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Pretreatment for Nerve Agent Exposure Chapter 6
PRETREATMENT FOR NERVE
AGENT EXPOSURE
MICHAEL A. DUNN, M.D., FACP*; BRENNIE E. HACKLEY, JR., PH.D.†; AND FREDERICK R. SIDELL, M.D.‡ INTRODUCTION
AGING OF NERVE AGENT–BOUND ACETYLCHOLINESTERASE
PYRIDOSTIGMINE, A PERIPHERALLY ACTING CARBAMATE COMPOUND
Efficacy
Safety
Wartime Use
Improved Delivery
CENTRALLY ACTING NERVE AGENT PRETREATMENTS
NEW DIRECTIONS: BIOTECHNOLOGICAL PRETREATMENTS
*Colonel, Medical Corps, U.S. Army; Director, Clinical Consultation, Office of the Assistant Secretary of Defense (Health Affairs), Washing- ton, D.C. 20301-1200; formerly, Commander, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Mary-land 21010-5425 †Scientific Advisor, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425 ‡Formerly, Chief, Chemical Casualty Care Office, and Director, Medical Management of Chemical Casualties Course, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425; currently, Chemical Casualty Consultant, 14Brooks Road, Bel Air, Maryland 21014 Medical Aspects of Chemical and Biological Warfare INTRODUCTION
Nerve agents are rapidly acting chemical com- cal as well and may impair physical and mental pounds that can cause respiratory arrest within performance. A pretreatment must be administered minutes of absorption. Their speed of action im- to an entire force under a nerve agent threat. Any poses a need for rapid and appropriate reaction by resulting performance decrement, even a compara- exposed soldiers, their buddies, or medics, who tively minor one, would make pretreatment use must administer antidotes quickly enough to save unacceptable in battlefield situations requiring lives. A medical defense against nerve agents that maximum alertness and performance for survival.
depends completely on postexposure antidote treat- In the late 1980s, the United States, following the example of Great Britain, stocked the compoundpyridostigmine for its combat units as a wartime • In the stress of a chemical environment, contingency pretreatment adjunct for nerve agent even well-trained military personnel will exposure.3 Several other Allies, including most not be uniformly successful in performing members of the North Atlantic Treaty Organization (NATO), did so as well. At the recommended dose, pyridostigmine is free of performance-limiting side • Aging, a change over time in the interac- effects. Unfortunately, pyridostigmine by itself is tion of nerve agents with the target enzyme ineffective as a pretreatment against subsequent acetylcholinesterase (AChE), renders oxime nerve agent exposure and thus it is not a true pre- treatment compound. Pyridostigmine pretreatment agent antidotes) much less effective.2 As does provide greatly improved protection against explained below, aging poses an especially soman exposure, however, when combined with difficult problem for treating effects from postexposure antidote therapy. For this reason, pyridostigmine is classified as a pretreatment adjunct.
Research workers have attempted to develop true Because of these limitations of postexposure pro- nerve agent pretreatments whose own neurotoxic- tection, military physicians have focused on the ity is balanced or diminished by coadministration possibility of protecting soldiers from nerve agents of a pharmacological antagonist to their undesir- by medical prophylaxis, or pretreatment, designed able properties (eg, the carbamate compound phy- to limit the toxicity of a subsequent nerve agent ex- sostigmine, which is administered in combination posure. A significant problem with pretreatments, with a cholinolytic compound, such as scopola- however, has been their own potential for adverse mine). The potential and the problems of this pre- effects. In general, the pharmacological pretreatments treatment approach are considered in this chapter, that protect humans from the toxic effects of nerve along with a new pretreatment concept that in- agents are themselves neuroactive compounds.
volves inactivating or binding nerve agents with Thus, their principal adverse actions are neurologi- scavenger macromolecules in the circulation.
AGING OF NERVE AGENT–BOUND ACETYLCHOLINESTERASE
Organophosphate nerve agents inhibit the active ylation of the AChE-bound nerve agent molecule pro- site of AChE, a key enzymatic regulator of cholin- ceeds depends on the nature of the nerve agent.
ergic neurotransmission. As noted in Chapter 5, Table 6-1 shows the aging half-time of each of the Nerve Agents, agent-bound AChE can be reacti- five chemical compounds commonly considered to vated by a class of antidote compounds, the oximes, be nerve agents: tabun (GA), sarin (GB), soman which remove the nerve agent molecule from the Aging is an irreversible reaction. After de- During the attachment of the agent with the en- alkylation, an AChE-bound nerve agent molecule zyme, a portion of the agent—the leaving group— can no longer be removed from the enzyme by breaks off. During a second, later reaction, one of an oxime. Thus, aging of enzyme-bound nerve the nerve agent’s alkyl groups leaves: this is the agent prevents oxime antidotes from reactivating process known as aging. The rate at which this dealk- AChE. This is an extremely difficult problem in the Pretreatment for Nerve Agent Exposure TABLE 6-1
AGING HALF-TIME OF NERVE AGENTS
Nerve Agent
RBC-ChE Source
Half-Time
Data sources: (1) Mager PP. Multidimensional Pharmacochemistry.
