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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Sep 10;105(37):14169–14174. doi: 10.1073/pnas.0807172105

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice

Fabio Pibiri *, Alan P Kozikowski †, Graziano Pinna *, James Auta *, Bashkim Kadriu *, Erminio Costa *,‡, Alessandro Guidotti *
PMCID: PMC2544596  PMID: 18784370

Abstract

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABAA receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life ≈5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for α5-containing GABAA receptors. Coadministration of HUP (50 μg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18–24 h. In HUP-pretreated mice, the ED50 of IMI to block DFP-induced mortality is ≈10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

Keywords: GABAA receptor, neurotoxicity

Organophosphate (OP) neurotoxins are among the most lethal chemical poisons ever developed and in the present political climate, should be considered possible threats to both civilians and military personnel in terrorist attacks (1, 2).

Most of the insecticides used worldwide are OP, and intoxication with these compounds represents a major public health concern in nonindustrialized countries (3, 4). OP neurotoxins target cholinergic neurotransmission by irreversibly inhibiting acetylcholinesterase (AChE) and thereby inducing life-threatening disorders, including bronchial spasms and gland hypersecretion, muscle fasciculation, cardiovascular impairment, tremors, convulsions, coma, and in the worst case, may even include death (5, 6). Subjects who survive severe OP-induced seizures will likely develop irreversible brain damage (7).

The current prophylactic strategy to treat OP poisoning recommended by military organizations relies on pyridostigmine (PYR) bromide tablets taken over several days (5). PYR is a reversible AChE blocker, which prevents the irreversible binding of OP to AChEs. In the event of OP poisoning, this pretreatment must be supported by a triple therapy based on atropine, oxime, and a benzodiazepine compound, usually diazepam (DZP).

Several limitations are associated with this conventional prophylactic treatment for subjects at risk for OP exposure (5). PYR prevents OP-induced hypercholinergic toxicity and lethality but because of its low blood–brain barrier (BBB) penetration, fails to protect against OP-induced convulsions (8, 9). However, the tertiary carbamate physostigmine (PHY) can cross the BBB. It thus protects brain AChEs from the irreversible binding of OP (10).

Thus, PHY weakens OP intoxication, thereby remaining the most effective treatment so far developed (8). However, PHY has a short half-life, and to reach a blood concentration sufficient to counteract OP toxicity, it requires doses that induce unwanted centrally mediated psychological or behavioral side effects (11–13). These side effects can be partly eliminated by a low dose of scopolamine (SCO) (13). However, the administration of a muscarinic blocker may lead to unwanted effects in persons under warm to hot weather conditions or in persons wearing protections against chemical agents. SCO administration blocking muscarinic cholinergic receptors of secretal glands may inhibit sweating and thereby enhance the risk of heat injury (14, 15).

An additional limitation on the use of AChE inhibitors is the incomplete protection from OP-induced seizures (5). Thus, this pretreatment must be followed rapidly by an administration of: (i) antimuscarinic agents, usually atropine (ATR), which prevents acetylcholine (Ach) from binding to muscarininc receptors [ATR cannot be given prophylactically because of its short half-life and at the doses required to overcome OP-induced hypercholinergic toxicity, it induces disorientation, confusion, and hallucinations (16–19)]; (ii) oxime, which reactivates AChEs blocked by OP before the “aging” of the binding to the enzyme (20); and (iii) anticonvulsants, usually DZP, which allosterically enhance GABA-mediated inhibition at various GABAA receptors. Unfortunately, DZP and congeners at the required anticonvulsant doses act at GABAA receptors expressing α1 subunits and cause sedation, amnesia, and cardiorespiratory depression (6). When given for protracted periods, these benzodiazepines cause not only tolerance to their anticonvulsant effects but also dependence (21). Hence, there is an urgent need to develop more effective and safe therapeutic strategies against OP poisoning.

