Effectiveness of Donepezil, Rivastigmine, and (?)Huperzine A
in Counteracting the Acute Toxicity of Organophosphorus
Nerve Agents: Comparison with Galantamine
Yasco Aracava, Edna F. R. Pereira, Miriam Akkerman, Michael Adler, and
Edson X. Albuquerque
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland
(Y.A., E.F.R.P., M.Ak., E.X.A.); and Neurobehavioral Toxicology Branch, Analytical Toxicology Division, U.S. Army Medical
Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland (M.Ad.)
Received August 11, 2009; accepted September 8, 2009
Galantamine, a centrally acting cholinesterase (ChE) inhibitor
and a nicotinic allosteric potentiating ligand used to treat
Alzheimer’s disease, is an effective and safe antidote against
poisoning with nerve agents, including soman. Here, the
effectiveness of galantamine was compared with that of the
centrally active ChE inhibitors donepezil, rivastigmine, and
(?)huperzine A as a pre- and/or post-treatment to counteract
the acute toxicity of soman. In the first set of experiments,
male prepubertal guinea pigs were treated intramuscularly
with one of the test drugs and 30 min later challenged with
1.5 ? LD50soman (42 ?g/kg s.c.). All animals that were
pretreated with galantamine (6–8 mg/kg), 3 mg/kg donepe-
zil, 6 mg/kg rivastigmine, or 0.3 mg/kg (?)huperzine A sur-
vived the soman challenge, provided that they were also
post-treated with atropine (10 mg/kg i.m.). However, only
galantamine was well tolerated. In subsequent experiments,
the effectiveness of specific treatment regimens using 8
mg/kg galantamine, 3 mg/kg donepezil, 6 mg/kg rivastig-
mine, or 0.3 mg/kg (?)huperzine A was compared in guinea
pigs challenged with soman. In the absence of atropine, only
galantamine worked as an effective and safe pretreatment in
animals challenged with 1.0 ? LD50soman. Galantamine
was also the only drug to afford significant protection when
given to guinea pigs after 1.0 ? LD50soman. Finally, all test
drugs except galantamine reduced the survival of the ani-
mals when administered 1 or 3 h after the challenge with 0.6
or 0.7 ? LD50soman. Thus, galantamine emerges as a
superior antidotal therapy against the toxicity of soman.
The nerve agents soman, sarin, VX, and tabun are organ-
ophosphorus (OP) compounds chemically related to, but far
more toxic than OP insecticides used worldwide in agricul-
ture and households. There are reports that some of these
agents have been used as weapons of mass destruction with
catastrophic results in the Second Sino-Japanese War, the
1980s Iraq-Iran conflict, and the 1990s terrorist attacks in
Japan (Romano and King, 2001).
Although OP compounds interact with a number of molec-
ular targets, acute signs of OP poisoning result primarily
from the irreversible inhibition of acetylcholinesterase
(AChE) (Newmark, 2007). Thus, overactivation of muscarinic
receptors by accumulated acetylcholine (ACh) causes miosis,
increased secretions, bronchoconstriction, hypotension, and
diarrhea. Overstimulation of nicotinic receptors triggers in-
tense skeletal muscle fasciculations and subsequent desen-
sitization of these receptors leads to muscle weakness. Cen-
tral nervous system (CNS)-related effects commonly seen in
severe cases of acute OP intoxication include anxiety, rest-
lessness, confusion, ataxia, tremors, seizures, impairment of
respiratory drive, and coma (Shih et al., 2003).
Approved treatment of OP poisoning relies on the use of
atropine to block muscarinic receptors, pralidoxime to reac-
tivate OP-inhibited AChE, and benzodiazepines to control
This work was supported by the Army Research Office [Contract
W911NF-06-1-0098]; and the National Institutes of Health National In-
stitute of Neurological Disorders and Stroke [Grant UO1NS059344]
Disclosures: Opinions or assertions contained herein are the private views
of the authors and should not be construed as official or as reflecting the views
of the U.S. Army, the Department of Defense or the federal government. The
use of galantamine as an antidote against OP poisoning is protected under
Albuquerque EX, Adler M, and Pereira EFR (2005) inventors; University of
Maryland, Baltimore, Assignee. International Patent Application PCT/US05/
33789. 2005 Sept 23.
