Potential therapeutic effects of oximes 1

Besides the expected cholinergic crisis, nerve agents induce seizures at lethal concentrations, which progress rapidly within 3-4 minutes to generalised convulsive status epilepticus GCS

Chapter 1

Introduction

Therapeutic Effects Against Nerve Agent Soman

1.1.1 Toxicology of Nerve Agent Poisoning

Nerve agents belong to a group of organophosphorus compounds with high acute mammalian toxicity It can be broadly divided into two subclasses, the G- and V- agents (Somani et al 1992, Marrs et al 1996) The G-agents are derivatives of phosphoric acids, with the leaving group linked directly to the phosphorus atom (Fest et al 1982) The leaving group is usually a fluorine atom except in the case of tabun, which has a nitrile functional group as the leaving group V-agents are phosphonothioates of the P=O type, with the leaving group linked to the phosphorus atom through a sulphur atom (Table 1a & b) Nerve agents are colourless liquids when pure, but take on a yellow-brown colour when present in an impure form Detailed physicochemical properties of nerve agents are tabulated in Table 2

Important physical characteristics contributing to their application as chemical weapons are their high volatility and lipophilicity Having a high volatility facilitates their dispersion in the battlefield while their lipophilicity enhances penetration through skin G-agents differ from V-agents in having higher

Nerve agents are degraded in water with the rate of degradation correlated to their water solubility Sarin, the most soluble nerve agent, has a half-life of only 15 minutes in water as compared to the half-life of 577 min for the more lipophilic soman For the same reason, sarin has a faster rate of detoxification

in the body compared to soman, which is comparatively more persistent in

vivo The persistence of soman in the body is unique among nerve agents

and has been postulated to be due to existence of storage depots for soman

in the body Stored soman is protected from degradation in tissues and is released slowly into the circulatory system, resulting in the reappearance of signs of intoxication and death (Wolthuis 1981, Van Helden et al 1983, Somani et al 1992)

Nerve agents exert their effect through irreversible inhibition of the synaptic nerve enzyme, acetylcholinesterase (AChE) (Somani et al 1992, Marrs et al 1996) Acetylcholinesterase (AChE) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) and terminates neural impulse transmission at the cholinergic synapses (Figure 1) When a critical proportion

of the synaptic AChEs are inactivated by phosphorylation, signs of OP poisoning would be manifested Inhibition of AChE will result in the accumulation of acetylcholine at neural synapses to produce depolarisation block (Karalliedde et al 1993) Intoxication will result in depression of the brainstem respiratory center leading to loss of central respiratory drive (Rickett

et al 1986) Peripheral neuromuscular blockade from prolonged ACh-receptor interactions leading to receptor desensitization (i.e., desensitization block), will exacerbate respiratory paralysis leading to respiratory insufficiency and

Table 1a Structures of Nerve Agents (G-Agents)

Abbreviation Common

Name

GA Tabun Ethyl N-dimethylphosphoramido –

P O F O

CH3

CH3

Table 1b Formulae of Nerve Agents (V-Agents)

O O

CH2C

H2O

CH2C

H3

N

CH3C

H3

CH3

P C

O O

CH2C

H3

N

CH3

CH3

Figure 1 Photomicrograph (Upper) Of Neuromuscular Junction And Schematic Diagram (Lower; Adapted From FOI Chemical Defence Handbook 1996) Illustrating Effects Of Nerve Agent Inhibition Of Acetylcholinesterase Leading

To An Excess Of Acetylcholine At Nerve Synapse If Left Untreated, Manifestations Of Cholinergic Toxicity As Indicated In Table 3 Would Ensue.