Newly tested reversible AChE inhibitors include huperzine A (HUP), an alkaloid that crosses the BBB, selectively inhibits AChE, and fails to bind to butyrylcholinesterase (BuChE) (5). HUP, isolated from Huperzia serrata, is currently approved in China for the treatment of Alzheimer's disease and other memory impairments (22) and has passed phase II clinical trials in the U.S. (http://clinicaltrials.gov/ct2/show/NCT00083590). At doses of 100–500 μg/kg, HUP increases the survival of laboratory animals exposed to lethal doses of OP (5, 10, 23, 24). However, at these high doses, HUP inhibits blood and brain AChEs by 50–70%, a value that limits its use in healthy subjects (5). Moreover, at a dose of 500 μg/kg, HUP produces untoward side effects such as chewing, salivation, tremors, and behavioral deficits (5, 10, 24). Because prophylactic treatment with HUP fails to prevent the development of seizures in OP-intoxicated animals, HUP treatment should be associated with a nonsedative benzodiazepine [i.e., imidazenil (IMI)].

IMI is a positive selective allosteric modulator of GABA action at α5-containing GABAA receptors, which, unlike DZP, is inactive at α1-containing GABAA receptors (25, 26). Thus, IMI elicits a potent anticonvulsant and anxiolytic action at doses that are virtually devoid of sedative and amnesic effects (6, 25–31). Of note, IMI also potently antagonizes diisopropyl fluorophosphate (DFP)-induced seizures and mortality in rodents (6, 32). This drug, at doses up to 60-fold greater than those producing anxiolytic and anticonvulsant actions, antagonizes the sedative and amnesic actions of DZP or alprazolam without causing tolerance (26–30, 33). Furthermore, in non-human primates, IMI is virtually devoid of tolerance and dependence liability (29, 30). The IMI clearance rate in vivo is slower (in rats the t½ is 180 min, and in non-human primates its t½ is >8 h) than that of DZP (26).

For a prophylactic treatment that is devoid of side effects and effective against DFP-induced toxicity, we used a combined treatment of HUP and IMI. We show that pretreatment with a low dose of HUP (50 μg/kg s.c., 15 min before DFP) in combination with IMI (2 mg/kg s.c., 30 min before DFP) is a safe prophylactic treatment against OP toxicity. This treatment not only prevents death but also protects against the seizures and neurodegeneration that follow OP intoxication. Our data also suggest that a combination of HUP and IMI could safely be used as a treatment after OP intoxication.

Results

Doses of HUP That Protect Mice Against DFP-Induced Mortality Fail to Abolish DFP-Induced Seizures.

DFP in doses of 0.3–12 μg/kg s.c. elicits a clear dose-dependent hypercholinergic toxicity with a LD50 of 2.7 ± 0.2 μg/kg s.c. (Fig. 1). DFP, at a dose of 6 μg/kg (≈2× LD50), elicits a cholinergic hyperactivity in 2–3 min. After 5–6 min, severe convulsions appear (range 4–5 of Racine scale), which evolve into a status epilepticus in 7–8 min. Mice generally die within 10 min (Table 1). Based on these characteristics, this DFP dose was selected to study the dose-dependent protective efficacy of pretreatment with HUP alone or in combination with IMI.

Fig. 1.

Fig. 1.

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Dose–response of DFP-induced lethality in mice. Results were obtained by using 6–10 mice per dose of DFP. Lethality was established 48 h after DFP intoxication.

Table 1.

Pretreatment combination with huperzine and imidazenil protects mice against DFP-induced seizures and lethality

Drug treatment Seizures
 Mortality,% Death, min
Grade 0–3, min Grade 4–5, min Status epilepticus, min Seizures end, h
Vehicle + DFP 4.2 ± 0.3 7.4 ± 0.6 8.4 ± 0.3 100 9.3 ± 0.9
HUP (100 μg/kg) + DFP 9.7 ± 1.2 14.3 ± 1.3 No 9 ± 2 0
HUP (50 μg/kg) + DFP 7.8 ± 0.2 12.3 ± 1.8 No 10 ± 2 0
HUP (25 μg/kg) + DFP 6.0 ± 0.9 8.5 ± 0.6 24 ± 3.6 >12 80 27 ± 5.6
HUP (50 μg/kg) + IMI + DFP 17.3 ± 2.3 No No 4 ± 2 0
HUP (25 μg/kg) + IMI + DFP 12.4 ± 0.9 No No 6 ± 2 0
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Huperzine (HUP) was injected s.c. 15 min before, and imidazenil (IMI, 2 mg/kg s.c.) was injected 30 min before the challenge with 2× LD50 of DFP (6 μg/kg s.c.). Mean ± SEM, n = 10–30 mice per group.

