Corey J. Hilmas

Galantamine is a novel post-exposure therapeutic against lethal VX challenge

The ability of galantamine hydrobromide (GAL HBr) treatment to antagonize O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX)-induced lethality, impairment of muscle tension, and electroencephalographic (EEG) changes was assessed in guinea pigs. Guinea pigs were challenged with 16.8 microg/kg VX (2LD50). One min after challenge, animals were administered 0.5 mg/kg atropine sulfate (ATR) and 25 mg/kg pyridine-2-aldoxime methochloride (2-PAM). In addition, guinea pigs were given 0, 1, 2, 4, 8 or 10 mg/kg GAL as a post-exposure treatment immediately prior to ATR and 2-PAM. Animals were either monitored for 24-h survival, scheduled for electroencephalography (EEG) recording, or euthanized 60 min later for measurement of indirectly-elicited muscle tension in the hemidiaphragm. Post-exposure GAL therapy produced a dose-dependent increase in survival from lethal VX challenge. Optimal clinical benefits were observed in the presence of 10 mg/kg GAL, which led to 100% survival of VX-challenged guinea pigs. Based on muscle physiology studies, GAL post-exposure treatment protected the guinea pig diaphragm, the major effector muscle of respiration, from fatigue, tetanic fade, and muscular paralysis. Protection against the paralyzing effects of VX was dose-dependent. In EEG studies, GAL did not alter seizure onset for all doses tested. At the highest dose tested (10 mg/kg), GAL decreased seizure duration when administered as a post-exposure treatment 1 min after VX. GAL also reduced the high correlation associated between seizure activity and lethality after 2LD50 VX challenge. GAL may have additional benefits both centrally and peripherally that are unrelated to its established mechanism as a reversible acetylcholinesterase inhibitor (AChEI).

Riot Control Agents

The toxicology of riot control agents (RCAs) has been the focus of numerous reviews, publications, and textbooks. RCAs comprise a diverse array of chemical compounds with similar toxicities since their introduction on the battlefield in the early part of the past century. They are all designed with the purpose of disabling a targeted group or individual through sensory irritation of the eyes, respiratory tract, and/or skin. By providing a minimal force alternative for controlling and managing individuals, RCAs are a desired public health and safety tool for military, domestic law enforcement, and personal use. The chemistry and physical properties of the most common RCAs in use today are discussed. The mechanism of action, toxicokinetics, and toxicity of these RCAs in animals and humans follow. Finally, we outline the process or risk assessment and characterize the safety, effectiveness, and risk of these agents in situations in which they may be used.

An Extraction Assay Analysis for Galanthamine in Guinea Pig Plasma and its Application to Nerve Agent Countermeasures

Galanthamine hydrobromide (GAL HBr), approved material for treatment of mild to moderate Alzheimer’s disease, is a centrally-acting reversible acetylcholinesterase inhibitor (AChEI) that is currently under evaluation as a therapeutic countermeasure against organophosphorus G- and V-Series nerve agents, which can induce rapid lethality in guinea pigs and humans. It has been shown that upon combination with atropine (ATR) and pyridine-2-aldoxime methochloride (2-PAM), a single dose of GAL administered before or soon after the acute exposure to a lethal dose of organophosphorus compounds can safely counteract toxicity in guinea pigs. To that end a new sample preparation extraction method analysis assay has been explored to enable future high-throughput, reproducible, and sensitive assays for the extraction of galanthamine in guinea pig plasma. Samples were prepared with diphenhydramine hydrochloride (DPH HCl) internal standard and recovered with 10 min liquid-liquid trichloromethane extractions. Samples were analyzed with a reversed phase liquid chromatographic column interfaced to a triple quadrupole mass spectrometer (LC/MS/MS) operating in the positive ion multiple reaction monitoring (MRM) Turbo Ionspray mode. Precursor to product ion (M+H)+ transitions of 288-to-213 m/z and 256-to-167 m/z for GAL and DPH were observed, respectively. Sample run times of 1.50 min were achieved.

A new device for the capture and transport of small nonhuman primates in scientific research

Conventional methods of capturing marmosets and other small nonhuman primates (NHPs) require prolonged physical contact between animals and their handlers. This causes NHPs to become stressed and exhausted and can put both animals and handlers at risk of injury or exposure to infectious diseases. The authors designed a self-contained device for the capture and transport of small NHPs. Food rewards encourage primates to enter the device independently, and handlers can then easily access the animals for routine veterinary or experimental procedures. Preliminary observations suggest that marmosets quickly become accustomed to the device and that the device causes less stress than capture by hand or by net.

Chapter 28 – Botulinum Toxin*

Botulinum neurotoxins (BoNTs) are perhaps the most potent substances known to humankind. BoNTs comprise a family of seven distinct neurotoxic proteins produced by immunologically discrete strains of anaerobic bacteria (Clostridia). Their high toxicity, stability, remarkable persistence, ease of production, and previous history of weaponization by the military make BoNTs likely candidates for use as terrorist or battlefield agents. These properties of the toxin highlight the need to guard against the contamination of food and beverages, develop sensitive detection systems, and procure effective post-exposure pharmacological treatments. Animals and humans intoxicated by BoNTs display symptoms characteristic of an acute, symmetric, and descending flaccid paralysis. Paralysis is the result of toxin blocking the release of acetylcholine at peripheral nerve synapses. Although untreated botulism is potentially fatal, the availability of antiserum has dramatically reduced the mortality rates for the common clinical manifestations of the disease. This chapter provides a structured and comprehensive insight into the history, pathogenesis, toxicokinetics, mechanisms of action, risk assessment, toxicity, and treatment of BoNTs.

