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Pretreatment for Nerve Agent ExposureChapter 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 MedicalResearch Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425; currently, Chemical Casualty Consultant, 14Brooks Road, Bel Air, Maryland 21014Medical Aspects of Chemical and Biological WarfareINTRODUCTION
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 ExposureTABLE 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 WarfarePyridostigmine 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 ExposureTABLE 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 WarfareTABLE 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
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Rethinking Innovation in Pharmaceutical R&D Health & Life Sciences The pharmaceutical industry is facing a challenge to be productive. One of thesolutions to this problem is for the industry to better harness innovation todeliver more and better drugs through the pipeline. But what is innovation and how can it deliver higher performance throughmore valuable drugs? Historically, p