Fig. 2 shows that HUP given 15 min before DFP (6 μg/kg s.c.) dose-dependently attenuates DFP-induced severe muscarinic effects and protects mice against DFP lethality (ED50, 34 ± 3.0 μg/kg). However, Table 1 shows that HUP doses (50 μg/kg s.c. or higher) that effectively protect against DFP-induced lethality fail to prevent DFP-induced recurrent seizures (range 4–5 of Racine scale). Seizures generally last for 8–12 h after a DFP challenge.

Fig. 2.

Fig. 2.

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Protective action of HUP and the combination of HUP with IMI against DFP-induced lethality. Mice were treated with DFP (6 μg/kg s.c., ≈2× LD50) 15 min after HUP (0.3–100 μg/kg s.c.) and 30 min after IMI (2 mg/kg s.c.). All values are the average of at least 6 animals per group. Lethality was established 48 h after DFP intoxication.

IMI Potentiates HUP Efficacy Against DFP-Induced Mortality.

Fig. 3 compares the potency of IMI with that of DZP in protecting mice pretreated with 25 μg/kg s.c. HUP from DFP-induced death. This dose of HUP alone protects only 20% of mice from DFP-induced death (Table 1). Although IMI or DZP alone fails to protect against DFP-induced lethality (Fig. 3), a synergistic protective interaction occurs between these benzodiazepines and HUP. IMI is ≈10-fold more potent than DZP in protecting HUP-pretreated mice from DFP-induced death (ED50 IMI, 0.08 mg/kg; ED50 DZP, 0.83 mg/kg; see Fig. 3). Moreover, in IMI-treated mice (2 mg/kg s.c. 30 min before DFP), the dose–response curve of HUP protection against DFP-induced mortality shifts 2-fold toward the left (ED50 16 ± 3 μg/kg; Fig. 2). At a dose of 50 μg/kg s.c. HUP fails per se to alter locomotion or memory retention (Fig. 4), whereas a dose of 100 μg/kg s.c. strongly impairs motility and memory (Fig. 4). IMI (2 mg/kg s.c.) either alone or in combination with HUP (50 μg/kg s.c.) fails to affect locomotion and mnemonic functions (Fig. 4). Of note, unlike DZP, IMI in combination with HUP at a dose that reduces DFP-induced lethality fails to induce sedation, amnesia, and muscle relaxation (6, 29).

Fig. 3.

Fig. 3.

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IMI is ≈10 times more potent than DZP in protecting against DFP-induced mortality (ED50 IMI, 0.08 mg/kg; ED50 DZP, 0.83 mg/kg). Mice were pretreated with HUP (25 μg/kg s.c., 15 min before DFP) and with various doses of DZP (▧) or IMI (♦) s.c. 30 min before DFP (6 μg/kg s.c.). Groups of mice were pretreated with DZP or IMI alone (▴), with HUP alone (■) or with vehicle alone (□) 15 min before the challenge with DFP. Each point is the average of 5 different mice.

Fig. 4.

Fig. 4.

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Locomotor activity (A) and contextual fear conditioning (B) in mice treated with HUP (100 or 50 μg/kg s.c.), IMI (2 mg/kg s.c.), and a combination of the two drugs. All drugs were injected 30 min before each test. Each group is the average of 5 different mice. (A) **, P < 0.01 when vehicle-treated group is compared with drug-treated groups (ANOVA followed by Newman–Keuls multiple comparison test). (B) *, P < 0.01 when vehicle-treated group is compared with drug-treated groups (one-way ANOVA followed by Newman–Keuls multiple comparison test). VH, vehicle; HUP 50, HUP 50 μg/kg s.c.; HUP 100, HUP 100 μg/kg s.c.; IMI, IMI 2 mg/kg s.c.

