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Home , Galantamine, Neurotoxicity, Paraoxon, Parathion, Pyridostigmine, Tacrine

Effective countermeasure against poisoning by organophosphorus and nerve agents

Edson X. Albuquerque*†, Edna F. R. Pereira*, Yasco Aracava*, William P. Fawcett*, Maristela Oliveira*, William R. Randall*, Tracey A. Hamilton‡, Robert K. Kan‡, James A. Romano, Jr.§, and Michael Adler¶

*Department of and Experimental Therapeutics, University of Maryland School of , 655 West Baltimore Street, Baltimore, MD 21201; ‡Comparative Pathology Branch and ¶Neurobehavioral Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010; and §U.S. Army Medical Research and Materiel Command, Fort Detrick, MD 21702

Communicated by John W. Daly, National Institutes of Health, Bethesda, MD, June 28, 2006 (received for review May 24, 2006) The nerve agents , , VX, and are deadly organ- difficulty in delivering these large molecules systemically may ophosphorus (OP) compounds chemically related to OP insecti- limit progress in this field. cides. Most of their acute results from the irreversible bromide, a quaternary that does inhibition of (AChE), the that inacti- not cross the blood–brain barrier appreciably and that reversibly vates the neurotransmitter . The limitations of avail- inhibits AChE and BuChE with similar potencies, has been able therapies against OP poisoning are well recognized, and more approved for use among military personnel who are under threat effective antidotes are needed. Here, we demonstrate that galan- of exposure to nerve agents. Pretreatment with pyridostigmine tamine, a reversible and centrally acting AChE inhibitor approved prevents OP-induced irreversible AChE inhibition in the pe- for treatment of mild to moderate Alzheimer’s disease, protects riphery, and it increases survival of animals acutely exposed to guinea pigs from the acute toxicity of lethal doses of the nerve lethal doses of nerve agents, provided that and oximes agents soman and sarin, and of , the active metabolite of are administered promptly after an OP exposure (5, 8, 9). When the . In combination with atropine, a single used acutely before an OP exposure, reversible inhibitors of dose of administered before or soon after acute AChE that are capable of crossing the blood–brain barrier, exposure to lethal doses of soman, sarin, or paraoxon effectively including , , and (hereafter and safely counteracted their toxicity. Doses of galantamine referred to as huperzine), afford better protection than pyri- needed to protect guinea pigs fully against the lethality of OPs dostigmine against OP toxicity, but generally this protection were well tolerated. In preventing the lethality of nerve agents, occurs at doses that produce significant incapacitation and galantamine was far more effective than pyridostigmine, a periph- impairment (10–13). erally acting AChE inhibitor, and it was less toxic than huperzine, Galantamine, a approved for treatment of mild to moderate a centrally acting AChE inhibitor. Thus, a galantamine-based ther- Alzheimer’s disease (14), has properties appropriate for an anti- apy emerges as an effective and safe countermeasure against OP dotal therapy against OP poisoning. Briefly, galantamine (i)isa poisoning. reversible AChE inhibitor that crosses the blood–brain barrier (14); (ii) has properties (15, 16); and (iii) prevents neu- galantamine ͉ guinea pig ͉ pyridostigmine ͉ soman ͉ sarin rodegeneration (17–19), a hallmark of OP poisoning (20). Thus, we used guinea pigs, the best nonprimate model to predict the effec- tiveness of antidotal therapies for OP poisoning in humans (21), to he organophosphorus (OP) compounds soman, sarin, VX, test the hypothesis that galantamine may be an effective and safe Tand tabun, referred to as nerve agents, are among the most countermeasure against OP intoxication. Because of their low lethal chemical weapons ever developed (1). Some of them were levels of circulating carboxylesterases, guinea pigs, like nonhuman used with catastrophic results in wars and also in terrorist attacks primates, are considerably more sensitive to OPs, and they respond in Japan in the 1990s (2). The majority of insecticides are also better than do rats or mice to antidotal therapies consisting of OPs, and intoxication with these compounds represents a major pretreatment with reversible AChE inhibitors and posttreatment public-health concern worldwide (3, 4). The possibility of further with atropine (21). terrorist attacks with nerve agents and the escalating use of OP Our results demonstrate that OP toxicity and lethality are insecticides underscore the urgent need to develop effective and