Profiling gene expression reveals insights into pulmonary response to aerosolized botulinum toxin type A exposure in mice.

Botulinum neurotoxin type A (BoNT/A) is traditional medicine and well known for its therapeutic use as an anesthetic and in cosmetic applications that work through the inhibition of acetylcholine exocytosis in neuronal cells. BoNT/A also has the potential to function as a biological weapon due to its high mortality rate and ease of dispersal. Emerging evidence suggests that BoNT/A exhibits biological effects on nonneuronal cells. In cytology experiments, BoNT/A induces global gene expression alterations. However, pulmonary effects from exposure to aerosolized BoNT/A have not been evaluated. This study investigated the global transcriptional profile of lung tissues after botulism inhalation. A mice model of inhaled botulism was established using intratracheal exposure to aerosolized BoNT/A and described through histological examination and flow cytometry. Transcriptomic analysis revealed that genes related to acute inflammatory responses were upregulated at 12-h postexposure. Increased expression of multiple anti-inflammatory marker genes and decreased expression of pro-inflammatory marker genes were observed at 48- to 72-h postexposure, underscoring a transcriptional shift toward a pro-reparative phenotype. Histological examination and cell proportions analysis mirrored these expression patterns. Accordingly, the orchestration of a quick phenotype transition prompted by BoNT/A may have the potential for promoting the resolution of the inflammatory lung. To our knowledge, this study represents the first research to investigate the pulmonary transcriptional responses of aerosolized BoNT/A exposure; the results may provide new insights in elucidating the molecular mechanism for pulmonary inhaled botulism and highlight the potential therapeutic application of BoNT/A in mitigating inflammatory conditions.

Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor ß1 (TGFß1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC(50) values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

Capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of botulinum neurotoxin a.

Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.

Botulinum neurotoxin serotypes A and B preparations have different safety margins in preclinical models of muscle weakening efficacy and systemic safety.

This preclinical study compared the muscle weakening efficacy, duration, and safety margin of the recently approved botulinum toxin type B (BTX-B; Myobloc/Neurobloc) to botulinum toxin type A (BTX-A; BOTOX((R))). Mice received a single hind limb intramuscular injection of BTX-B (1-150U/kg) or BTX-A (1-120U/kg). An observer who was masked to treatment assessed the magnitude and duration of muscle weakening efficacy on a 0-4 scale using the digit abduction scoring assay. Safety margins were determined as the ratio of the IM median lethal dose to the IM dose that produced half-maximal muscle weakness in the DAS. BTX-A produced muscle weakness at lower doses than BTX-B (IM ED(50): 6.2+/-0.6 vs. 20.8+/-1.4U/kg, respectively) (p<0.0001). BTX-A at 29U/kg and BTX-B at 67U/kg produced comparable peak DAS scores of approximately 4 indicating maximal muscle weakness. At these doses, the duration of BTX-A was longer, with a return to baseline by day 36 compared to a return to baseline by day 14 with BTX-B. The mean dose that was lethal in 50% of mice was lower for BTX-A than BTX-B (81.4+/-3.5 vs. 104.6+/-1.9U/kg, respectively) (p<0.001) and the safety margin was higher (13.9+/-1.7 vs. 5.4+/-0.3, respectively (p<0.001). These results indicate that the BTX-A:BTX-B dose ratio for muscle weakening efficacy is different from the ratio for systemic effects following IM injections and suggest that no single dose ratio is adequate to compare these preparations. The in vivo differences found are consistent with the different clinical profiles reported for these two products.

Black tea extract, thearubigin fraction, counteract the effects of botulinum neurotoxins in mice.

Botulinum neurotoxin type A (BoNT/A, 1.5 nM) completely inhibited indirectly evoked twitches in in vitro mouse phrenic nerve-diaphragm preparations within 40 - 45 min. Black tea extract, thearubigin fraction (TRB), mixed with BoNT/A blocked the inhibitory effect of the toxin. The protective effect of TRB extended to botulinum neurotoxins types B and E (BoNT/B and BoNT/E) and tetanus toxin, but not to tetrodotoxin. TRB was also effective against oral toxicity of BoNT/A, B and E. Thus, TRB may be of potential benefit in protecting the paralytic actions of botulinum neurotoxins (BoNTs), but its use is limited by mixing with the toxin.

Efficacy of certain quinolines as pharmacological antagonists in botulinum neurotoxin poisoning.

Various 4- and 8-aminoquinolines, which are effective antimalarial agents, were examined as potential pretreatment compounds for prolongation of the time to 50% block of nerve-elicited muscle twitches in isolated mouse diaphragms exposed to botulinum type A neurotoxin. The 4-aminoquinolines (chloroquine, amodiaquine) and quinacrine, an acridine derivative similar to chloroquine, prolonged the time required for botulinum type A neurotoxin to block neuromuscular transmission by more than 3-fold; 8-aminoquinolines (primaquine and WR242511) had no antibotulinum type A neurotoxin activity. Pyrimethamine, an antimalarial drug lacking the quinoline ring structure, was also ineffective. Rank order potencies based on equimolar effective concentrations for the test compounds were quinacrine > amodiaquine > chloroquine > quinine or quinidine. Maximum protection from botulinum type A neurotoxin-induced neuromuscular block was achieved when muscles were exposed to drug prior to or simultaneously with the toxin. A delay of more than 20 min abolished the protective ability of the antimalarial agents, presumably owing to the release of the toxin from endosomes in quantities sufficient to initiate neuromuscular block. All of the test compounds except quinine and quinidine depressed muscle contractions when concentrations exceeded 20 microM. In addition, amodiaquine at 50 microM induced muscle contracture. A combination of agents at low concentrations that act at different steps of botulinum type A neurotoxin poisoning potentiated the prolongation of time to 50% block in an approximately additive fashion. Thus N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (2 microM) and quinacrine (5 microM), when administered in combination, produced up to a 4-fold increase in time to 50% block. A similar level of protection with quinacrine alone required a 4-fold increase in the aminoquinoline concentration. Although the mechanism of protection by these antimalarial agents is probably through the raising of endosomal pH, the possibility that some of these drugs could also act by inhibiting toxin-induced channel formation cannot be ruled out.