Prophylactic treatment against OP exposure could be given before intoxication. Hence, we studied whether IMI prolongs the duration of HUP protection against DFP-induced lethality. As shown in Fig. 5, if given 15 min before DFP exposure, a dose of 50 μg/kg HUP is fully protective against DFP-induced lethality. However, its potency is reduced to 75% if given 30 min before DFP and entirely loses its efficacy if given 1 h before DFP intoxication. Nonetheless, IMI [as expected by its long half-life in rodents (26)] potentiates the protective action of HUP. Also, IMI prolongs the efficacy of HUP at 1 h pretreatment, delaying occurrence of death from 3 to 6 h (Fig. 5).

Fig. 5.

Fig. 5.

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Time course of the protective action of HUP and the combination of HUP with IMI against DFP-induced lethality. Mice were treated with DFP (6 μg/kg s.c., ≈2× LD50) at various times after HUP (50 μg/kg s.c.) alone or in combination with IMI (2 mg/kg s.c.). All values are the average of at least 6 animals per group. Lethality was established 24 h after DFP intoxication.

Combination of IMI and HUP Protects Mice Against DFP-Induced Seizures, Neurotoxicity, and Cognitive Impairment.

A combination of IMI (2 mg/kg s.c. 30 min before DFP) and HUP (25 or 50 μg/kg s.c., 15 min before DFP) not only protects against DFP-induced mortality, but also against DFP-induced seizures (Table 1). Seizure onset is delayed from 8 to 10 min in mice receiving only HUP, to 15–20 min when HUP is given with IMI, and in these mice, seizures never reached level 4–5 on the Racine scale (Table 1). The window for recurrent seizures was considerably reduced from 8–12 h to 3–4 h in mice receiving HUP in combination with IMI (Table 1).

Clear signs of DFP-induced neurotoxicity (TUNEL-positive nuclei) in the cortex and hippocampus of mice pretreated with HUP (50 μg/kg s.c., 15 min before DFP) appear 48 h after a DFP challenge (Fig. 6). In contrast, these brain areas do not show signs of nuclear neuronal damage in HUP- and IMI- (30 min before DFP) treated mice (Fig. 6), suggesting that these drugs together offer powerful neuroprotection.

Fig. 6.

Fig. 6.

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DFP-induced TUNEL gray-positive reaction is not present in neurons of the cingulate cortex (Top), CA1 (Middle), and dentate gyrus (Bottom) of the hippocampus in mice pretreated 48 h before with a combination of HUP (50 μg/kg s.c., 15 min before DFP) and IMI (2 mg/kg s.c., 30 min before DFP). (A) Vehicle-treated mice. (B) HUP-pretreated mice intoxicated with DFP showing TUNEL-positive neurons in all of the three index areas. (C) HUP + IMI-pretreated mice intoxicated with DFP showing almost complete absence of TUNEL-positive neurons. Photomicrographs are representative of results obtained from groups of 5 mice. (Scale bars: 40 μm.)

One week after DFP intoxication, the combination of HUP and IMI prevents cognitive performance deficits (Fig. 7B) that develop in mice treated with HUP alone (Fig. 7B). Of note, these neuroprotective doses of IMI and HUP fail to produce behavioral dysfunctions (Fig. 4).

Fig. 7.

Fig. 7.

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Locomotor activity (A) and contextual fear conditioning (B) in mice treated with vehicle, HUP (50 μg/kg s.c., 15 min before DFP) + DFP (6 μg/kg s.c.), and HUP (50 μg/kg s.c., 15 min before DFP) + IMI (2 mg/kg s.c., 30 min before DFP). The experiments were carried out 1 week after DFP intoxication. Each group is the average of 5 different mice. *, P < 0.01 when vehicle-treated group is compared with drug-treated groups (one-way ΑΝΟVA followed by Newman–Keuls multiple comparison test). VH, vehicle; HUP 50, HUP 50 μg/kg s.c.; IMI, IMI 2 mg/kg s.c.

HUP and IMI in Posttreatment Prevent DFP-Induced Toxicity.