Article, publication date, and citation information can be found at
ABBREVIATIONS: ACh, Acetylcholine; AChE, acetylcholinesterase; APL, allosteric potentiating ligand; BuChE, butyrylcholinesterase; ChE,
cholinesterase; OP, organophosphorus; CNS, central nervous system; ANOVA, analysis of variance; NMDA, N-methyl-D-aspartate.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
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JPET 331:1014–1024, 2009
Vol. 331, No. 3
Printed in U.S.A.
OP-induced convulsions (Newmark, 2007). However, AChE
inhibited by some OPs, particularly soman, is refractory to
reactivation by clinically available oximes (Kassa, 2002). In
addition, reduction of the incidence and severity of OP-
induced convulsions is not sufficient to prevent the develop-
ment of neuropathology (Filliat et al., 1999). Consequently,
the fatality rate from OP insecticide poisoning remains ex-
tremely high despite the use of these treatments, exceeding
100,000 deaths annually (Buckley et al., 2004). The preva-
lence of delayed neurotoxic effects is also alarming among
farmers and workers who frequently handle OP pesticides
and among the first responders who treated the civilian
population exposed to sarin during the 1995 terrorist attack
in Japan (Nishiwaki et al., 2001; Buckley et al., 2004). These
reports underscore the urgent need for the development of a
medical countermeasure to increase the preparedness of the
first responders to attend the general population in the event
of a terrorist attack and to better treat an accidental/occupa-
tional exposure to OP compounds.
Pretreatment with pyridostigmine, a reversible cholinest-
erase (ChE) inhibitor, prevents the OP-induced irreversible
enzyme inhibition and, thereby, increases the survival of
laboratory animals acutely exposed to lethal doses of nerve
agents, provided that atropine and pralidoxime chloride are
administered promptly after the exposure (Jones et al., 1985).
The ultimate usefulness of pyridostigmine is limited by two
factors. First, as a quaternary base, pyridostigmine does not
cross the blood-brain barrier appreciably and, therefore, does
not protect CNS AChE from the irreversible inhibition by
OPs. Second, in addition to inhibiting AChE, pyridostigmine
blocks butyrylcholinesterase (BuChE), an endogenous scav-
enger for OP compounds (Doctor et al., 1991). Thus, centrally
acting reversible inhibitors that are more selective for AChE
than for BuChE may offer advantages in the medical man-
agement of OP intoxication.
Potent, reversible AChE inhibitors capable of crossing the
blood-brain barrier, including physostigmine and tacrine, af-
ford protection against OP toxicity, but, in general, at doses
that produce some level of CNS impairment (Deshpande
et al., 1986; Fricke et al., 1994). In contrast, galantamine, a
centrally acting reversible AChE inhibitor approved for
treatment of mild-to-moderate Alzheimer’s disease (Corey-
Bloom, 2003), has been shown to counteract the acute toxicity
and lethality of soman and sarin with no apparent central or
peripheral toxicity (Albuquerque et al., 2006).
A number of unique actions of galantamine contribute to
its effectiveness and safety as a medical countermeasure
against OP poisoning. First, the selective AChE inhibition by
galantamine (Thomsen and Kewitz, 1990) should help to
preserve the scavenger capacity of plasma BuChE for OPs.
Second, galantamine crosses the blood brain barrier readily,
and, thereby, can protect brain AChE from OP-induced irre-
versible inhibition. Third, acting as a nicotinic allosteric po-
tentiating ligand (APL), galantamine has neuroprotective
actions (see Pereira et al., 2002; Albuqueraque et al., 2009).
The present study was designed to examine whether the
centrally acting reversible ChE inhibitors donepezil and riv-
astigmine, both of which are currently approved for treat-
ment of Alzheimer’s disease, and (?)huperzine A can be used
safely and effectively as pre- or post-treatments to curtail the
acute toxicity of soman in guinea pigs, the best nonprimate
model of OP poisoning (Inns and Leadbeater, 1983). Results
presented herein indicate that, like galantamine, all three
drugs can prevent the lethality of 1.5?LD50soman provided
that the animals are treated with atropine immediately after
the nerve agent challenge. In contrast to galantamine, how-
ever, donepezil, rivastigmine, and (?)huperzine A are toxic
at their respective therapeutic doses and are not effective as
post-treatments in soman-intoxicated animals.