San Diego, Calif: Academic Press; 1984: 52–53. (2) Doctor BP, Blick DW, Caranto G, et al. Cholinesterases as scavengers fororganophosphorus compounds: Protection of primate perfor- mance against soman toxicity. Chem Biol Interact. 1993;87:285– 293. (3) Sidell FR, Groff WA. The reactivatibility of cholinest-erase inhibited by VX and sarin in man. Toxicol Appl Pharm. 1974;27:241–252. (4) Talbot BG, Anderson DR, Harris LW,Yarbrough LW, Lennox WJ. A comparison of in vivo and in vitro rates of aging of soman-inhibited erythrocyte acetylcholinest-erase in different animal species. Drug Chem Toxicol. 1988;11:289– 305. (5) Hill DL, Thomas NC. Reactivation by 2-PAM Cl of Hu-man Red Blood Cell Cholinesterase Poisoned in vitro by Cyclohexyl- methylphosphonofluoridate (GF). Edgewood Arsenal, Md: Medi-cal Research Laboratory; 1969. Edgewood Arsenal Technical case of poisoning with soman, which ages within 2 of exposure (mg•min). For example, a PR of 1.0 would indicate a completely ineffective antidote, Aging appears to be a key limiting factor in the because it means that the LD50 or LCt50 is the same efficacy of postexposure oxime therapy for soman for subjects who received an antidote and those who poisoning. One method for assessing the relative did not. A PR of 5, on the other hand, indicates that efficacy of antidotes and other countermeasures is the LD50 or LCt50 for subjects who received an an- the determination of their protective ratios. The tidote is 5-fold higher than that for subjects who protective ratio (PR) of an antidote is the factor by did not receive one. A PR of 5 or greater is consid- which it raises the LD50 or the LCt50 of a toxic nerve ered to represent a reasonable level of effectiveness agent challenge. Readers will remember that LD50 for medical countermeasures against nerve agents.
is defined as the dose (D) of liquid or solid nerve This value was determined through threat analysis agent that is lethal (L) to 50% of the subjects ex- of battlefield conditions and consideration of the posed to it; LD50 is also described as the median fact that trained and equipped soldiers will be able lethal dose. LCt50 is the term used to describe the to achieve at least partial protection against nerve median lethal concentration for an aerosol or va- agent attacks by rapid donning of masks and use por agent, expressed as concentration (C) • time (t) PYRIDOSTIGMINE, A PERIPHERALLY ACTING CARBAMATE COMPOUND
Pyridostigmine is one of a class of neuro- (CNS). Pyridostigmine has been used for many active compounds called carbamates. Its chemical years in the therapy of neurological disorders, structure and that of a related carbamate, physo- especially myasthenia gravis, a disease of neuro- stigmine, are shown below. Like the nerve agents, muscular transmission. In patients with myasthe- carbamates inhibit the enzymatic activity of AChE.
nia gravis, inhibition of synaptic AChE is clinically As a quaternary amine, pyridostigmine is ionized under normal physiological conditions and pen- As an inhibitor of AChE, pyridostigmine in large etrates poorly into the central nervous system doses mimics the peripheral toxic effects of the or- Medical Aspects of Chemical and Biological Warfare Pyridostigmine
Physostigmine
ganophosphate nerve agents. At first it might seem fore based on a series of animal efficacy studies5–7 paradoxical that carbamate compounds should help conducted with several species in a number of coun- protect against nerve agent poisoning, but two criti- tries that found evidence that pyridostigmine pre- cal characteristics of the carbamate–enzyme bond treatment strongly enhances postexposure antidote explain the usefulness of the carbamates.
First, carbamoylation, the interaction between Data from one experiment are shown in Table 6- carbamates and the active site of AChE, is freely 2. In this study7 with male rhesus monkeys, pre- and spontaneously reversible, unlike the normally treatment with orally administered pyridostigmine irreversible inhibition of AChE by the nerve agents.
inhibited circulating red blood cell AChE (RBC- No oxime reactivators are needed to dissociate, or AChE) by 20% to 45%. (Inhibition of RBC-AChE by decarbamoylate, the enzyme from a carbamate com- pyridostigmine is a useful index of its inhibition of pound. Carbamates do not undergo the aging reac- AChE in peripheral synapses). Monkeys that had no pyridostigmine pretreatment were not well pro- Second, carbamoylated AChE is fully protected tected from soman by the prompt administration from attack by nerve agents because the active site of atropine and 2-pyridine aldoxime methyl chlo- of the carbamoylated enzyme is not accessible for ride (2-PAM Cl). The PR of 1.64 in these monkeys is binding of nerve agent molecules. Functionally, typical of the most effective known postexposure sufficient excess AChE activity is normally present antidote therapy in animals not given pretreatment in synapses so that carbamoylation of 20% to 40% before a soman challenge. In contrast to this low of the enzyme with pyridostigmine does not sig- level of protection, however, the combination of nificantly impair neurotransmission.
pyridostigmine pretreatment and prompt post- When animals are challenged with a lethal dose challenge administration of atropine and 2-PAM Cl of nerve agent, AChE activity normally decreases resulted in greatly improved protection (PR > 40 rapidly, becoming too low to measure. In pyrido- when compared with the control group; PR = 24 stigmine-pretreated animals with a sufficient quan- when compared with the group given atropine and tity of protected, carbamoylated enzyme, sponta- neous decarbamoylation of the enzyme regenerates Limitation of the number of animals available for enough AChE activity to sustain vital functions, soman challenge at extremely high doses made ac- such as neuromuscular transmission to support curate calculation of a PR indeterminate in this ex- respiration. Prompt postexposure administration periment. The PR was well in excess of 40, clearly of atropine is still needed to antagonize acetyl- meeting the requirement for effectiveness of 5-fold choline (ACh) excess, and an oxime reactivator improved protection. In a later study,8 four of five must also be administered if an excess of nerve rhesus monkeys receiving pyridostigmine pretreat- agent remains to attack the newly uncovered ment and postexposure therapy of atropine and 2- AChE active sites that were protected by pyrido- PAM Cl survived for 48 hours after being challenged with soman at a level 5-fold higher than its LD50.