Table 2 Physicochemical Properties of Classical Nerve Agents Properties

Appearance Colorless liquid (pure); Yellow-brown liquid (impure)

Vapour Pressure (mm Hg) at 25 O C

Volatility (mg/m 3 ) * at 25 O C

Solubility in water (% w/v) at

Besides the expected cholinergic crisis, nerve agents induce seizures at lethal concentrations, which progress rapidly (within 3-4 minutes) to generalised convulsive status epilepticus (GCSE), the most common and most potentially damaging form of status epilepticus (SE) GCSE is clinically defined as 30 minutes of continuous tonic-clonic seizure, with full loss of consciousness The time interval of 30 minutes was set to render the definition clinically useful and is partly justified on the basis of loss of self-compensatory physiological

changes in blood pressure, hyperthermia and cerebral damage (Shorvon et al 1992) Similarities between convulsive status in rodent and human have been previously demonstrated by (Treiman et al 1990), with similar 5 phases of electrocorticographic changes in various animal models of generalised convulsive seizures (i.e., kainic acid, bicuculline, lithium/pilocarpine and soman), which reflects a common underlying neurochemical mechanism in its initiation and maintenance (Meldrum et al 1973, Ben-Ari et al 1985, Koplovitz

et al 1998, Tang et al 2000)

From reported studies, the frequency and degree of neuropathology correlated well with the duration of nerve agent-induced seizures (McDonough

et al 1996) Seizures that were not terminated or progressed for at least 40 min produced severe neuropathology (Figures 2 and 3) and long-term behavioural deficits in all poisoned animals (Petras et al 1994, J.H McDonough et al 1986, 1995, 1996) with the pattern of injury resembling those described for status epilepticus (Oxbury et al 1971, Meldrum 1973, Corsellis et al 1983, Shorvon et al 1992)

Nerve agent induced-seizure is hypothesised by some research groups (McDonough et al.1997, Lallement et al 1999) to occur through two distinct neurochemical stages, an initial cholinergic phase followed by an established

Table 3 Main Effects of Nerve Agents at Various Sites in the Body

Receptor Target Organ Symptoms and signs _

Muscarinic Eye Long-lasting and painful miosis

conjuctival congestion Ciliary spasm,

Nose Nasal discharge/Rhinorrhea

Increased bronchial secretion Bronchoconstriction, Bronchospasm

Gastrointestinal Vomiting, abdominal cramps, diarrhea

Glandular Activity Excessive sweating, salivation

Bladder Frequent involuntary micturition

Nicotinic Skeletal muscle Weakness, fasciculations and paralysis

Autonomic ganglia Sympathetic effects, pallor, tachycardia,

hypertension

Central CNS Giddiness, anxiety, restlessness, headache,

tremor, confusion, failure to concentrate convulsions, respiratory depression, hypothermia

status epilepticus phase driven by glutamate neurochemistry. The role of

glutamate in sustaining established status epilepticus is validated through the use of NMDA receptor antagonists, which are effective in terminating established status in experimental animals (Shih 1990, Sparenborg et al

1992, McDonough et al 1993, Carpentier et al 1994) However, the ensuing neuropathology does not appear to be due to a direct neurotoxic action of nerve agent, nor to high levels of acetylcholine or hypoxic-anoxic injury mechanism (McDonough et al 1987) Instead, some research groups have postulated that excessive release of glutamate during nerve agent-mediated status epilepticus trigger NMDA-mediated excitotoxicity to produce the observed profound neuropathology (McDonough et al 1987, 1993, Shih et al 1990) However, besides activation of the glutamate system, delayed (>40 min following seizure onset) increases in extracellular dopamine levels has also been reported by other research groups (Fosbraey et al 1990, Jacobsson et al 1997, 1999) As dopamine serves as a major source of free radicals in the brain, massive release of dopamine has also been suggested

to generate oxidative stress on striatal neurons leading to increased neuronal loss in the striatum region (Pazdernik et al 2001)

In addition, besides the initial cholinergic hyperexcitation during nerve agent poisoning, sustained cholinergic excitation of locus coeruleus (LC) norepinephrine-containing neurons has also been reported by other research

Figure 2 Photomicrographs (25x) of MAP-2 Stained Brain Sections showing normal pattern of MAP-2 immunoreactivity at Piriform Cortex (A) and Hippocampus (B) of Control and following Soman Treatment, Arrows Indicate Loss of MAP-2 Staining in Piriform Cortex (C) and Hippocampus (D) Results were obtained from researcher’s own studies MAP refers to Microtubule-Associated Protein and is determined to be a Marker of Seizure-related Brain Damage (Ballough G.P H et al 1995)