counteracted when galantamine is administered before or soon safe antidotes against OP poisoning. after the acute exposure of atropine-treated guinea pigs to the The acute toxicity of OPs results primarily from their action nerve agents soman and sarin, or to paraoxon, the active as irreversible inhibitors of acetylcholinesterase (AChE) (5). In metabolite of the OP insecticide parathion. We also provide the periphery, acetylcholine accumulation leads to persistent evidence that the antidotal therapy consisting of galantamine muscarinic receptor stimulation that triggers a syndrome whose and atropine is more effective and less toxic than alternative symptoms include , profuse secretions, bradycardia, bron- treatments. choconstriction, , and diarrhea. It also leads to overstimulation followed by desensitization of nicotinic recep- Results tors, causing severe skeletal muscle fasciculations and subse- Combination Therapy Consisting of Galantamine and Atropine Effec- quent weakness. Central nervous system-related effects include tively Prevents the Acute Toxicity of Lethal Doses of Soman, Sarin, and , restlessness, , , , , car- Paraoxon: Comparison with Pyridostigmine and Huperzine. Clear diorespiratory paralysis, and coma. signs of hyperexcitation, including miosis, increased Current therapeutic strategies to decrease OP toxicity include atropine to reduce the muscarinic syndrome, oximes to reacti- vate OP-inhibited AChE, and benzodiazepines to control OP- Conflict of interest statement: No conflicts declared. triggered seizures (5). The limitations of these treatments are Freely available online through the PNAS open access option. well recognized (4), and alternative therapies have been sought. Abbreviations: AChE, acetylcholinesterase; BuChE, ; FJ-B, Fluoro-Jade Among these therapies are phosphotriesterases and butyrylcho- B; OP, organophosphorus. linesterase (BuChE), that act as OP scavengers (6, 7). †To whom correspondence should be addressed. E-mail: [email protected]. However, potential adverse immunological reactions and the © 2006 by The National Academy of Sciences of the USA

13220–13225 ͉ PNAS ͉ August 29, 2006 ͉ vol. 103 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605370103 Downloaded by guest on September 30, 2021 However, a synergistic interaction occurred between galantamine and Ն6mg͞kg atropine; increasing the dose of galantamine from 5to8mg͞kg decreased the dose of atropine needed to protect the animals from the toxicity of soman (Fig. 1C). Doses of galantamine and atropine required to treat OP intoxication may be optimized by using response-surface methods (22). The acute toxicity of paraoxon was also effectively counter- acted by therapy consisting of galantamine and atropine. All guinea pigs treated with atropine (10 mg͞kg, i.m.) immediately after their exposure to Ն1.8 mg͞kg paraoxon developed life- threatening symptoms and were euthanized. In contrast, all atropine-treated animals survived with no signs of toxicity when they received galantamine (8 mg͞kg, i.m.) 30 min before their exposure to 2 mg͞kg paraoxon (Fig. 1D). Further, galantamine͞ atropine-treated animals that survived the challenge with 3 mg͞kg paraoxon (Fig. 1D) displayed only brief, mild signs of intoxication that included increased chewing and slight tremors. The effectiveness of the antidotal therapy consisting of ga- lantamine͞atropine surpassed that of a combination of pyri- Fig. 1. Pretreatment with galantamine prevents the acute toxicity of lethal dostigmine and atropine in preventing acute OP toxicity. Only a doses of OPs: Comparison with pyridostigmine and huperzine. In all experi- fraction of animals pretreated with pyridostigmine (26–65 ␮g͞ ments, guinea pigs received an i.m. of selected doses of galantamine, kg) and posttreated with 10 mg͞kg atropine survived the chal- pyridostigmine, or huperzine followed 30 min later by a single s.c. injection of lenge with 1.5ϫ LD50 soman (Fig. 1E). The effectiveness of this 1.5ϫ LD50 (42 ␮g͞kg) or 2.0ϫ LD50 (56 ␮g͞kg) soman, 1.5ϫ LD50 sarin (56 ␮g͞kg), or the indicated doses of paraoxon. At 1 min after the OP challenge, therapy increased as the dose of pyridostigmine was raised to 52 all animals received atropine (1–10 mg͞kg, i.m.). (A–C) Dose–response rela- ␮g͞kg (Fig. 1E). Increasing the dose of pyridostigmine to 65 tionships for galantamine or atropine to maintain 24-h survival of animals ␮g͞kg, however, decreased the effectiveness of the treatment, challenged with nerve agents. (D) Dose–response relationship for paraoxon- most likely because the potential benefit of increasing the induced decrease in 24-h survival of atropine-treated guinea pigs that were protection of