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.

Therapeutic efficacy of equine botulism heptavalent antitoxin against all seven botulinum neurotoxins in symptomatic guinea pigs.

Botulism neurotoxins are highly toxic and are potential agents for bioterrorism. The development of effective therapy is essential to counter the possible use of these toxins in military and bioterrorism scenarios, and to provide treatment in cases of natural intoxication. Guinea pigs were intoxicated with a lethal dose of botulinum neurotoxin serotypes A, B, C, D, E, F or G, and at onset of the clinical disease intoxicated animals were treated with either BAT® [Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)] or placebo. BAT product treatment significantly (p<0.0001) enhanced survival compared to placebo for all botulinum neurotoxin serotypes and arrested or mitigated the progression of clinical signs of botulism intoxication. These results demonstrated the therapeutic efficacy of BAT product in guinea pigs and provided supporting evidence of effectiveness for licensure of BAT product under FDA 21 CFR Part 601 (Subpart H Animal Rule) as a therapeutic for botulism intoxication to serotypes A, B, C, D, E, F or G in adults and pediatric patients.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products.

Challenges in searching for therapeutics against Botulinum Neurotoxins.

INTRODUCTION: Botulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes. Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed. Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged.

A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin.

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication.

Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism.

Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.

A platform stratifying a sequestering agent and a pharmacological antagonist as a means to negate botulinum neurotoxicity.

Botulinum neurotoxicity is characterized by peripheral neuromuscular blockade/flaccid paralysis that can lead to respiratory failure and ultimately death. Current therapeutic options provide relief in a pre-exposure scenario, but there are no clinically approved postexposure medical countermeasures. Here, we introduce a platform that utilizes a combination of a toxin sequestering agent and a pharmacological antagonist to ablate botulinum neurotoxicity in a well-defined mouse lethality assay. The platform was constructed to allow for ready exchange of sequestering agent and/or pharmacological antagonist for therapeutic optimization. As such, we attempted to improve upon the pharmacological antagonist, a potassium channel blocker, 3,4-diaminopyridine, through a prodrug approach; thus, a complete kinetic decomposition pathway is described. These experiments provide the first proof-of-principle that a synergistic combination strategy can be used to reduce toxin burden in the peripheral using a sequestering antibody, while restoring muscle action via a pharmacological small molecule antagonist.

Current strategies for designing antidotes against botulinum neurotoxins.

INTRODUCTION: Botulinum neurotoxins (BoNTs) are proteins responsible for the deadly paralytic disease botulism. Extreme toxicity, ease of production and lack of antidotes against BoNT makes it a category A biothreat agent, according to the United States Center of Disease Control and Prevention. The only available therapy for BoNT is an equine antitoxin antibody or/and a protracted respiratory support system. Even then, antibody treatment can only prevent further exposure of the toxin and cannot rescue already intoxicated neurons. AREAS COVERED: In this article, the authors provide a summary of the current status of inhibitors and antitoxins used against BoNTs. In particular, the authors focus on new strategies used in the development of novel therapeutics. They also outline the major steps involved in BoNT's mechanism of action and identify specific inhibitors for each step. EXPERT OPINION: Several previous efforts have resulted in less than satisfactory results that are due, in part, to a lack of sustained effort in addition to a poor understanding of the unique structural features of the toxin. BoNT is a double-edged sword with both toxic effects and therapeutic benefits, excluding vaccination as a preventative measure. The long lasting intracellular endopeptidase activity, which causes an extended period of muscle paralysis, necessitates the need to identify effective inhibitor(s) against BoNT, and this could ultimately lead to new therapeutic options.

Differences in immunological responses of polyclonal botulinum A1 and A2 antitoxin against A1 and A2 toxin.

Botulinum type A antitoxin in standard and therapeutic preparation is a polyclonal antibody purified from immunized sera with subtype A1 toxin. To investigate the difference between immunological responses of antitoxin against toxin among different subtypes, we examined the response of polyclonal A1 and A2 antitoxins with A1 and A2 toxins. In the mouse neutralization test, the A1 antitoxin had equivalent potency against both the A1 and A2 toxins. However, the neutralization titer of the A2 antitoxin was 4-9 fold higher against the A2 toxin than against the A1 toxin. Since the titers of the antitoxins were calculated relative to the standard antitoxin, we assumed that the difference between the antibody titers against the test toxins was due the standard antitoxin having different reactivities with the toxins. The binding volume of the A2 toxin against the standard and A1 antitoxins was 3-4% of the binding volume of the A1 toxin. The neutralization curve of the standard antitoxin was parallel against both the A1 and A2 toxins. However, the curve of the A2 antitoxin was not parallel against the A1 and A2 toxins. Furthermore, binding analysis comparing these antitoxins and toxins showed that the A1 antitoxin had a higher binding affinity and slower dissociation speed with the A1 toxin than with the A2 toxin. In contrast, the A2 antitoxin showed a higher binding affinity with the A2 toxin than with the A1 toxin. These findings indicated that antitoxin reacted strongly against the same subtype, and showed a weak response against the toxin of different subtypes. We propose that the existing standard type A antitoxin should be used for reaction with A1 toxins, and that it is necessary to establish a new standard antitoxin for each subtype of toxin.