Because it is difficult to predict when a person will be exposed to toxic levels of nerve agents (for example, in case of terrorist attacks), we studied whether posttreatment with HUP and IMI could also effectively counteract the acute toxicity of OPs. In posttreatment, it is imperative to add ATR to reduce the acute muscarinic syndrome that follows a few minutes after an administration of DFP (Table 2). Posttreatment with ATR (10 mg/kg i.p.) and HUP (50 μg/kg s.c.) given soon after intoxication with DFP protects all affected mice from DFP-induced mortality (Table 2). Furthermore, when mice are treated immediately after DFP injection with HUP, ATR, and IMI (2 mg/kg s.c.), these mice are also protected from the seizures that follow DFP intoxication (Table 2).

Table 2.

Posttreatment combination with huperzine, atropine, and imidazenil protects mice against DFP-induced seizures and lethality

Drug treatment Seizures
 Mortality,% Death
Grade 0–3, min Grade 4–5, min Status epilepticus, min Seizures end, h
Vehicle + DFP 4.2 ± 0.3 7.4 ± 0.6 8.4 ± 0.3 100 9.3 ± 0.9 min
DFP + HUP (100 μg/kg) 4.7 ± 0.8 8.4 ± 0.8 9.1 ± 0.1 100 9.9 ± 0.2 min
DFP + HUP (100 μg/kg) + IMI 5.3 ± 0.8 8.7 ± 0.1 9.5 ± 0.9 100 10 ± 0.9 min
DFP + ATR 6.1 ± 0.9 9.7 ± 0.9 11 ± 0.8 100 4 h
DFP + ATR + HUP (25 μg/kg) 6.1 ± 0.5 9.1 ± 0.4 No 100 10 h
DFP + ATR + HUP (50 μg/kg) 6.8 ± 0.5 8.9 ± 0.4 No >12 0
DFP + ATR + HUP (100 μg/kg) 7.8 ± 0.9 9.9 ± 0.3 No >12 0
DFP + ATR + HUP (50) + IMI 6.9 ± 0.3 no No >12 0
DFP + ATR + HUP (100) + IMI 8.6 ± 0.5 no No >12 0
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Huperzine (HUP), imidazenil (IMI, 2 mg/kg s.c.), and atropine (ATR, 10 mg/kg i.p.) were injected 1 min after the challenge with 2× LD50 of DFP (6 μg/kg s.c.). Mean ± SEM, n = 10–30 mice per group.

Discussion

The present work reports on the remarkable efficacy and low side effect liability of prophylactic treatment with IMI in combination with HUP against DFP-induced neurotoxicity and lethality in mice.

By reducing DFP-induced cholinomimetic toxicity, HUP pretreatment in doses of 100 μg/kg s.c. or higher effectively prevents OP-induced lethality (Table 1). However, at the dose of 100 μg/kg s.c., HUP causes a significant decrease in locomotor activity and reduces mnemonic function (Fig. 4). In rodents, higher doses of HUP (200 μg/kg or higher) induce dangerous unwanted side effects, including profuse salivation, bronchial hypersecretion, arterial hypotension, tremors, and convulsions (5, 10, 24).

Of note, HUP alone, even if administered prophylactically in doses that protect mice from DFP-induced death, provides only marginal protection against DFP-induced seizures (Table 1). Moreover, HUP fails to protect DFP-induced cortical and hippocampal neurotoxicity (Fig. 6), likely caused by the protracted occurrence and duration of seizures (Table 1). This evidence underscores the relevance of optimizing prophylactic action against OP toxicity using HUP by adding an anticonvulsant to this drug.

To abolish DFP-induced seizures and neurotoxicity, we investigated whether the combination of HUP with DZP or IMI improves the protective action of HUP. Pretreatment administration of these benzodiazepines combined with HUP provides full protection against DFP-induced lethality (Fig. 3) and exerts a potent anticonvulsant action (Table 1). Of note, IMI is 10 times more potent than DZP in counteracting DFP-induced lethality, and unlike DZP, it fails to produce sedation, amnesia, or respiratory depression and fails to induce tolerance and dependence after repeated treatments (6–29). Moreover, the half-life of IMI (≈3–4 h in rodents) is longer lasting than that of DZP (26).