AChE from the actions of OPs is counteracted and pretreated with saline or galantamine. (E) Effects of increasing doses of eventually outweighed by the simultaneous pyridostigmine- pyridostigmine or huperzine in maintaining 24-h survival of soman- induced inhibition of BuChE, an enzyme that serves as an challenged, atropine-treated animals. Each group had 8–12 animals. Percent endogenous scavenger of OPs (6). survival represents the percent of animals that were kept alive because they ͞ presented no life-threatening symptoms. The safety of the antidotal therapy consisting of galantamine atropine was greater than that of a combination of huperzine and atropine. Approximately 80% of the animals challenged s.c. with ϫ chewing, hypersalivation, muscle fasciculations, difficulty in 1.5 LD50 soman survived if they were pretreated with 100–200 breathing, and loss of motor coordination, were evident at 5–15 ␮g͞kg huperzine and posttreated with 10 mg͞kg atropine; the min after the s.c. injection of 1.5ϫ LD50 soman (42 ␮g͞kg of body minimum dose of huperzine needed to provide 100% survival of weight) or sarin (63 ␮g͞kg) in prepubertal male guinea pigs. soman-challenged, atropine-treated guinea pigs was 300 ␮g͞kg Ն ␮ ͞ Although an i.m. injection of atropine (6–16 mg͞kg) immedi- (Fig. 1E). However, at doses 300 g kg, huperzine triggered ately after the OP challenge attenuated the muscarinic signs, all transient, albeit incapacitating side effects that included profuse animals showed tremors and intense convulsions within 15–30 secretions, muscle fasciculations, abnormal gait, tremors, and min after the challenge. Atropine-treated, OP-challenged guinea respiratory distress. The stereotypic behavior of animals treated pigs were euthanized when they developed life-threatening with huperzine was quantitatively analyzed in an open-field symptoms, and at 24 h after the exposure to nerve agents, only arena, as described below. 11% of the animals (7 of 65) remained alive. All guinea pigs that were pretreated with 5–12 mg͞kg Galantamine Maintains Long-Term Survival of OP-Challenged, Atro- pine-Treated Guinea Pigs and Has No Significant Effect on Gross galantamine⅐HBr (hereafter referred to as galantamine) and Behavior of the Animals: Comparison with Huperzine. Even though posttreated with 10 mg͞kg atropine survived the s.c. injection of all animals survived the first 24 h after the soman challenge when 1.5ϫ LD soman or sarin, with no toxic signs either before or 50 they were pretreated with 6 mg͞kg galantamine and posttreated after the OP exposure. The ED values of galantamine for 24-h 50 with6mg͞kg atropine, only 80% of them remained alive after

survival of animals exposed to soman or sarin were 1.82 Ϯ 0.37 PHARMACOLOGY Ϯ ͞ the 3rd day post-OP exposure (Fig. 2A). In contrast, during the or 2.2 0.50 mg kg, respectively (Fig. 1 A and B). The optimal entire observation period, survival remained at 100% in animals dosage of galantamine changed as the OP levels increased. For ͞ ͞ pretreated with 5–8 mg kg galantamine and posttreated with 10 example, in animals posttreated with 10 mg kg atropine, the mg͞kg atropine. Increasing the dose of atropine to 16 mg͞kg ϫ ED50 for galantamine to prevent the lethality of 2.0 LD50 reduced the acute and long-term of doses of galan- Ϯ ͞ Ϯ ϭ soman was 5.1 0.66 mg kg (mean SEM; n 8–10 animals tamine Ͻ8mg͞kg (Fig. 2A). Thus, 10 mg͞kg atropine ensured Ն per group), with 100% 24-h survival being achieved with 8 the highest long-term effectiveness of galantamine against the ͞ mg kg (Fig. 1A). Effective doses of galantamine were well toxicity of 1.5ϫ LD50 soman. tolerated; only animals that received 16–20 mg͞kg galantamine Within 1 week after a single i.m. injection of saline, galan- showed mild adverse symptoms, which lasted 10–15 min and tamine (8 mg͞kg), or atropine (10 mg͞kg), guinea pigs gained included increased chewing, hypersalivation, fasciculations, and weight at similar rates, i.e., 2.51 Ϯ 0.11% per day, 2.30 Ϯ 0.05% tremors. per day, and 2.37 Ϯ 0.03% per day (Fig. 2B). In contrast, guinea Muscarinic blockade by atropine contributed to the antidotal pigs that received a single i.m. injection of huperzine (300 ␮g͞kg) effectiveness. Regardless of whether animals were pretreated with gained weight at a rate of 1.72 Ϯ 0.17% per day (Fig. 2B), which 5or8mg͞kg galantamine, 50% reduction of the lethality of those is significantly slower than that measured for saline-injected nerve agents was achieved with similar doses of atropine (mean Ϯ animals (P Ͻ 0.01 compared with saline-injected animals ac- SEM ϭ 5.7 Ϯ 0.47 mg͞kg and 5.2 Ϯ 0.13 mg͞kg, respectively). cording to ANOVA followed by Dunnett’s post hoc test).