Materials and Methods
Animal Care and Treatments. Male albino guinea pigs [Crl-
(HA)Br] were purchased from Charles River Laboratories (Wilming-
ton, MA) and were 33 to 35 days old (320–350 g) on arrival at the
animal-care unit. They were housed in a controlled animal-care unit
with constant temperature (21 ? 0.5°C) and a 12-h light/dark cycle.
Animals were handled daily and used between 5 and 7 days after
arrival. Guinea pigs were chosen for this study because they are
similar to humans and nonhuman primates in their sensitivity to OP
compounds. Like humans and nonhuman primates, guinea pigs have
low levels of circulating carboxylesterases, the enzymes that hydro-
lyze and inactivate OP compounds. Thus, the LD50values for most
OP nerve agents in mice and rats are much higher than in monkeys
or guinea pigs. For example, the subcutaneous LD50for the OP nerve
agent soman in mice and rats is approximately 4- and 10-fold higher
than in guinea pigs and monkeys, respectively (Maxwell et al., 2006).
In addition, several lines of evidence indicate that guinea pigs are
the best small-animal model to predict the effectiveness of medical
therapies against OP intoxication in humans. Pyridostigmine bro-
mide, a peripherally acting carbamate that reversibly inhibits ChE,
provides better protection against OP toxicity in non-human pri-
mates and guinea pigs than in rats or mice (Dirnhuber et al., 1979).
In addition, pralidoxime, a reactivator of OP-bound AChE, affords
significant protection against soman intoxication in rats and mice,
but not in primates and guinea pigs (Inns and Leadbeater, 1983).
Thus, in an attempt to standardize testing of antidotes against OP
intoxication, it is advised that initial studies be performed in guinea
pigs and that results be confirmed subsequently in non-human pri-
mates. The use of male guinea pigs at prepubertal ages facilitates the
comparison of the results obtained herein using centrally acting
AChE inhibitors with those published earlier using pyridostigmine.
Donepezil, rivastigmine, or (?)huperzine A was injected intra-
muscularly in a hind limb in volumes not exceeding 0.5 ml/kg, before
or after subcutaneous injection of soman (0.6, 1.0, or 1.5 ? LD50). As
reported in numerous studies (e.g., Inns and Leadbeater, 1983; Lal-
lement et al., 1997), the reference 1.0 ? LD50for soman in prepu-
bertal male guinea pigs is 28 ?g/kg s.c. Whenever stated, atropine
sulfate (10 mg/kg i.m.) was injected into the other hind limb. The
subcutaneous injection of soman was made between the shoulder
blades, and the injection volume did not exceed 0.5 ml/kg. Treatment
of animals with nerve agents was performed according to procedures
set forth by the U.S. Army Medical Research Institute of Chemical
On the day of the experiments, a vial containing an aliquot (0.3–
0.5 ml) of the U.S. Army-issued stock solution of soman (1.88–1.90
mg/ml) was diluted with sterile saline to appropriate concentrations
and kept on ice for the duration of the experiment. At the end of the
experiments, any remaining diluted OP was decontaminated with
10% sodium hydroxide before disposal. Solutions of each test drug
were prepared on the day of the experiment, and injections were
made by use of disposable tuberculin syringes with 25- or 26-gauge
needles. After all treatments, animals were kept warm with a heat
lamp for 8 h and provided food and water ad libitum. The guinea pigs
were observed every 15 min for the first hour and every 30 min for
the next 7 h after the OP challenge. Onset of convulsions was the
time between the OP challenge and appearance of discontinuous,
involuntary skeletal muscular contractions interrupted by intervals
of relaxation. A group of three investigators was in charge of observ-
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Address correspondence to: Dr. Edson X. Albuquerque, 655 W. Baltimore St.,
Baltimore, MD 21201. E-mail: firstname.lastname@example.org
Aracava et al.