CHAPTER 30 – Botulinum Toxin

Publisher Summary Botulism is a disease caused by anaerobic, spore-forming bacteria found in soil. The disease results from the actions of chemical toxins produced by these bacteria. The most common forms of human botulism include food-borne, infant, and wound. The main etiology of botulism in humans is food-borne; this form is caused by eating foods contaminated with botulinum spores, which germinate and multiply into bacteria to produce neurotoxin in the food. Commonly contaminated foods include improperly preserved home-processed foods such as honey, corn, green beans, and beets. Botulinum neurotoxins (BoNTs) comprise a family of seven distinct neurotoxic proteins produced by immunologically discrete strains of the anaerobic bacteria. BoNTs are the most potent substances known to humankind. Due to their extremely high potency, ease of production and previous history of weaponization, the BoNTs have been designated as category A threat agents. The toxins are highly lethal, easy to isolate, and easy to deliver by terrorists. There are currently seven known antigenic serotypes of botulinum toxin, designated with the letters A through G, whereby antitoxin to one type does not cross-neutralize any of the others. Only early administration of antitoxin antibody in cases of suspected botulism will minimize the neurologic damage but will not reverse any existing paralysis. Paralysis could persist for weeks to months, and the available treatment consists of supportive care including fluids, total parenteral nutrition (TPN), and mechanical ventilation.

CHAPTER 31 – Anthrax

Publisher Summary Anthrax is a virulent, contagious, and potentially fatal disease. Depending on the route of exposure, anthrax can cause a different disease, including inhalational, cutaneous, and oral/ingestional forms. Anthrax infection involves a complex set of steps in its pathogenesis from spore uptake by immune cells, germination, transport to local lymph nodes, production of deadly toxins, systemic spread, and ultimately death of the host. Themes common among all anthrax infections are: uptake by macrophages and other immune cells, germination to the vegetative form at or near the site of inoculation prior to transit to target tissues, time course of transport to target organs, organs targeted for toxicity, overwhelming septicemia, and release of soluble factors responsible for death. Two types of B. anthracis have been identified: a spore form and a vegetative form. Some of the most common findings in human inhalational anthrax concern the respiratory tract. Pathological findings from inhalational anthrax patients in a bioterrorism-related outbreak cited hemorrhage and necrosis in mediastinal lymph nodes, hemorrhage, or inflammation of the pleurae and interhilar septae, and prominent intraalveolar macrophages or inflammation in the lung parenchyma. Treatment of any form of anthrax infection is generally the same: aggressive antibiotics and supportive care. Intravenous ciprofloxacin or doxycycline is recommended for treatment of anthrax, usually as part of a cocktail of antibiotics.

Chapter 29 – Anthrax

The fear of Bacillus anthracis has been heightened in recent years as a result of the terrorist events in 2001 and the increase in acts of terrorism worldwide. There are few infectious agents that are more notorious than B. anthracis. B. anthracis would be an ideal weapon for a terror attack because of its potent and lethal nature in the course of causing anthrax disease. Once inside the host, the organism can proliferate and spread rapidly, resulting in systemic infection and release of soluble toxin factors; anthrax produces a high mortality rate if left undiagnosed and untreated. To complicate matters, the bacillus responsible for anthrax infection can survive long-term outside of its host as a dormant spore. Additionally, host animals and humans infected by the bacterium and its anthrax toxins can display general symptoms characteristic of numerous infections or disease states, prolonging the time required for immediate diagnosis to initiate early treatment. This chapter provides a structured and comprehensive insight into the history, pathogenesis, toxicity, toxicokinetics, mechanisms of action, risk assessment, and treatment of anthrax. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the US Food and Drug Administration.

A nerve clamp electrode design for indirect stimulation of skeletal muscle.

A nerve clamp electrode was developed to indirectly stimulate skeletal muscle innervated by α motor neurons as an alternative to conventional electrodes. The stimulating electrode device consists of a spring coil-activated nerve clamp mounted inside a 1-mL syringe barrel. Supramaximal pulses were generated by a Grass stimulator and delivered to the nerve segment via the nerve clamp electrode. The salient feature of the electrode is its ability to produce muscle contractions indirectly through stimulation of the attached nerve. Indirect muscle stimulation is critical for studying the paralytic actions of presynaptic-acting toxins such as botulinum neurotoxins (BoNT), a potent inhibitor of acetylcholine (ACh) release from α motor neurons. This device enables stimulation of muscle contraction indirectly as opposed to contraction from direct muscle stimulation. The electrode is able to stimulate indirect muscle contraction when tested on ex vivo preparations from rodent phrenic nerve-hemidiaphragm muscle in similar fashion to conventional electrodes. In addition, the electrode stimulated external intercostal nerve-muscle preparations. This was confirmed after applying BoNT serotype A, a potent inhibitor of ACh release, to induce muscle paralysis. Alternative methods, including suction and bipolar loop electrodes, were unsuccessful in stimulating indirect muscle contraction. Therefore, this novel electrode is useful for physiological assessment of nerve agents and presynaptic actions of toxins that cause muscle paralysis. This electrode is useful for stimulating nerve-muscle preparations for which the length of nerve is a concern.