Albuquerque et al. PNAS ͉ August 29, 2006 ͉ vol. 103 ͉ no. 35 ͉ 13221 Downloaded by guest on September 30, 2021 Fig. 3. Efficacy of galantamine as a pre- or posttreatment for OP poisoning is dose- and time-dependent. (A) Twenty-four-hour survival of animals that received a single i.m. injection of 8 or 10 mg͞kg galantamine at 1, 2, 3, 4, or 5 h before the s.c. injection of 1.5ϫ LD50 soman that was followed 1 min later by an i.m. injection of 10 mg͞kg atropine. (B and C) Twenty-four-hour survival of animals that received a single i.m. injection of specific doses of galantamine at different times after their challenge with 1.5ϫ LD50 soman or 2–3 mg͞kg paraoxon, respectively. Each group had 8–10 animals.

anism that appears to contribute to the effectiveness of huper- zine in preventing OP toxicity (24), is known to increase stereo- typy in rodents (25). Each guinea pig, immediately after receiving an i.m. injection of saline, galantamine (8 mg͞kg), or huperzine (300 ␮g͞kg), was placed in an open-field arena equipped with infrared sensors. At the dose tested, galantamine had no significant effect on the overall locomotor activity of guinea pigs (Fig. ). However, huperzine increased the locomotor activity of the animals, and this effect became significant at 30 min after the treatment (Fig. Fig. 2. Long-term effectiveness and acute toxicity of different antidotal 2C). At this time, a distinct pattern of locomotor stereotypy, therapies against OP poisoning. (A) Seven-day survival of guinea pigs treated including repetitive routes of locomotion in the open-field arena, with galantamine at 30 min before and atropine at 1 min after their challenge was also significantly higher in huperzine- than in saline-treated with 1.5ϫ LD50 soman. Each group had 8–12 animals. (B) Seven-day follow-up animals (Fig. 2C). These effects of huperzine resemble those of of the weight of animals subjected to different treatments. Weights are the NMDA receptor antagonists , , and expressed as percent of the weights measured 1 h before a treatment. Control dizolcipine (25). groups consist of animals that received a single i.m. injection of atropine, ͞ galantamine, huperzine, or saline. The soman atropine groups consist of Galantamine Can Be Safely Used Pre- or Posttreatment to Counteract animals treated with galantamine or huperzine at 30 min before and atropine Acute OP Toxicity: Therapeutic Windows of Time. An effective at 1 min after soman (n ϭ 5–8 animals per treatment). (C) Graphs of the average total distance traveled and stereotypy of guinea pigs in an open-field antidotal therapy should afford long-lasting protection for first arena at the indicated times after they received an i.m. injection of saline, responders who will attend a population acutely exposed to toxic galantamine, or huperzine (n ϭ 6 animals per treatment). In B and C, results levels of OPs. Thus, experiments were designed to determine are presented as the mean Ϯ SEM. Asterisks indicate that results from huper- how long before an exposure to OPs an acute pretreatment with zine- and saline-treated animals are significantly different at P Ͻ 0.05 (ANOVA galantamine would remain effective in preventing their toxicity. followed by Dunnett’s post hoc test). All atropine-treated guinea pigs that received 8 mg͞kg galan- tamine up to 1 h before soman survived with no signs of toxicity ͞ (Fig. 3A). As the interval between the injections of galantamine Although galantamine atropine-treated, soman-challenged an- and soman increased beyond 1 h, the survival decreased (Fig. imals lost, on average, 10% of their body weight at 24 h after the 3A). Increasing the dose of galantamine to 10 mg͞kg prolonged OP exposure (Fig. 2B), their rate of weight gain during the the time within which the antidotal therapy remained effective Ϯ Ϯ remaining recovery period (2.72 0.26% per day; mean SEM; (Fig. 3A). ϭ n 5 animals) was not significantly different from that of Considering the difficulty of predicting when a person will be saline-treated animals that were not challenged with soman. exposed to toxic levels of OPs under battlefield conditions, in the ͞ Galantamine atropine was equally effective in maintaining the case of a terrorist attack, or during