resulting in repeated pouching system failures and leaks. DESIGN: Prospective case series. SUBJECTS AND SETTING: Ten consecutive patients who presented to the outpatient stoma clinic with actively contractile stomas that shortened spasmodically resulting in leaks were offered treatment with BoNT-A if treatment with other measures had been unsuccessful. METHODS: Following an observed reduction in the peristalsic shortening of a stoma after intradermal injection of BoNT-A for hyperhidrosis, we conducted a prospective case series of 10 patients with pouch adhesive failures attributed to spasmodic shortening of the stoma. Ten patients, 3 with urostomies and 7 with ileostomies, were offered BoNT-A injection. The first was treated cautiously with 15 units of BoNT-A injected into the muscularis layer, followed by an additional 25 units injected 1 month later. Subsequent patients received doses varying from 50 to 100 units. Ongoing treatments ranged 50 to 100 units every 3 to 6 months. RESULTS: Seventy percent (n = 7) of patients reported a useful reduction in leakage and pouching system seal failures. In these 7 patients, the frequency of pouch changes changed from an average of 2.18 to 0.44 per day (over all 10 patients this was a change from an average of 2.35 per day to 1.16 per day). No adverse side effects were reported. CONCLUSION: Findings from this clinical case series suggest that BoNT-A may be a promising treatment in the management of patients with leaks caused by actively contracting stomas.

Intradermal botulinum toxin a for peristomal hyperhidrosis: a case study.

BACKGROUND: Peristomal hyperhidrosis can interfere with pouch adherence, resulting in pouch leakage and peristomal skin damage. CASE: A patient with autonomic dysregulation resulting in excessive sweating (hyperhidrosis) experienced difficulty with adherence of her ileostomy appliance. Two hundred units of botulinum toxin A (BTX-A) were injected in the dermis of the surrounding skin in order to improve adherence of her pouching system and alleviate moisture of her peristomal skin. RESULTS: Following BTX-A injection, the typical wear time of her pouching system improved from less than 24 hours to 120 hours. Peristomal moisture-associated skin damage resolved almost completely. These effects lasted 3 months. A repeat intradermal BTX-A injection had a similar positive effect. CONCLUSION: Peristomal hyperhidrosis can be controlled with BTX-A intradermal injections, improving patient pouch adherence and alleviating moisture-associated skin damage.

Re-assembled botulinum neurotoxin inhibits CNS functions without systemic toxicity.

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a "protein-stapling" technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications.

Effective results with botulinum toxin in cerebral palsy.

This study evaluated the improvement in clinical measures and quality of life (QOL) among patients with cerebral palsy treated with botulinum toxin type A. Fifty-seven parents of cerebral palsy patients who used botulinum toxin during the time of the study were enrolled. The QOL questionnaires included the following: Child Caregiver Questionnaire, Pediatrics Outcomes Data Collection Instrument, and clinical evaluations. The questionnaires were administered before the first use of botulinum toxin and approximately 1 year later, a mean interval of 13.8 months. Treatment resulted in clinical improvement in tone, upper limb function, and Gross Motor Function Classification System score. Better outcomes were observed in patients younger than 6.5 years. QOL questionnaires revealed a tendency toward improvement in the comfort dimension of the Child Caregiver Questionnaire as well as in the upper extremities and physical functions, transfers and basic mobility, and global function and symptom of the Pediatrics Outcomes Data Collection Instrument. The QOL measures correlated with clinical evaluations. Patients with low cognitive ability and refractory epilepsy had the worst results. Children and adolescents have reduced spasticity and experience good results in the clinical measurements and in QOL after treatment with botulinum toxin.

The osmolyte trimethylamine N-oxide (TMAO) increases the proteolytic activity of botulinum neurotoxin light chains A, B, and E: implications for enhancing analytical assay sensitivity.

Botulism, the disease caused by botulinum neurotoxins (BoNTs), secreted by the spore-forming, anaerobic bacteria Clostridium botulinum, has been associated with food poisoning for centuries. In addition, the potency of BoNTs coupled with the current political climate has produced a threat of intentional, malicious poisoning by these toxins. The ability to detect and measure BoNTs in complex matrixes is among the highest research priorities. However, the extreme potency of these toxins necessitates that assays be capable of detecting miniscule quantities of these proteins. Thus, signal-boosting strategies must be employed. A popular approach uses the proteolytic activity of the BoNT light chain (LC) to catalyze the cleavage of synthetic substrates; reaction products are then analyzed by the analytical platform of choice. However, BoNT LCs are poor catalysts. In this study, the authors used the osmolyte trimethylamine N-oxide (TMAO) to increase the proteolytic activities of BoNT LCs. Their data suggest that concentrated solutions of TMAO induce complete folding of the LCs, resulting in increased substrate affinity and enhanced enzyme turnover. The authors observed increases in catalysis for BoNT serotypes A, B, and E, and this increased proteolytic activity translated into substantial increases in analytical assay sensitivity for these medically relevant toxins.

Low pH-induced pore formation by the T domain of botulinum toxin type A is dependent upon NaCl concentration.

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.