C Piriform Cortex – Soman Treated D Hippocampus – Soman Treated

B Hippocampus - Control

A Piriform Cortex - Control

Figure 3 Cellular Pathology in Soman (1.6 x LD50) Treatment in Piriform Cortex (PC)

- Control (A), 1 day Post-Soman (C), 1 week Post-Soman (E), and in Hippocampus Hilus Dentate region - Control (B) 1 day (D), 1 week (F) Revealed By Cresyl Violet Staining (100x mag) Note massive neuronal degeneration at layer II and III and gradual loss of hilus polymorphic cells Extensive gliosis observed in both regions (highlighted by arrows) 1 week after soman Results were obtained from researcher’s own studies

A Piriform Cortex - Control

C Piriform Cortex – 1 day post soman treatment

E Piriform Cortex – 1 week post soman treatment

B Hippocampus - Control

D Hippocampus – 1 day post soman treatment

F Hippocampus – 1 week post soman treatment

groups (Mohamed et al 1992) Statistically significant reductions in neuronal norepinephrine (NE) content were observed in all brain regions 5-10 minutes after the onset of nerve agent-induced seizure activity This depletion in neuronal NE store was attributed to excessive release of NE by the authors and the absence of NE depletion in nerve agent poisoned animals that did not convulse was suggested by the authors to indicate an important innate anticonvulsant role for NE through 2A-postsynaptic heteroreceptor during the initial propagation phase of nerve agent seizures (Szot et al 2004) Their assertions are supported by a vast amount of literature indicating that a loss

of endogenous NE is proconvulsant (Weiss et al 1990, Mishra et al 1994, Weinshenker et al 2001b) and also by subsequent reports of synergistic enhancement of atropine anticonvulsant and neuroprotective actions by co-application of clonidine in a soman-poisoned rat model (Buccafuso et al 1986,

1987, 1988a, 1988b, 1996, Loke et al 2001, 2004) There could hence be more than one neurochemical mechanisms responsible for induction and propagation of neuropathology following nerve agent mediated seizures

1.1.2 Medical Countermeasures For Nerve Agent Poisoning

Medical countermeasure for nerve agent poisoning can be divided into four stages:

1.1.2.1 Pre-treatment, 1.1.2.2 Administration of Anticholinergic Drugs, 1.1.2.3 Anticonvulsant and Neuroprotection Therapy, 1.1.2.4 Administration of Oxime Reactivators

The nature of medical therapy in each stage will be further elaborated in the

following sections:

Pre-treatment for nerve agent intoxication is based on the assumption that AChE inhibited by reversible anticholinesterases will not be further attacked

by nerve agents Since only a small amount of acetylcholinesterase is required for maintaining neurotransmission at synapses, manifestation of cholingeric toxicity is averted (Kirby et al 1992, Sidell 1997) Current recommendations for pre-treatment required either single or repeated low doses of the carbamate known as pyridostigmine which is available in tablet forms Pyridostigmine is poorly absorbed through the gastrointestinal tract with a bioavailability of the administered dose at 5-10% Despite its poor bioavailability, enough of the drug is absorbed to cause an inhibition of 25-79% of serum butyrylcholinesterase activity A single dose of 30 mg pyridostigmine is able to achieve 25% reversible inhibition of

acetylcholinesterase (AChE) within 5h of administration (Sharab et al 1991)

The time taken by the drug to reach maximum concentration in the plasma is 60-120 min while the elimination half-life is 3-4 hr Consequently, the action of the drug is characterised by gentle onset, smooth course, comparatively prolonged duration and gradual decline of its action Symptoms developed within 15 minutes - 2 hr (average of 1.6 hr) and last up to 24 hr The drug penetrates poorly through the blood-brain barrier, hence it should not cause central nervous system effects unless high doses are taken Repeated doses

of 30 mg three times daily (one tablet every 8h) will produce sufficient

inhibition of plasma cholinesterase (20-40%) for protection against nerve agents without causing significant adverse effects