Pyridostigmine pretreatment shows its strongest Efficacy
benefit (compared with atropine and oxime therapyalone) in animals challenged with soman and tabun Exposure of humans to soman is virtually un- and provides no benefit against challenge by sarin known in Western countries, with the exception of or VX.9–11 Table 6-3 shows the PRs obtained in ani- a single laboratory accident.4 The decision to pro- mals given atropine and oxime therapy after chal- vide military forces with pyridostigmine is there- lenge with the five nerve agents with and without Pretreatment for Nerve Agent Exposure TABLE 6-2
EFFECT OF THERAPY ON LD50 IN MONKEYS EXPOSED TO SOMAN
Mean LD50(µg/kg) [95% CL]
Mean Protective Ratio [95% CL]
*Indeterminate because of small number of subjects; PR relative to the atropine plus 2-PAM Cl group > 24 (617 ÷ 25.1)CL: confidence limit (based on a separate slopes model)LD50: the dose that is lethal to 50% of the exposed population PR: factor by which the LD50 of a nerve agent challenge is raised (in this experiment, the LD50 for group given therapy divided by 2-PAM Cl: 2-pyridine aldoxime methyl chlorideAdapted from Kluwe WM. Efficacy of pyridostigmine against soman intoxication in a primate model. In: Proceedings of the SixthMedical Chemical Defense Bioscience Review. Aberdeen Proving Ground, Md: US Army Medical Research Institute of Chemical De-fense; 1987: 233.
pyridostigmine pretreatment.9 As shown, pyrido- until it was determined that SCUD missiles fired stigmine pretreatment is essential for improved against them did not have chemical loads. Later, survival against soman and tabun challenge. With U.S. ground forces attacking into Iraq and Kuwait sarin or VX, depending on the animal system stud- used pyridostigmine only as long as the corps-level ied, pyridostigmine causes either no change or a commanders on the ground considered the Iraqi minor decrease in PRs, which still indicate strong efficacy of atropine and oxime therapy for exposure U.S. and Allied decisions to use pyridostigmine to these agents. The data for GF show no benefit followed established doctrine, taking into account from pyridostigmine pretreatment for mice and a Iraqi capabilities and intentions. Iraq was known small benefit for guinea pigs. The only published to have substantial stocks of sarin and VX, for which data8 on protection of primates from GF show a PR pyridostigmine pretreatment is unnecessary, as dis- of more than 5 with pyridostigmine pretreatment cussed above. However, Iraq was also known to be and atropine/oxime therapy, but a control group keenly interested in acquiring any compounds that treated with atropine/oxime alone for comparison might defeat Allied protection, such as soman. The was not included. Clinical experts from all coun- security of Warsaw Pact stocks of soman, for ex- tries who have evaluated pyridostigmine have con- ample, was a growing concern in 1990.
cluded from these data that it is an essential pre- In 1990, it was also known that Iraq had begun treatment adjunct for nerve agent threats under large-scale production of GF, a laboratory com- combat conditions, where the identity of threat pound that had not earlier been manufactured in agents is virtually never known with certainty.
weapons quantity. International restrictions on the Pyridostigmine was used to protect soldiers from purchase of chemical precursors of the better- an actual nerve agent threat in the Persian Gulf War.
known nerve agents may have led Iraq to acquire NATO Allies using pyridostigmine followed their cyclohexyl alcohol, which it then was able to use to national policies on chemical protection. British produce GF. Very limited data on medical protec- soldiers, for example, were ordered to take pyrido- tion against GF were not reassuring. Although GF’s stigmine for over a month while they were posi- aging time with AChE was reported to be relatively tioned near the Iraqi border. U.S. forces followed long (see Table 6-1), unpublished information from the doctrine of only using pyridostigmine when a Allied countries suggested that postexposure atro- nerve agent threat was assessed to be imminent by pine/oxime therapy in rodents exposed to GF did the responsible division- or corps-level commander.
not protect against the effects of GF poisoning. As Thus, soldiers of the U.S. XVIII Airborne Corps took confirmed by the later studies shown in Table 6-3, pyridostigmine for several days in January 1991 atropine/oxime therapy only provided rodents Medical Aspects of Chemical and Biological Warfare TABLE 6-3
EFFECT OF THERAPY WITH AND WITHOUT PYRIDOSTIGMINE PRETREATMENT ON
PROTECTIVE RATIOS IN ANIMALS EXPOSED TO NERVE AGENTS
Protective Ratio
Nerve Agent
Animal Tested
Atropine + Oxime
Pyridostigmine + Atropine + Oxime
*Two doses of pyridostigmine were used.
Data sources: (1) Joiner RL, Dill GS, Hobson DW, et al. Task 87-35: Evaluating the efficacy of antidote drug combinations againstsoman or tabun toxicity in the rabbit. Columbus, Oh: Battelle Memorial Institute; 1988. (2) Koplovitz I, Harris LW, Anderson DR,Lennox WJ, Stewart JR. Reduction by pyridostigmine pretreatment of the efficacy of atropine and 2-PAM treatment of sarin and VXpoisoning in rodents. Fundam Appl Toxicol. 1992;18:102–106. (3) Koplovitz I, Stewart JR. A comparison of the efficacy of HI6 and 2-PAM against soman, tabun, sarin, and VX in the rabbit. Toxicol Lett. 1994;70:269–279. (4) Sultan WE, Lennox WJ. Comparison of theEfficacy of Various Therapeutic Regimens, With and Without Pyridostigmine Prophylaxis, for Soman (GD) Poisoning in Mice and Rabbits.