After the coadministration of HUP and IMI, in our histological studies of toxicity, we failed to detect the signs of neurotoxicity that were found in the brain of mice pretreated with HUP alone (Fig. 6). Moreover, HUP in combination with IMI also fails to affect motility and memory functions (Fig. 4). Thus, HUP and IMI provide an effective combination therapy that is devoid of side effects and can efficaciously be used as a medication for life-threatening exposure to OP. The half-life of HUP and IMI in humans is ≈5 and ≈12 h, respectively (26, 34). Moreover, even after repeated treatment, HUP and IMI do not develop tolerance toward their protective effects (5, 26). If applied to military personnel or civilians at risk for nerve toxin exposure, the prophylactic treatment against OP toxicity reported in the present work is particularly significant and should guarantee potent and efficacious protection for the various symptoms of OP toxicity. At the same time, this treatment fails to modify cognitive performance and alertness (Fig. 4). It is noteworthy that the prophylactic efficacy of this treatment combination is not achieved by administering HUP alone or in combination with DZP or other commercially available benzodiazepines whose pharmacological profile includes sedative effects among other unwanted features.

The precise mechanism that accounts for the efficacy of treatment with HUP and IMI against DFP intoxication is still to be fully elucidated. HUP is a specific reversible blocker of the AChEs and does not interact with the BuChE and carboxylesterase (CarbE) present in the periphery, so these two proteins can act as endogenous scavengers and detoxify DFP in the bloodstream before it reaches critical concentration levels in the brain (35). Additionally, this drug has been shown to dose-dependently inhibit the binding of two antagonists at the N-methyl-d-aspartate (NMDA) glutamate receptors, thus showing a possible antagonistic action toward this receptor subtype (36). It is well established that this receptor subtype triggers neuronal damage caused by seizures (36).

IMI acts as a nonsedative and potent antiepileptic agent endowed with a long duration of action (37), and unlike DZP and other benzodiazepines, it protects against OP agent-induced seizures without producing sedation, tolerance, or dependence liability (6). Because of these characteristics, IMI is an excellent candidate as the drug of choice to counteract OP-induced seizures. IMI acts specifically at α5-containing GABAA receptors (25), which are the receptor subtypes involved in its anticonvulsant action (38). IMI is inactive at α1-containing GABAA receptors, which accounts for the lack of sedative and amnesic actions and most likely for its failure to develop tolerance and dependence during protracted treatment (6, 26–31). More importantly, because it is devoid of the unwanted side effects of classical benzodiazepines (37), IMI can be safely used as a prophylactic treatment.

Relevant to accidental or unforeseen exposure to OP, the present work also demonstrates that HUP and IMI in combination with ATR (administered as a single dose immediately after DFP exposure) fully counteracts DFP-induced neurotoxicity, including lethality (Table 2).

In conclusion, this article demonstrates the efficacy of a combination treatment with IMI and HUP that can safely be used against the toxicity of OP compounds. The therapeutic strategy of using this combination of IMI and HUP offers an excellent prophylactic tool against OP exposure because of its long-lasting action and the lack of unwanted side effects when given to healthy subjects. The prophylactic use of this treatment is particularly advisable for those workers who are at risk for being accidentally exposed to OP substances, including farmers or military personnel during war actions (1, 2). Of course, this treatment would become extremely useful for civilians in the case of terrorist attacks.

Materials and Methods

Animals and Drugs.

Adult male Swiss–Webster mice (Harlan Breeders), 25–30 g of body weight, maintained under a 12-h dark/light cycle and food and water ad libitum were used for these studies. Mice were housed in groups of five per cage (24 × 17 × 12 cm). The Internal Review Board at the University of Illinois at Chicago approved all animal experiment protocols for Animal Welfare. DFP and diazepam were purchased from Sigma–Aldrich. HUP was kindly offered by A. P. Kozikowski from the Department of Medicinal Chemistry and Pharmacology (University of Illinois, Chicago, IL). HUP and DFP were dissolved in sterile saline, and IMI and DZP were dissolved in vegetable oil. The drugs were injected s.c. between the shoulder blades of the animals. All injections (100 μl/10 g) were performed by using disposable tuberculin syringes with 26-gauge needles.