The life history of a botulinum toxin molecule.

There is an emerging literature describing the absorption, distribution, metabolism and elimination of botulinum toxin. This work reveals that the toxin can be absorbed by both the oral and inhalation routes. The primary mechanism for absorption is binding and transport across epithelial cells. Toxin that enters the body undergoes a distribution phase, which is quite short, and an elimination phase, which is comparatively long. During the distribution phase, botulinum toxin migrates to the peri-neuronal microcompartment in the vicinity of vulnerable cells, such as cholinergic nerve endings. Only these cells have the ability to selectively accumulate the molecule. When the toxin moves from the cell membrane to the cell interior, it undergoes programmed death. This is coincident with release of the catalytically active light chain that paralyzes transmission. Intraneuronal metabolism of light chain is via the ubiquitination-proteasome pathway. Systemic metabolism and elimination is assumed to be via the liver. The analysis of absorption, distribution, metabolism and elimination of the toxin helps to create a life history of the molecule in the body. This has many benefits, including: a) clarifying the mechanisms that underlie the disease botulism, b) providing insights for development of medical countermeasures against the toxin, and c) helping to explain the meaning of a lethal dose of toxin. It is likely that work intended to enhance understanding of the fate of botulinum toxin in the body will intensify. These efforts will include new and powerful analytic tools, such as single molecule-single cell analyses in vitro and real time, 3-dimensional pharmacokinetic studies in vivo.

Transsynaptic inhibition of spinal transmission by A2 botulinum toxin.

Type A botulinum toxin blocks not only ACh release from motor nerve terminals but also central synaptic transmission, including glutamate, noradrenaline, dopamine, ATP, GABA and glycine. Neurotoxins (NTXs) are transported by both antero- and retrogradely along either motor or sensory axons for bidirectional delivery between peripheral tissues or the CNS. A newly developed type A2 NTX (A2NTX) injected into one rat foreleg muscle was transported to the contralateral muscle. This finding was consistent with the NTX traveling retrogradely via spinal neurons and then transsynaptically through motor neurons to the contralateral motor neurons within the spinal cord and on to the soleus muscle. In the present study we found that toxin injection into the rat left soleus muscle clearly induced bilateral muscle relaxation in a dose-dependent fashion, although the contralateral muscle relaxation followed the complete inhibition of toxin-injected ipsilateral muscles. The toxin-injected ipsilateral muscle relaxation was faster and stronger in A2NTX-treated rats than A1LL (BOTOX). A1LL was transported almost equally to the contralateral muscle via neural pathways and the bloodstream. In contrast, A2NTX was mainly transported to contralateral muscles via the blood. A1LL was more successfully transported to contralateral spinal neurons than A2NTX. We also demonstrated that A1LL and A2NTX were carried from peripheral to CNS and vice versa by dual antero- and retrograde axonal transport through either motor or sensory neurons.

Mouse compound muscle action potential assay: an alternative method to conduct the LD50 botulinum toxin type A potency test.

PURPOSE: We performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD50), which is used for quality control. METHODS: Prevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1-100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4-40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23-25 and 25-27 g) and measurement times (3-5 and 5-7 min) after anesthetic induction were tested to assess robustness. RESULTS: This assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2-40 U/mL (R² = 0.982). The accuracy of 14 determinations was within the range of 89.8-118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3-14.4%. The range of quantification and the LOQ were 4-40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude. CONCLUSIONS: These results suggest that the mouse CMAP assay is an alternative method to the standard LD50 potency test and meets the requirement of the three Rs (particularly refinement and reduction).

Type A1 but not type A2 botulinum toxin decreases the grip strength of the contralateral foreleg through axonal transport from the toxin-treated foreleg of rats.