handling of insecticides, ϫ rates of weight gain of guinea pigs challenged with 1.5 LD50 experiments were also designed to determine whether posttreat- sarin or 3 mg͞kg paraoxon at 2.53 Ϯ 0.20% per day or 2.66 Ϯ ment with galantamine could effectively counteract the acute 0.21% per day (mean Ϯ SEM; n ϭ 3–5 animals per group), toxicity of OPs. All animals treated with 8 or 10 mg͞kg galan- respectively. The acute toxicity of huperzine was not reflected in tamine up to 5 min after the soman challenge survived (Fig. 3B) the rates of weight gain of animals that survived the OP with no signs of intoxication; the rate of weight gain and gross challenge when treated with huperzine͞atropine (Fig. 2B). behavior of these animals were indistinguishable from those of In an attempt to quantify potential untoward behavioral saline-treated animals that were not exposed to soman. Galan- effects of the doses of galantamine and huperzine needed to tamine was no longer effective when given 10 min after 1.5ϫ prevent acute OP poisoning, the overall ambulatory activity of LD50 soman. Posttreatment with galantamine͞atropine also guinea pigs was examined in an open-field arena. Previous prevented the acute toxicity of supralethal doses of paraoxon studies reported that other centrally acting AChE inhibitors, (Fig. 3C). The therapeutic window of time within which post- including physostigmine, decrease locomotor activity and ste- treatment with galantamine remained effective in sustaining reotypic behavior of rodents in the open field (23). Further, 100% survival of the animals decreased as the dose of the OP inhibition of the NMDA type of glutamate receptors, a mech- increased (Fig. 3C).

13222 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605370103 Albuquerque et al. Downloaded by guest on September 30, 2021 Fig. 5. Differential sensitivity of brain and blood AChE activities to inhibition by galantamine in vivo and in vitro.(A and B) Concentrations of galantamine measured in blood (A) and brain (B) samples obtained at various times after treatment of guinea pigs (n ϭ 4–6 animals per time point) with galantamine (8 Fig. 4. Soman-induced is not present in the hippocam- mg͞kg, i.m.) is plotted on a logarithmic scale against time. (C) AChE activity pus, pyriform cortex, and amygdala of guinea pigs pre- or posttreated with measured in samples from saline-treated animals was taken as 1 and used to galantamine. (A and B) Representative photomicrographs of the hippocampal normalize the enzyme activity measured in samples obtained at various times CA1 field, the pyriform cortex, and the amygadala of guinea pigs that were after treatment of animals with galantamine (8 mg͞kg, i.m.). Normalized inhi- euthanized 24 h after an i.m. injection of saline (A)or8mg͞kg galantamine bition (1 Ϫ normalized activity) is plotted against the time at which samples were (B). No FJ-B-positive were seen in the brains of these animals. (C) Large obtained. Asterisks indicate that results from galantamine- and saline-treated numbers of FJ-B-positive neurons were seen in all three index areas of the animals are significantly different at P Ͻ 0.001 (***)orϽ 0.01 (**) (ANOVA brain of a guinea pig that survived for 24 h after the challenge with 1.5ϫ LD50 followed by Dunnett’s post hoc test). In A–C, the first point corresponds to results soman. (D and E) FJ-B-positive neurons were rarely seen in brain sections of obtained at 5 min after the treatment. (D) Increasing concentrations of galan- animals that received galantamine (8 mg͞kg, i.m.) at 30 min before (D)or5 tamine were added in vitro to brain homogenates and blood samples obtained min after (E) soman. In C–E, all animals received atropine (10 mg͞kg, i.m.) at from naive animals. AChE activity in untreated samples was taken as 1, and it was 1 min after the OP, and they were euthanized at 24 h after the OP challenge. used to normalize activity measured in galantamine-treated samples. The graph Photomicrographs are representative of results obtained from each group, of normalized AChE activity . galantamine concentrations was fitted with the which had five animals. Hill equation. Results are presented as the mean Ϯ SEM (n ϭ 4–6 animals per galantamine concentration).