Aberdeen Proving Ground, Md: US Army Chemical Systems Labororatory; 1983. ARCSL Technical Report 83103. (5) Anderson DR,Harris LW, Woodard CL, Lennox WJ. The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman orVX in rats. Drug Chem Toxicol. 1992;15:285–294. (6) Jones DE, Carter WH Jr, Carchman RA. Assessing pyridostigmine efficacy byresponse surface modeling. Fundam Appl Toxicol. 1985;5:S242–S251. (7) Lennox WJ, Harris LW, Talbot BG, Anderson DR. Relation-ship between reversible acetylcholinesterase inhibition and efficacy against soman lethality. Life Sci. 1985;37:793–798. (8) CapacioBR, Koplovitz I, Rockwood GA, et al. Drug Interaction Studies of Pyridostigmine With the 5HT3 Receptor Antagonists Ondansetron andGranisetron in Guinea Pigs. Aberdeen Proving Ground, Md: US Army Medical Research Institute of Chemical Defense; 1995.
USAMRICD Training Report 95-05. AD B204964. (9) Inns RH, Leadbeater L. The efficacy of bispyridinium derivatives in the treat-ment of organophosphate poisoning in the guinea pig. J Pharm Pharmacol. 1983;35:427–433. (10) Kluwe WM. Efficacy of pyridostigmineagainst soman intoxication in a primate model. In: Proceedings of the 6th Medical Chemical Defense Bioscience Review. Aberdeen Prov-ing Ground, Md: US Army Medical Research Institute of Chemical Defense; 1987: 227–234. (11) Stewart JR, Koplovitz I. The effect ofpyridostigmine pretreatment on the efficacy of atropine and oxime treatment of cyclohexylmethylphosphonofluoridate (CMPF)poisoning in rodents. Aberdeen Proving Ground, Md: US Army Medical Research Institute of Chemical Defense; 1993. Unpub-lished manuscript. (12) Koplovitz I, Gresham VC, Dochterman LW, Kaminskis A, Stewart JR. Evaluation of the toxicity, pathology,and treatment of cyclohexylmethlyphosphonofluoridate (CMFF) poisoning in rhesus monkeys. Arch Toxicol. 1992;66:622–628.
Pretreatment for Nerve Agent Exposure with PRs in the range of 1.4 to 2.7. The only pri- remains that animals pretreated with pyrido- mate data available showed that rhesus monkeys stigmine that receive atropine and oxime therapy given pyridostigmine pretreatment and atropine/ promptly after an otherwise lethal soman exposure oxime therapy uniformly survived a 5-LD50 chal- are able to maintain adequate respiration and survive.
lenge with GF.8 Concern about Iraq’s new GF capa- The major deficiency of pyridostigmine pretreat- bility, added to its known interest in acquiring ment is also related to its poor penetration into the soman, made Allied use of pyridostigmine a rea- brain. Animals that survive challenge with a supra- lethal dose of nerve agent because of pyridostigmine The fact that pyridostigmine inhibits AChE has pretreatment frequently show severe histological raised one theoretical problem with its use: if 20% evidence of brain injury, prolonged convulsions, to 40% of AChE has been inhibited by pyrido- and long-lasting performance impairments.15 Al- stigmine, would a subsequent low-level exposure though centrally acting carbamate pretreatment to a nerve agent, which might be well tolerated with compounds, such as physostigmine, offer a degree no pretreatment, be converted to a toxic dose if it of protection against nerve agent–induced brain raised the total percentage of AChE inhibition into injury, pretreatment with known brain-protecting a toxic range? In practice, it has not been possible compounds such as physostigmine, the benzodiaz- to clearly demonstrate such additive toxicity in epine anticonvulsants, and benactyzine has not animal experiments, perhaps because the increase been acceptable because of their known decremen- in nerve agent toxicity from initial signs to lethal- tal effects on performance. Postexposure anticon- ity rises very sharply over a narrow exposure range.
vulsant therapy appears to be the most practical, A minor additive toxicity effect would there- readily available approach to minimizing nerve fore be difficult to detect. The signs of mild nerve agent–induced brain injury and promoting rapid agent exposure are easily managed with antidote recovery of normal function after severe nerve agent therapy, and the benefit of a pretreatment in life- exposure (for further discussion, see Chapter 5, threatening exposures is so great as to clearly war- rant pyridostigmine pretreatment for soldierswhose exact extent of nerve agent exposure is not The fact that an ionized, hydrophilic carbamate Pyridostigmine has had a good safety record over compound such as pyridostigmine is effective as a the years of its administration to patients with my- pretreatment adjunct against soman suggests that asthenia gravis. Known adverse reactions have been its critical sites of action and, therefore, the critical limited to infrequent drug rashes after oral admin- sites where soman exerts its lethal effects, are out- istration and the complete set of signs of periph- side the blood–brain barrier. As noted in Chapter eral cholinergic excess, which have been seen only 5, Nerve Agents, respiratory arrest after lethal nerve when the dosage in patients with myasthenia gravis agent exposure appears to be a summation of the was increased to AChE inhibition levels well be- agent’s effects on tracheobronchial secretions and yond the 20% to 40% range desired for nerve agent bronchoconstriction with obstruction, impairment pretreatment. The effects of excessive pyrido- of neuromuscular transmission with respiratory stigmine—miosis, sweating, intestinal hypermotil- muscle insufficiency, and direct depression of cen- ity, and salivation—could clearly degrade soldiers’ tral respiratory drive. Electrophysiological monitor- ing suggests that of these processes, central respi- When the recommended adult dose of 30 mg of ratory drive may be the most susceptible to nerve pyridostigmine bromide, one tablet orally every 8 hours, has been followed, no significant decrements The effectiveness of pyridostigmine pretreatment have been found in the performance of a variety of may not be conclusive evidence against the impor- military tasks. A review of British studies reported16 tance of central mechanisms in respiratory arrest; that pyridostigmine caused no changes in memory, it appears that there is at least partial permeability manual dexterity, vigilance, day and night driving of the blood–brain barrier to polar compounds such ability, or in psychological tests for