Pyridostigmine alone, without atropine and oxime reactivator therapy, is ineffective against agent intoxication There is also no evidence that pyridostigmine is of any therapeutic benefit against nerve agents with slower aging times, i.e., sarin and VX challenge (Somani et al 1992, Worek et al 1995a) On the contrary, it has been reported that pyridostigmine reduced the efficacy of atropine and 2-PAM against sarin and VX poisoning in rodents (Koplovitz et al 1992) Hence its use should only be recommended when the threat is either soman or tabun In addition, as a positively charged chemical moiety, pyridostigmine has difficulty penetrating the blood-brain barrier due to its limited solubility in lipids Consequently, pyridostigime offers only protection against peripheral anticholinesterase activity and has limited protection against the central effects of nerve agent poisoning Consequently, it was observed that while pyridostigimine prophylaxis could provide protection against the lethal effects of soman in animals, they do not protect against soman-induced performance decrements in the same animals (Blick et al

1994)

Atropine belongs to the class of anticholinergics, which act as reversible antagonists of acetylcholine at muscarinic receptors at synapses to minimise manifestations of cholinergic toxicity (Somani et al 1992, Marrs et al 1996)

slower compared to other tertiary ammonium-based anticholinergics, it possesses sufficient central ameliorative effects in nerve agent poisoning Atropine should be given as soon as possible after onset of agent effects Intra-muscular administration, through auto-injector devices such as the US Army Mark I Kit, is the preferred means in the field although intra-venous administration provides more rapid effects With intra-muscular injection, peak concentrations are obtained after 30 minutes while the initial distribution half-life following intravenous administration is 1 minute Atropine has an elimination half-life of 2.6-4.3 h and an apparent volume of distribution (VD) of 1-1.7 l/kg while its clearance rate is 5.9-6.8 ml/kg per min The elimination half-life is longer in children under 2 years of age and in the elderly where half-life may be prolonged to 10-30 h due to reduced clearance (Heath et al 1992)

Although the adequacy of atropinization is usually determined by drying of secretions, there are nonetheless some basic recommendations (Somani et al 1992) In cases of mild intoxication, 2 mg of atropine suffice as an initial dose and if the observed mild to moderate dyspnea (i.e., difficulty in breathing) is not resolved within 10-15 minutes, further doses of 2-4 mg of atropine is advised Patients with severe dyspnea and distress should be treated with 4-6

mg atropine at the onset An additional 4-6 mg of atropine should be administered if signs of moderate to severe dyspnea are still observed after 5-

10 minutes With severely exposed individuals, initial dose of atropine should

be 6 mg or higher Additional atropine should be administered for any increase in severity or lack of regressions of toxic signs While it is relatively

easy to dose atropine in the early phase of poisoning, it is more difficult to decide on the dose of atropine in the intensive care unit (ICU) In this phase, the patient is under sedation with benzodiazepines and opiates, which by itself influences pupil size There may be tachycardia due to SIRS (Systemic Inflammatory Response Syndrome) and the circulation may be instable Stabilisation of cardiovascular system with catecholamines could be instituted while atropine is gradually reduced to 0.5-2.0 mg/h Over-atropinisation in the intensive care unit is discouraged as it leads to longer stay in the intensive care unit and development of cholinergic hyper-sensitization In the absence

of pupil size for titration of atropine dose in the ICU, the absence of inhibitory activities in blood, recovery of blood cholinesterase to >30% of baseline levels and repetitive nerve stimulation could be used to determine the need for continuous atropine administration

Systemic administration of atropine is not expected to reverse miosis unless given in large amounts (Worek et al 1994) Consequently, eye pain resulting from nerve agent intoxication is usually relieved by local instillation of atropine However, as blurring of vision results from such therapy, atropine is only indicated in the event of severe pain in the eye (Somani et al 1992) In addition, while atropine therapy is effective in suppressing nerve agent’s pressor (direct or reflex) effects (Bataillard et al 1990), rapid administration of atropine to a cyanotic and hypoxic person will precipitate ventricular fibrillation,

a potentially fatal arrhythmia leading to collapse of the cardiovascular system (Worek 1995b) Hence, intravenous atropine therapy is contraindicated until