No Signs of Were Observed in the Brains of Atropine- Treated Guinea Pigs That Received Galantamine Before or After the Doses of Galantamine That Effectively Prevent OP-Induced Toxicity Challenge with Soman. Neurodegeneration in three areas of the and Lethality Are Clinically Relevant. To help establish the clinical brain, the pyriform cortex, the amygdala, and the hippocampus, relevance of the doses of galantamine needed to counteract OP is characteristic of OP intoxication. The components of the poisoning, plasma and brain concentrations of the drug were antidotal therapy regimen, by themselves, were not neurotoxic. determined by HPLC at various times after treatment of guinea No signs of were detected at 24 h after an i.m. pigs with 8 mg͞kg galantamine. This dose was selected because injection of saline (Fig. 4A),8mg͞kg galantamine (Fig. 4B), or (i) in association with atropine, it afforded full protection against

10 mg͞kg atropine (data not shown). Galantamine is a critical OP-induced toxicity and lethality, and (ii) it was half of the PHARMACOLOGY component of the antidotal therapy regimen because atropine minimum dose at which galantamine triggered mild side effects. alone was unable to prevent the well described neuronal death In guinea pigs, as in humans (26), plasma levels of galantamine triggered by 1.5ϫ LD50 soman (Fig. 4C). Large numbers of declined with first-order kinetics. After an i.m. injection of 8 shrunken neurons that were labeled with Fluoro-Jade B (FJ-B), mg͞kg galantamine, plasma and brain levels of the drug peaked an anionic fluorescein derivative that binds with high affinity to between 5 and 30 min and decayed with half-times of 71.7 Ϯ 14.4 degenerating cells, were consistently seen in the hippocampus, min and 57.8 Ϯ 4.31 min, respectively (Fig. 5 A and B). As shown amygdala, and pyriform cortex of atropine-treated guinea pigs in Fig. 3A, full protection against acute toxicity was achieved ͞ that survived for 24 h after the challenge with 1.5ϫ LD50 soman when 8 mg kg galantamine was administered to guinea pigs up (Fig. 4C). In contrast, staining with FJ-B was rarely seen in brain to 1 h before soman, a time when plasma and brain levels of the sections of soman-challenged, atropine-treated animals that drug were 0.90 Ϯ 0.01 ␮g͞ml and 0.80 Ϯ 0.04 ␮g͞g, respectively were given 8 mg͞kg galantamine at 30 min before or 5 min after (Fig. 5 A and B). Because the molecular weight of galantamine the OP (Fig. 4 D and E). Further, the edema observed in the is 287.4, these findings suggest that the minimal plasma concen- hippocampus and the marked parenchymal spongy state of the tration of galantamine needed to prevent OP toxicity and amygdala and pyriform cortex of soman-exposed, atropine- lethality is Ϸ2.8 ␮M. Doses of galantamine recommended for treated animals were absent in animals that received galan- treatment of patients with Alzheimer’s disease are between 8 tamine 30 min before or 5 min after the (Fig. 4 C–E). and 24 mg͞day (14), and peak plasma concentrations of 0.2–3

Albuquerque et al. PNAS ͉ August 29, 2006 ͉ vol. 103 ͉ no. 35 ͉ 13223 Downloaded by guest on September 30, 2021 ␮M have been detected in healthy human subjects treated orally galantamine against different insults (17–19, 34, 35) may be or s.c. with a single dose of 10 mg of galantamine (26, 27). Thus, important determinants of the antidotal effectiveness. Because doses of galantamine needed to prevent OP toxicity generate no cognitive impairment has been detected in soman-challenged peak plasma concentrations similar to those achieved with doses animals when neuronal loss in their brains remains below a clinically used to treat Alzheimer’s disease. certain threshold (32), galantamine is likely to maintain normal In agreement with the concept that galantamine-induced cognitive performance of OP-exposed subjects. AChE inhibition is reversible, the degree of AChE inhibition in Inhibition of brain AChE by Ͼ60–70% has been shown to brain and blood from galantamine-treated guinea pigs decreased trigger severe incapacitating effects, including seizures (36). as the galantamine levels declined in both compartments. Inhi- Maximal degrees of inhibition of AChE activities observed in bition of AChE became negligible at6hafterthetreatment (Fig. guinea pigs treated with doses of galantamine that effectively 5C), when plasma and brain levels of the drug were Ͻ0.1 ␮g͞ml counteracted OP intoxication were Ϸ70% in blood and 25% in and 0.1 ␮g͞g, respectively (Fig. 5 A and B). Maximal inhibition brain. All other centrally acting AChE inhibitors studied to date, of blood AChE activity was Ϸ70% (Fig. 5C), observed at 30 min including huperzine, acutely prevent OP toxicity when used at after the treatment when the plasma levels of galantamine had doses that decrease blood AChE activity by