cognitive and as pyridostigmine, specifically in the regions of the psychomotor skills. No significant changes in sen- fourth ventricle and brainstem, where respiratory sory, motor, or cognitive functioning at ground centers are located. In addition, an increase in level, at 800 ft, and at 13,000 ft were noted in 12 blood–brain barrier permeability occurs rapidly subjects in another study17 after their fourth 30-mg after soman administration.13,14 The key observation Medical Aspects of Chemical and Biological Warfare The flight performance of subjects taking nicity in animals that were given pyridostigmine pyridostigmine in two studies18,19 was not affected, have had negative results (Hoffman-LaRoche, pro- no impairment in neuromuscular function was prietary information).33 In a study34 in which noted in a study20 in which subjects took pyrido- pyridostigmine was administered to rats, either stigmine for 8 days, and cardiovascular and pul- acutely or chronically, in doses sufficient to cause monary function were normal at high altitudes in an average 60% AChE inhibition, ultrastructural pyridostigmine-treated subjects in another study.21 alteration of a portion of the presynaptic mitochon- However, one study22 noted a slight decrement in dria at the neuromuscular junction resulted, as well performance in subjects taking pyridostigmine as alterations of nerve terminal branches, postsyn- when they tried to perform two tasks at the same aptic mitochondria, and sarcomeres. These morpho- time; these subjects also had a slight decrement on logical findings, which occurred at twice the AChE a visual probability monitoring task. Two studies23,24 inhibition level desired in humans, have not been found an increase in sweating and a decrease in skin correlated with any evidence of functional impair- blood flow in pyridostigmine-treated subjects sub- ment at lower doses, but they emphasize the need to limit enzyme inhibition to the target range of 20% Although there has been wide experience with to 40%. Pyridostigmine has been used by pregnant long-term administration of pyridostigmine to pa- women with myasthenia gravis at higher doses and tients with myasthenia gravis, until recently there for much longer periods than it was used during was no comparable body of safety data in healthy the Persian Gulf War and has not been linked to young adults. Short-term pyridostigmine adminis- fetal malformations.35 Because safety in pregnancy tration (one or two 30-mg doses) has been con- has not been completely established, the Food and ducted in peacetime in some countries, including Drug Administration considers pyridostigmine a the United States, to screen critical personnel, such Class C drug (ie, the risk cannot be ruled out).
as aircrew, for unusual or idiosyncratic reactions, Several studies have sought information on such as drug rash. The occurrence of such reactions pyridostigmine use under certain conditions: sol- appears to be well below the 0.1% level, and no mili- diers in combat who frequently take other medica- tary populations are now routinely screened with tions; wounding and blood loss; and use while un- administration of a test dose of pyridostigmine.
dergoing anesthesia. The possible interaction of Pyridostigmine for military use by the United pyridostigmine with other commonly used battle- States is approved only as a wartime contingency field medications was reviewed by Keeler.36 There measure. After the Persian Gulf War, there was appears to be no pharmacological basis for expect- much discussion about the use of pyridostigmine ing adverse interactions between pyridostigmine under an Investigational New Drug (IND) applica- and commonly used antibiotics, anesthetics, and tion.25–32 The Food and Drug Administration (FDA) analgesic agents. In a study37 of pyridostigmine- waived informed consent for its use to make the treated swine, for example, the autonomic circula- best medical treatment available in a specific com- tory responses to hemorrhagic shock and resusci- bat situation.26 The FDA based this waiver on (a) tation appeared normal. One potentially important data from animal studies conducted in both the effect of pyridostigmine deserves consideration by United States and other NATO countries that found field anesthesiologists and anesthetists using that pyridostigmine increases survival when used muscle relaxants for anesthesia induction: depend- as pretreatment against challenge by certain nerve ing on the duration of muscle-relaxant administra- agents (data on efficacy in humans challenged by tion, there may be either up- or down-regulation of nerve agents is not experimentally obtained), and postsynaptic ACh receptors.36 Clinical assessment (b) a long history of safety when the drug was used of the status of neuromuscular transmission using for approved indications at doses severalfold higher a peripheral nerve stimulator should provide a ba- than the doses administered in the military. Rarely sis for adjusting the dose of both depolarizing and considered in postwar discussions was the ethical nondepolarizing muscle relaxants to avoid an un- issue of nonuse: If pyridostigmine had not been desirable duration of muscle paralysis.
used, and Iraq had used nerve agents causing largenumbers of casualties, should the military have Wartime Use
been held responsible for withholding this drug? A limited number of animal studies of toxicologi- Pyridostigmine bromide tablets, 30 mg, to be cal abnormalities and teratogenicity and mutage- taken every 8 hours, are currently maintained in war Pretreatment for Nerve Agent Exposure thought to be at risk for nerve agent exposure. Data
on safety and possible adverse responses were col-
lected from the unit medical officers caring for the
41,650 soldiers of the XVIII Airborne Corps who
took from 1 to 21 doses of pyridostigmine during
January 1991.39 Most major unit commanders con-
tinued the medication for 6 to 7 days, with over
34,000 soldiers taking it for that time. There was
nearly total compliance with the regimen by these
soldiers, who were fully aware of the nerve agent
threat. They were able to perform their missions
without any noticeable impairment, similar to find-
ings with peacetime volunteers participating in
studies.16 However, they reported a higher than ex-
pected incidence of side effects, as noted in Table 6-4.