The adverse effects of botulinum LL toxin and neurotoxin produced by subtype A1 (A1LL and A1NTX) are becoming issues, as the toxins could diffuse from the toxin-treated (ipsilateral) to contralateral muscles. We have attempted to produce neurotoxin from subtype A2 (A2NTX) with an amino acid sequence different from that of neurotoxin subtype A1. We measured the grip strength on the contralateral foreleg as an indicator of toxin spread from the ipsilateral to contralateral muscles. Doses of 0.30 log U or above of A1LL and A1NTX reduced the contralateral grip strength, whereas a dose of 0.78 log U of A2NTX was required to do so. We investigated the route of toxin spread using denervated, colchicine-treated, and antitoxin-treated rats. A1LL was transported via axons at doses higher than 0.30 log U and via both axons and body fluid at about 0.80 log U or a higher dose. Interestingly, A2NTX was transported via body fluid at about 0.80 log U or a higher dose, but not via axons to the contralateral side. It was concluded that A1LL and A1NTX decreased the grip strength of the toxin-untreated foreleg via both axonal transport and body fluids, while A2NTX was only transported via the body fluid.

An alternative in vivo method to refine the mouse bioassay for botulinum toxin detection.

Botulism is a rare, life-threatening paralytic disease of both humans and animals that is caused by botulinum neurotoxins (BoNT). Botulism is confirmed in the laboratory by the detection of BoNT in clinical specimens, contaminated foods, and cultures. Despite efforts to develop an in vitro method for botulinum toxin detection, the mouse bioassay remains the standard test for laboratory confirmation of this disease. In this study, we evaluated the use of a nonlethal mouse toe-spread reflex model to detect BoNT spiked into buffer, serum, and milk samples. Samples spiked with toxin serotype A and nontoxin control samples were injected into the left and right extensor digitorum longus muscles, respectively. Digital photographs at 0,8, and 24 h were used to obtain objective measurements through effective paralysis scores, which were determined by comparing the width-to-length ratio between right and left feet. Both objective measurements and clinical observation could accurately identify over 80% of animals injected with 1 LD(50) (4.3 pg) BoNT type A within 24 h. Half of animals injected with 0.5 LD(50) BoNT type A and none injected with 0.25 LD(50) demonstrated localized paralysis. Preincubating the toxin with antitoxin prevented the development of positive effective paralysis scores, demonstrating that (1) the effect was specific for BoNT and (2) identification of toxin serotype could be achieved by using this method. These results suggest that the mouse toe-spread reflex model may be a more humane alternative to the current mouse bioassay for laboratory investigations of botulism.

Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics.

Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents.

Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD(50)) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD(50) of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.

Localization of the sites and characterization of the mechanisms by which anti-light chain antibodies neutralize the actions of the botulinum holotoxin.

The recombinant, catalytically active light chain of botulinum toxin type A was evaluated as a potential vaccine candidate. Previous studies have shown that the light chain can elicit protective immunity in vivo. [Kiyatkin N, Maksymowych AB, Simpson LL. Induction of immune response by oral administration of recombinant botulinum toxin. Infect Immun 1997;65(11):4586-91], but the underlying basis for this observation was not determined. In the present study, antibodies directed against the light chain were shown to act at three different sites in the body to produce neutralization. Firstly, these antibodies acted to block toxin absorption into the body. This was demonstrated in vitro, in studies on binding and transport of toxin across epithelial monolayers, and in vivo, in studies on inhalation poisoning. Secondly, anti-light chain antibodies acted to promote clearance of toxin from the general circulation. This was demonstrated in vivo in studies on toxin levels in blood and in parallel studies on toxin accumulation in liver and spleen. Finally, anti-light chain antibodies acted to protect cholinergic nerves from botulinum toxin action. This was demonstrated in two types of in vitro assays: rate of paralysis of murine phrenic nerve-hemidiaphragm preparations and extent of binding to Neuro-2a cells. When taken together, these data show that anti-light chain antibodies can evoke three layers of protection against botulinum toxin.

Botulinum neurotoxin serotype A inhibitors: small-molecule mercaptoacetamide analogs.

Botulinum neurotoxin elicits its paralytic activity through a zinc-dependant metalloprotease that cleaves proteins involved in neurotransmitter release. Currently, no drugs are available to reverse the effects of botulinum intoxication. Herein we report the design of a novel series of mercaptoacetamide small-molecule inhibitors active against botulinum neurotoxin serotype A. These analogs show low micromolar inhibitory activity against the isolated enzyme. Structure-activity relationship studies for a series of mercaptoacetamide analogs of 5-amino-3-phenylpyrazole reveal components essential for potent inhibitory activity.