Ͼ70% (10–13). peaked. The effectiveness of galantamine in patients with Alz- However, brain AChE activity is inhibited to a similar extent heimer’s disease has been correlated with 40–70% inhibition of (Ϸ70%) by these (13). Therefore, a high degree of AChE in blood (28). reversible and selective AChE inhibition in the blood appears to Maximal AChE inhibition in the brains of galantamine- be necessary to counteract the peripheral toxic effects of OPs treated animals was significantly different from that observed in acutely. A low degree of reversible inhibition of brain AChE may their blood (Fig. 5C). Measured peak concentrations of galan- be sufficient to protect a significant pool of the enzyme from tamine were 1.6 Ϯ 0.13 ␮g͞ml in the plasma and 1.38 Ϯ 0.11 ␮g͞g OP-induced irreversible inhibition, and it may be critical to limit in the brain. These concentrations resulted in Ϸ70% and 25% the occurrence of untoward side effects of centrally acting inhibition of AChE in the blood and brain, respectively. Mea- reversible AChE inhibitors. sured peak levels of galantamine in the plasma correspond to Development of effective and safe antidotes against OP 5.6 Ϯ 0.5 ␮M. Considering 80% of the brain weight as water, toxicity will help improve the treatment of the victims of a measured peak levels of galantamine in brain tissue would terrorist attack with nerve agents, and it will help reduce the correspond to 3.8 Ϯ 0.3 ␮M. Based on the concentration– mortality associated with OP insecticide poisoning worldwide. response relationships obtained for galantamine-induced inhi- The demonstration that an acute galantamine-based therapy bition of guinea pig blood and brain AChE in vitro (Fig. 5D), it effectively and safely counteracts OP poisoning is, therefore, of is estimated that 5.6 ␮M galantamine would inhibit blood AChE utmost relevance for farm workers and others who handle OP activity by 68%, and 3.8 ␮M galantamine would inhibit brain insecticides, for the general population under threat of OP AChE activity by 25%. In vitro, galantamine inhibited guinea pig exposure in terrorist attacks, and for soldiers, who, despite the blood and brain AChE with EC50 values of 1.8 Ϯ 0.38 ␮M and Geneva Protocol, may be exposed to deadly nerve agents in the 16.9 Ϯ 9.8 ␮M, respectively (mean Ϯ SEM; Fig. 5D). In humans, course of battle. blood AChE activity is also 10-fold more sensitive to inhibition by galantamine than is brain AChE activity (29). Materials and Methods Animal Care and Treatments. Male albino guinea pigs [Crl(HA)Br; Discussion Charles River Laboratories, Wilmington, MA] weighing 350– The present study demonstrates the remarkable potential of 420 g (5–6 weeks old) were used. Galantamine, pyridostigmine, galantamine to improve antidotal therapy for even the most or huperzine was injected in one hindlimb, and atropine was deadly OPs. In combination with atropine, well tolerated, clin- injected in the other. The nerve agents, diluted in sterile saline, ically relevant doses of galantamine, administered acutely either and paraoxon, diluted in DMSO, were injected s.c. between the before or soon after an exposure to the nerve agents soman and shoulder blades of the animals. All injections (Ϸ0.5 ml͞kg) were sarin or paraoxon, fully counteract the toxicity and lethality of performed by using disposable tuberculin syringes with 25- to these compounds. Although atropine alone attenuates the mus- 26-gauge needles. Handling and disposal of nerve agents were carinic syndrome resulting from the exposure of guinea pigs to according to the rules set forth by the U.S. Army. All conditions the OPs, it does not afford significant protection against their for animal maintenance conformed to the regulations of the lethality. Association for Assessment and Accreditation of Laboratory The exact mechanisms that account for the superiority of Animal Care, complied with the standards of the Animal galantamine as a countermeasure against OP poisoning are yet Welfare Act, and adhered to the principles of the 1996 Guide for to be fully elucidated. However, it can be postulated that the the Care and Use of Laboratory Animals (37). Atropine sulfate, effectiveness of galantamine is related both to the higher potency pyridostigmine bromide, (Ϯ)-huperzine A, and paraoxon were with which it inhibits AChE compared with BuChE (30), an purchased from Sigma–Aldrich (St. Louis, MO). Soman and action that should help preserve the scavenger capacity of sarin were obtained from the U.S. Army Medical Research and plasma BuChE for OPs, and to the protection of brain AChE Development Command (Fort Detrick, MD). Galantamine⅐HBr from OP-induced irreversible inhibition. The finding that galan- was a generous gift from Alfred Maelicke (Galantos, Mainz, tamine was essential to counteract soman-induced neurodegen- Germany). eration in