Fig. 6-1. A pyridostigmine blister pack containing 21
Gastrointestinal changes included flatus, loose 30-mg tablets, along with the carrying sleeve. This is the stools, and abdominal cramps that were noticeable nerve agent pyridostigmine pretreatment set (NAPPS) but not disabling. Together with urinary urgency, that was used by designated military personnel duringthe Persian Gulf War.
many soldiers reported a sense of awareness thatthey were taking a medication. In most soldiers,these changes were noticed within hours of taking stocks of U.S. combat units. The compound is pack- the first tablet. In many, the effects subsided after a aged in a 21-tablet blister pack called the nerve day or two of administration, and in others they agent pyridostigmine pretreatment set (NAPPS, persisted as long as pyridostigmine was adminis- Figure 6-1). One NAPPS packet provides a week of tered. Some units adopted a routine of taking pyridostigmine pretreatment for one soldier.3 pyridostigmine with meals, which was thought to The decision to begin pretreatment with pyrido- stigmine is made by commanders at army division Soldiers taking pyridostigmine during this pe- level or the equivalent, based on assessment of the riod were also experiencing a wide range of other nerve agent threat by their chemical, intelligence, wartime-related stresses, such as repeatedly don- and medical staff officers.3 Because of the lack ofdata on long-term administration of pyridostigmineto healthy adults, current doctrine calls for a maxi- TABLE 6-4
mum pretreatment period of 21 days, with reassess- EFFECTS OF PYRIDOSTIGMINE
ment at frequent intervals of the need for continued PRETREATMENT* ON U.S. SOLDIERS
pretreatment. A senior commander’s judgment about IN THE PERSIAN GULF WAR
the severity of a nerve agent threat beyond 21 daysdetermines whether pretreatment should continue.
Incidence (%)
Pyridostigmine is poorly absorbed when taken N=41,650
orally; its bioavailability is 5% to 10%.38 Ideally, twodoses of pyridostigmine, taken 8 hours apart, should be administered prior to any risk of nerve agent ex-posure.3 However, some benefit would be expected even if the first pyridostigmine dose is taken an hour before nerve agent exposure. Because excessive AChE inhibition can impair performance, no more than one 30-mg tablet should be taken every 8 hours. If a doseis forgotten or delayed, administration should sim- ply be resumed on an 8-hour schedule as soon aspossible, without making up missed doses.
*Dose was 30 mg pyridostigmine bromide, administered orally In Operation Desert Storm in 1991, pyrido- Adapted from Keeler JR, Hurst CG, Dunn MA. Pyridostigmine stigmine was administered under combat condi- used as a nerve agent pretreatment under wartime conditions.
tions for the first time to U.S. and Allied soldiers Medical Aspects of Chemical and Biological Warfare ning and removing their chemical protective suits Later in the Persian Gulf War, more than 200,000 and masks in response to alarms, sleep deprivation, service members took pyridostigmine for 1 to 4 days and anticipation of actual combat. Because there during the ground attack into Iraq and Kuwait.
was no comparable group of soldiers undergoing Their medical experience, as personally reported to identical stresses without taking pyridostigmine, us by many unit medical officers, was similar to that it is not clear to what extent pyridostigmine reported above. It is now clear that pyridostigmine itself was responsible for the symptoms noted can be used effectively in large military populations above. The findings are thus a worst-case estimate under combat conditions without impairing mis- for effects attributable to pyridostigmine use in sion performance. Soldiers must have a clear un- derstanding of the threat and the need for this medi- Among these soldiers, fewer than 1% sought cation, however. Otherwise, it seems unlikely that medical attention for symptoms possibly related to they would have the same degree of willingness to pyridostigmine administration (483 clinic visits).
accept the gastrointestinal and urinary symptoms Most of these had gastrointestinal or urinary dis- noted above or to comply with an 8-hour dosage turbances. Two soldiers had drug rashes; one of them had urticaria and skin edema that responded In a group of 213 soldiers in Israel who took to diphenhydramine. Three soldiers had exacerba- pyridostigmine (30 mg every 8 h), 75% reported at tions of bronchospasm that responded to bron- least one symptom. Included among these symp- chodilator therapy. Because the units of the XVIII toms were excessive sweating (9%), nausea (22.1%), Airborne Corps had been deployed to a desert en- abdominal pain (20.4%), diarrhea (6.1%), and uri- vironment for 5 months before pyridostigmine was nary frequency (11.3%). In a smaller group of 21 used, most soldiers with significant reactive airways soldiers, pseudocholinesterase (also called butyro- disease had already developed symptoms and had cholinesterase, which is discussed later in this chap- been evacuated earlier. The consensus among medi- ter) activity was the same in the 12 who were symp- cal personnel more recently arrived was that they tomatic and the 9 who were not symptomatic.40 saw more pyridostigmine-related bronchospasm in An Israeli soldier who developed cholinergic their soldiers, who had not been present in theater symptoms after taking pyridostigmine was re- ported41 to have a genetic variant of serum butyro- Because of increased exposure to the work-of- cholinesterase. The variant enzyme has low breathing requirements of being masked, as well as binding affinity for pyridostigmine and other car- inhaled dust, smoke, and particles, it was unclear bamates. The authors of the report suggested that whether pyridostigmine was a major causative fac- persons who are homozygous for the variant en- tor in those who had bronchospasm at the onset of zyme could therefore show exaggerated responses hostilities. Two soldiers from the XVIII Airborne to anticholinesterase compounds. The soldier had Corps had significant blood pressure elevations, with a history of prolonged apnea after receiving succi- diastolic pressures of 110 to 120 mm Hg, that mani- nylcholine premedication for surgery. Persons with fested as epistaxis or persistent bleeding after a cut similar histories of severe adverse responses to cho- and subsided when pyridostigmine was stopped.