Fear Conditioning.

Fear conditioning was performed by using a computerized fear-conditioning system as reported in detail in ref. 39. Mice were placed into the training chamber (10 × 10× 12 cm, clear plastic) and allowed to explore for 3 min. After this time, they received an electric foot shock (2 s, 0.5 mA) delivered through the floor grid three times every 2 min. After the last shock, mice were allowed to explore the context for an additional 1 min before removal from the training chamber and placed back into the home cage. Freezing behavior was measured 24 h after training (for 5 min) in which the percentage of time spent in the context in which they were shocked was assessed. Freezing involved the absence of all movement except for respiratory-related movements while the mouse was in a stereotypical crouching posture (40).

Measurement of Locomotor Activity in a Novel Cage.

A computerized AccuScan 12 animal activity monitoring system (Columbus Instruments) assisted by VERSAMAX software (AccuScan Instruments) monitored locomotor activity in mice (41). Each activity cage consisted of a Perspex box (20 × 20 × 20 cm) surrounded by horizontal and vertical infrared sensor beams. The locomotor activity of these mice was recorded between 1:00 and 3:00 p.m. in the facility room where they had been housed.

Measurement of Seizure Activity.

The Racine scale was used (42) with the introduction of minor modifications to this seizure model. Behaviors were represented by the following numbers: 0, behavioral arrest (motionless), hair raising, excitement, and rapid breathing; 1, mouth movement of lips and tongue, vibrissae movements and salivation; 2, head clonus and eye clonus; 3, forelimb clonus, “wet dog shakes”; 4, clonic rearing; 5, clonic rearing with loss of postural control and uncontrollable jumping.

Neuronal Damage Evaluation.

Forty-eight hours after the acute DFP challenge, mice were anesthetized with Nembutal (50 mg/kg i.p.) and perfused with 4% paraformaldehyde in PBS (pH 7.4). Brains were removed and postfixed in 4% paraformaldehyde for at least 72 h in PBS (pH 7.4) at 4°C and then transferred to 30% sucrose in PBS (pH 7.4) at 4°C for cryoprotection. Fixed brains were then embedded in frozen section medium, and 20-μm thick sections were cut in a cryostat at 4°C and mounted on glass slides for TUNEL assays.

Tissues were permeabilized with 0.1% Triton X-100 in freshly prepared 0.1% sodium citrate for 2 min on ice, rinsed briefly, and incubated in proteinase K (15 mg/ml) for 10 min at room temperature and washed three times with PBS buffer for a total of 30 min. Endogenous peroxidase was quenched with 0.3% (vol/vol) hydrogen peroxide in methanol for 30 min, and the tissue was rinsed with PBS for 10 min. After equilibration in PBS buffer, sections were incubated with terminal deoxynucleotide transferase (TdT)-containing digoxigenin-dUTP at 37°C for 1 h in a humidified chamber. The TdT reaction was terminated by three washes in PBS buffer for 10 min. Antidigoxigenin peroxidase (converter-POD) was added to the tissue sections, and the sections were incubated for an additional 30 min at 37°C in a humidified chamber. Two controls per assay were performed: incubating sections with DNase I served as positive control, and omission of the terminal transferase from the reaction mixture served as the negative control. Staining was developed with diaminobenzidine–nickel ammonium sulfate, and light microscopic examination of the sections was performed at 40× magnification.

Acknowledgments.

We thank Drs. Maria Luisa Barbaccia (University of Rome, Tor Vercata, Italy) and Norton H. Neff (Ohio State University College of Medicine) for constructive criticisms and suggestions in the preparation of the article. This work was supported by National Institute of Mental Health/National Institutes of Health Grants MH5680 (to A.G.) and MH062090 (to E.C.) and a Regione Autonoma della Sardegna, Italy, “Master and Back” postdoctoral fellowship (to F.P.).

Footnotes

The authors declare no conflict of interest.