1.1.2.3 Anticonvulsant and Neuroprotection Therapy in Nerve Agent

Poisoning

Studies have also indicated a strong relationship between control of seizures and protection against the lethal effects of nerve agent exposure (McDonough, et al 1995, 1996, Shih et al 1998, 2003) A 38% increase in 24

hr mortality rate was observed in animals in which seizures were not terminated while the 10-day prognosis was even worse with a 57% increase

in mortality rate when seizures was not successfully terminated These animal studies indicated clearly that seizure control exerts significant influence on morbidity rates during nerve agents poisoning, and are in good agreement with clinical status epilepticus literature that suggested a similar relationship between the duration of convulsive status and increased morbidity rates (Oxbury et al 1971) Hence, status epilepticus in itself, has clinically been considered an emergency situation and seizures arrest has since been regarded as one of the immediate concerns in the treatment of nerve agent casualties and is no longer considered merely as an adjunct or supportive follow-on therapy (Dunn et al 1989)

Consequently, in November 1990, the anticonvulsant drug diazepam, was fielded in the form of autoinjector devices for immediate field treatment of nerve agent-induced seizures Each autoinjector device contains 10 mg of the benzodiazepine drug, diazepam, and is intended for co-administration with atropine and oxime reactivators at the onset of severe nerve agent intoxication Further development resulted in the introduction of a water soluble prodrug version of diazepam, Avizafone Although Avizafone, as a

water-soluble drug, is more suitable for intramuscular administration than the lipophilic diazepam, its anticonvulsant action is not superior to diazepam This

is due to incomplete conversion of this prodrug to diazepam in vivo leading

effectively to reduce plasma load of the active diazepam component thus negating its superior pharmacokinetic properties (Lallement et al 2000)

Diazepam was a logical initial choice of drug for management of nerve agent seizures as it is an approved medication by the U.S Food and Drug Administration (FDA) for clinical management of status epilepticus However, from subsequent animal studies, diazepam anticonvulsant efficacy in rats when administered late (>30 min) post-seizure onset is reduced and seizure activity recurred several hours later (McDonough JH et al 1995, Shih et al 1999a) The loss of diazepam’s anticonvulsant effects has been attributed to rapid modulation of GABAA receptors during status epilepticus that induces pharmacoresistance to benzodiazepine drugs, which acts by enhancing GABAA-mediated inhibition of neuronal excitability (Kapur et al 1996, Mazarati et al 1998, Jones et al 2002, Naylor et al 2005) Increasing diazepam dose to circumvent this pharmacoresistance is potentially dangerous due to benzodiazepine-potentiation of nerve agent-induced respiratory depression and is hence considered a medical risk for field management of nerve agent casualties with established status epilepticus conditions Similar pharmacoresistance to other classes of benzodiazepine and barbiturate drugs are either reported or expected on the basis of similar mechanism of action on GABAA receptor-mediated inhibition

Most of current research for alternate neuroprotectant drugs is centred on methyl-D-aspartate (NMDA) receptor antagonist compounds Current generation of NMDA drugs, while exhibiting neuroprotective effects during established status, have several drawbacks that rendered human applications non-viable Key drawbacks include potent psychomimetic properties, hyper-reactive behaviour and memory impairment They also have narrow therapeutic window, being neurotoxic at high doses by facilitating apoptotic death in healthy neurons (Shih et al 1990, Faber et al 2002) Hence, while work with NMDA receptor antagonists has proceeded for the past two decades, none is approved for clinical application New generations of non-competitive NMDA antagonist drugs are currently being evaluated as neuroprotectants against nerve agent-induced status epilepticus These included GK-11 or Gascyclidine, non-competitive NMDA receptor antagonist, from France (Lallement et al 1999) and HU-211, a dexanabinol that is a functional antagonist of the NMDA receptor but lacks cannabimimetic activity, from Pharmos, Israel (Filbert et al 1999) Both are currently in early phases of clinical trials and none is approved for human application till date

n-On the other hand, anti-cholinergics drugs, such as atropine sulfate, besides being used as the primary means for attenuating hyper-cholinergic toxicity during nerve agents poisoning, are also able, at supra-high doses, to terminate early seizures initiated by nerve agents (Shih et al 1999a, 1999b) However, while atropine alone has no anticonvulsant properties when administrated late (i.e 40 minutes) after seizure onset, tertiary anticholinergics, with their enhanced lipophilic and hence enhanced ability to penetrate the blood-brain