linergic medications should be carefully assessed Another soldier who took two pyridostigmine tab- concerning their potential deployability to combat, lets together to make up a missed dose experienced where they might face either a nerve agent threat mild cholinergic symptoms, self-administered an or the potential need for resuscitative surgery in- atropine autoinjector, and recovered fully after sev- volving emergency induction of anesthesia36 using eral hours. There were no hospitalizations or medi- cal evacuations attributable to pyridostigmine Since the Persian Gulf War, veterans of that among XVIII Airborne Corps soldiers. In other units, conflict have experienced a range of illnesses at least two female soldiers, both weighing approxi- in themselves, in their spouses, and in children mately 45 to 50 kg, noted increased salivation, mus- conceived after the conflict. Combinations of symp- cular twitching, severe abdominal cramps, and toms have included fatigue, skin rash, muscle and sweating that prompted medical observation. The joint pain, headache, loss of memory, shortness symptoms subsided after pyridostigmine was of breath, and gastrointestinal and respiratory stopped. This experience suggests that cholinergic symptoms, which could be explained by a variety symptoms may occur in a small number of persons of conditions, but do not fit readily into a single Pretreatment for Nerve Agent Exposure The possible interaction of multiple, potentially Improved Delivery
toxic compounds has generated interest in thecontext of these problems. With respect to pyrido- The currently stocked 30-mg pyridostigmine bro- stigmine, one report43 was published of neurotox- mide tablets were purchased for wartime contin- icity in chickens that received pyridostigmine together gency use because of their ready availability.
with large parenteral doses of the insect repellent Clearly, the need to maintain an 8-hour schedule of DEET (diethyltoluamide) and the insecticide pyridostigmine pretreatment under the conditions permethrin. The relevance of this report is doubt- of actual or anticipated combat stress is a major ful, because systemic administration of the two in- practical deficiency in our medical defense against teracting compounds to the chickens was at least 10,000-fold in excess of their maximum potential The United States is considering the development absorption from skin or clothing of soldiers.
of sustained-release forms of pyridostigmine that Both the National Institutes of Health and the would permit maintenance of an adequate level of National Institute of Medicine of the National Acad- AChE inhibition with once-daily oral administra- emy of Sciences established expert panels to evalu- tion. To date, however, no sustained-release prepa- ate these problems and to suggest an etiology or ration has shown sufficient promise to warrant ad- etiologies. Both panels held public hearings, which vanced testing. Unfortunately as well, efforts to included testimony from veterans with the symp- provide transdermal delivery of pyridostigmine toms. The initial reports44,45 of these panels found with skin patches have had disappointing results, no evidence to suggest that pyridostigmine use was as would be expected because of the polar nature CENTRALLY ACTING NERVE AGENT PRETREATMENTS
The inability of pyridostigmine to provide pro- Neuroactive compounds that penetrate the CNS tection against nerve agent–induced CNS injury has generally cause some degree of performance im- led to two different pharmacological approaches to pairment in experimental animals, as well as a vari- protection. The first involves improving postex- able incidence of symptoms, such as nausea and posure treatment with brain-protecting anticonvul- light-headedness, in humans. Even a slight degree sant compounds, such as benzodiazepines. While of impaired performance of critical battlefield tasks these compounds have a clear-cut, intrinsic poten- would be life-threatening in itself and therefore tial for functional impairment and incapacitation, would be unacceptable in a pretreatment to be ad- their administration to casualties who are already ministered to all combatants. A possible solution to incapacitated by nerve agents will not increase the this problem is antagonism of the undesirable ef- total number of casualties. In fact, clinical observa- fects of carbamates, which are generally cholinergic tion of nonhuman primates suggests that postex- in nature, by simultaneous administration of a posure therapy with the benzodiazepines diazepam cholinolytic pretreatment adjunct, such as atropine, and midazolam actually decreases the time to re- scopolamine, or trihexyphenidyl (Artane, manufac- covery of consciousness after soman intoxication.46 tured by Lederle Laboratories, Wayne, NJ). Animals An alternative to postexposure therapy is pro- treated with what has been called a behavior-defi- tection of the CNS with pretreatment compounds cit-free combination of physostigmine and a choli- that penetrate the blood–brain barrier, such as phy- nolytic compound, for example, show excellent pro- sostigmine, a tertiary amine that freely enters the tection against subsequent nerve agent challenge and CNS. Physostigmine is often used as a model com- rapid clinical recovery of normal function.49 pound for reproducing in laboratory animals the In theory, it is possible to offset the side effects clinical signs of nerve agent intoxication. This non- of physostigmine and achieve a performance-defi- polar compound carbamoylates CNS AChE and cit-free effect by careful titration with a cholinergic protects experimental animals from nerve agent blocking drug. The severely limiting factor in challenge more effectively than does pretreatment developing a physostigmine combination pretreat- with pyridostigmine.47 Another centrally acting car- ment for practical use is an unacceptable degree bamate compound, cui-xing-ning, with character- of interindividual variation in the bioavailability of istics that are apparently similar to those of phy- this short half-life compound when administered sostigmine, has been evaluated in China.48 to humans.50,51 At present, it would appear necessary Medical Aspects of Chemical and Biological Warfare to define, for each recipient, an acceptable dose through in individual drug delivery of a well- ratio for physostigmine and a cholinolytic adjunct to matched, centrally acting pair of carbamate and avoid performance deficits. The effort required for adjunct compounds, the possibility of developing protecting a total force is clearly beyond our cur- centrally acting pretreatments would merit further rent capability. In the event of a technological break- NEW DIRECTIONS: BIOTECHNOLOGICAL PRETREATMENTS
Until recently, medical defense against nerve ministration of either of these human bioproducts, agents has focused on preventing or reversing the with a potential plasma half-life of up to 12 days binding of the agents to AChE, as well as on limit- for BuChE, may be able to provide similar protec- ing the effects of the agents on neurotransmission tion against nerve agent challenge for humans. The by administration of pharmacological antagonists main obstacles to development of these products such as atropine. An intriguing new concept for at the present time appear to be the high cost of dealing with nerve agent toxicity involves taking production of the quantities involved and the pos- advantage of naturally occurring macromolecules, sible need for frequent parenteral administration of such as a circulating nerve agent scavenger or a me- tabolizing enzyme, that would, respectively, bind Another biotechnological protective strategy un- to or catalyze the hydrolysis of nerve agents. These der active study is the production of monoclonal macromolecules have the potential of providing antibodies with high affinity for nerve agents.60,61 If protection against all effects of nerve agents with a human-derived monoclonal antibody of the im- minimal side effects, since they would stoichio- munoglobulin G (IgG) class could be produced, metrically bind or metabolize a nerve agent before theoretically it would have the advantage of being its distribution to the site of toxic effect.