the brain supports the notion that AChE-related and͞or -unrelated actions of this drug in the central nervous Histopathological Analyses. Guinea pigs were anesthetized at system contribute to its effectiveness. Neuronal loss in the brains appropriate times after their treatments and transcardially per- of OP-intoxicated animals correlates to some extent with the fused with 0.9% saline (70 ml͞min) until blood was cleared and intensity and duration of OP-triggered seizures (31–33). Yet, subsequently perfused with 10% formalin. Their brains were neurodegeneration and consequent cognitive impairment in- then removed, placed in 10% formalin for no longer than 48 h, duced by OPs can be significantly reduced by therapeutic dehydrated, and embedded in paraffin. Sections 5 ␮m thick were interventions that, although unable to suppress OP-triggered cut and then dried in an incubator at 37°C for 12 h before they seizures, effectively decrease glutamate (32). The were stained with FJ-B (38). After it was mounted, the tissue was ability of the galantamine-based therapy to prevent OP-induced examined under an epifluorescence microscope with blue (450– convulsions and the well reported neuroprotective effects of 490 nm) excitation light and a filter for fluorescein isothiocya-

13224 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605370103 Albuquerque et al. Downloaded by guest on September 30, 2021 nate. Photomicrographs were taken with a digital microscope Behavioral Assays. Locomotor activity and stereotypy of guinea camera (AxioCam; Zeiss, Jena, Germany). pigs were analyzed in an open-field arena equipped with infrared sensors (AccuScan Instruments, Columbus, OH), as described Analysis of Galantamine Concentrations in the Brain and Plasma of by June et al. (41). Counts obtained from the total number of Guinea Pigs. At various times after treatment with galantamine (8 interruptions of the infrared beams were automatically compiled mg͞kg, i.m.), animals were anesthetized with CO2. Blood (5–10 every 5 min and processed for measures of total distance traveled ml) was collected by cardiopuncture with a plastic heparinized and stereotypy. system and kept in dry ice. Immediately after cardiopuncture, the animals were exsanguinated by carotid artery transection. We thank Dr. Harry L. June and Dr. Jacek Mamczarz (Department of Their brains were removed, superfused with 0.9% saline, and Psychiatry, University of Maryland School of Medicine) for guidance and assistance with the behavioral experiments. We are grateful to Dr. snap frozen in liquid nitrogen. Frozen blood samples and brains Ϫ Robert Bloch (Department of Physiology, University of Maryland were kept at 80°C until further processing. Brain and plasma School of Medicine), Dr. David Burt (Department of Pharmacology and levels of galantamine were measured by using a modified HPLC Experimental Therapeutics, University of Maryland School of Medi- method (39). cine), Col. George Korch (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD), Col. Dr. David Moore (Strategic Radiometric Enzymatic Assay. Pulverized brain tissue was mixed Research Program Development, Office of the Commander, U.S. Army with buffer containing antiproteases (0.5 unit͞ml aprotinin, 30 Medical Research Institute of Chemical Defense), and Dr. Susan ␮ ͞ ͞ Wonnacott (Department of Biology and Biochemistry, University of g ml leupeptin, 1 mg ml bacitracin, 2 mM benzamidine, and 5 Bath, Bath, U.K.) for helpful comments. We are also indebted to Ms. mM N-ethylmaleimide) and sonicated for 20 s on ice. Aliquots Mabel Zelle for technical and editorial support and to Ms. Christina M. of the resulting suspensions and of blood samples were used for Tompkins and Ms. Denise M. Fath for excellent assistance with the determination of protein concentration (micro BCA protein histological techniques. This work was supported by U.S. Army Medical assay; Pierce, Rockford, IL). Measurements of AChE activity Research and Development Command Contract DAMD-17-95-C-5063, were performed in the presence of the BuChE inhibitor tetrai- Battelle Scientific Services Contract TCN 03132, U.S. Public Health sopropyl pyrophosphoramide (1 mM) with a modified two-phase Service Grant NS25296 from the National Institutes of Health, and National Institutes of Environmental Health Sciences͞National Insti- radiometric assay (40) using 20 pM [3H]acetylcholine iodide ͞ ϭ tutes of Health Training Grant T32 ES07263 (all to E.X.A.). The use of [specific activity, 76 Ci mmol (1 Ci 37 GBq); PerkinElmer galantamine as an antidote against OP poisoning is protected under the Life Sciences, Boston, MA], which produced Ϸ200,000 cpm International Patent Application PCT͞US05͞33789 filed on September when totally hydrolyzed by eel AChE (2 units). 23, 2005.

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