able to bind and thus protect against a soman chal- The first evidence that circulating macromol- lenge in man after administration of about 2 g of ecules have potential for protecting animals from antibody protein, similar to the amount of poly- nerve agents came from study of the remarkably clonal antibody routinely administered in 10 mL of broad range of toxic doses of the nerve agents in standard immune serum globulin. The 6-week different animal species. For example, the LD50 of plasma half-life of IgG in man would make the use soman in mice and rats is about 10-fold higher than
the LD50 in monkeys or guinea pigs.52 An enzyme, Nerve agents, like other reactive small molecules, plasma carboxylesterase, binds to and thus inacti- pass through a high-energy transition state during vates soman and other nerve agents in the G series their reaction with water or with tissue targets such (but not VX). The different amounts of this enzyme as AChE. By preparing antigens with a geometry in the blood of various species can adequately ex- that spatially mimics the transition states of these plain their differential sensitivity to the G-series small molecules,62 researchers have raised antibod- ies which not only bind to the nerve agent molecules In addition to carboxylesterase, blood contains but also catalyze their hydrolysis.63 These catalytic two forms of cholinesterase, AChE in the red cells antibodies have a major advantage over the other (RBC-AChE) and butyrocholinesterase (BuChE; also bioproducts noted above in that they could continue called pseudocholinesterase and plasma cholines- to inactivate multiple nerve agent molecules. For terase) in the plasma. Both of these forms of cho- this reason, the preparation of catalytic antibodies linesterase bind and inactivate nerve agents. In to nerve agents, if successful, may result in the de- preloading experiments in which exogenous AChE velopment of a superior, long-term nerve agent pre- from fetal bovine serum or BuChE from equine or human sources was administered to animals (non- Enzymes found in hepatocytes,64 neuronal cells,65 human primates, mice, or rats) intravenously or and plasma also hydrolyze nerve agents, albeit com- intramuscularly, a stoichiometric degree of protec- paratively weakly. Study of the requirements for tion against subsequent nerve agent challenge was hydrolysis at the enzyme active sites could poten- provided.54–57 Investigators supported by the U.S.
tially lead to the design of more efficient hydrolytic Army Medical Research Institute of Chemical De- enzymes that could be used as catalytic scavengers.66 fense have recently cloned and expressed the genes The major reason for interest in biotechnolog- for both human AChE and human BuChE.58,59 Ad- ically derived nerve agent pretreatments lies in their Pretreatment for Nerve Agent Exposure unique mechanism of action as potential circulat- ed with levels of agent below the limits of their cir- ing nerve agent scavengers and hydrolytic catalysts.
culating protection without requiring the use of Animals protected against nerve agent challenge masks or protective clothing. The operational with these compounds have shown no evidence advantage that these soldiers would have over op- of toxicity or performance impairment from ponents encumbered by chemical protective equip- the nerve agents.54–56 Thus, soldiers pretreated ment adds considerable appeal to exploring the with these products might be able to function potential of these newer nerve agent countermea- normally in a chemical environment contamina- nerve agent–induced injury—may be overcome by for nerve agent casualties, particularly those with postexposure administration of anticonvulsants.
potentially lethal exposures to soman, has been While centrally acting pretreatments offer more ef- of great concern. Development of pyridostigmine, fective protection than does pyridostigmine, their a peripherally active carbamate compound, as development is limited because of their potential a nerve agent pretreatment adjunct has substanti- for impairing soldier performance. New research ally improved the ability of the U.S. military to pro- may provide a revolutionary advance in protection tect its soldiers from the lethal effects of nerve against nerve agents with biotechnologically de- agents. A major deficiency of this pretreatment pro- rived pretreatments that bind or inactivate nerve gram—that it does not protect the CNS against 1. Soldier’s Manual of Common Tasks. Skill Level 1. Washington, DC: Department of the Army; 1994: 507–510. Report 2. Michel HO, Hackley BE Jr, Berkowitz L, et al. Aging and dealkylation of soman (pinocolylmethyl- phosphonofluoridate)-inactivated eel cholinesterase. Arch Biochem Biophys. 1967;121:29–34.
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