References

  • 1.Coupland R, Leins KR. Science and prohibited weapons. Science. 2005;308:1841. doi: 10.1126/science.1115436. [DOI] [PubMed] [Google Scholar]
  • 2.Romano JA, King JM. Psychological casualties resulting from chemical and biological weapons. Mil Med. 2001;166:21–22. [PubMed] [Google Scholar]
  • 3.Karalliedde L, Senanayake N. Organophosphorus insecticide poisoning. Br J Anaesth. 1989;63:736–750. doi: 10.1093/bja/63.6.736. [DOI] [PubMed] [Google Scholar]
  • 4.Buckley NA, Roberts D, Eddleston M. Overcoming apathy in research on organophosphate poisoning. BMJ. 2004;329:1231–1233. doi: 10.1136/bmj.329.7476.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lallement G, et al. Review of the value of huperzine as pretreatment of organophosphate poisoning. Neurotoxicology. 2002;23:1–5. doi: 10.1016/s0161-813x(02)00015-3. [DOI] [PubMed] [Google Scholar]
  • 6.Auta J, Costa E, Davis J, Guidotti A. Imidazenil: A potent and safe protective agent against diisopropyl fluorophosphate toxicity. Neuropharmacology. 2004;46:397–403. doi: 10.1016/j.neuropharm.2003.09.010. [DOI] [PubMed] [Google Scholar]
  • 7.Lemercier G, Carpentier P, Sentenac-Roumanou H, Morelis P. Histological and histochemical changes in the central nervous system of the rat poisoned by an irreversible anticholinesterase organophosphorus compound. Acta Neuropathol. 1983;61:123–129. doi: 10.1007/BF00697391. [DOI] [PubMed] [Google Scholar]
  • 8.Miller SA, Blick DW, Kerenyi SZ, Murphy MR. Efficacy of physostigmine as a pretreatment for organophosphate poisoning. Pharmacol Biochem Behav. 1993;44:343–347. doi: 10.1016/0091-3057(93)90472-6. [DOI] [PubMed] [Google Scholar]
  • 9.Kerenyi SZ, Murphy MR, Hartgraves SL. Toxic interactions between repeated soman and chronic pyridostigmine in rodents. Pharmacol Biochem Behav. 1990;37:267–271. doi: 10.1016/0091-3057(90)90332-c. [DOI] [PubMed] [Google Scholar]
  • 10.Grunwald J, Raveh L, Doctor BP, Ashani Y. Huperzine A as a pretreatment candidate drug against nerve agent toxicity. Life Sci. 1994;54:991–997. doi: 10.1016/0024-3205(94)00501-x. [DOI] [PubMed] [Google Scholar]
  • 11.Coelho F, Birks J. Physostigmine for Alzheimer's disease. Cochrane Database Syst Rev. 2001;2:CD001499. doi: 10.1002/14651858.CD001499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Philippens IH, Wolthuis OL, Busker RW, Langenberg JP, Melchers BP. Side effects of physostigmine as a pretreatment in guinea pigs. Pharmacol Biochem Behav. 1996;55:99–105. doi: 10.1016/0091-3057(96)83115-7. [DOI] [PubMed] [Google Scholar]
  • 13.Philippens IH, et al. Subchronic physostigmine pretreatment in guinea pigs: Effective against soman and without side effects. Pharmacol Biochem Behav. 1998;59:1061–1067. doi: 10.1016/s0091-3057(97)00511-x. [DOI] [PubMed] [Google Scholar]
  • 14.Meshulam Y, Davidovici R, Wengier A, Levy A. Prophylactic transdermal treatment with physostigmine and scopolamine against soman intoxication in guinea pigs. J Appl Toxicol. 1995;15:263–266. doi: 10.1002/jat.2550150406. [DOI] [PubMed] [Google Scholar]
  • 15.Philippens IH, Melchers BP, Olivier B, Bruijnzeel PL. Scopolamine augments the efficacy of physostigmine against soman poisoning in guinea pigs. Pharmacol Biochem Behav. 2000;65:175–182. doi: 10.1016/s0091-3057(99)00171-9. [DOI] [PubMed] [Google Scholar]
  • 16.Anderson DR, et al. Efficacy comparison of scopolamine and diazepam against soman-induced debilitation in guinea pigs. Fundam Appl Toxicol. 1994;22:588–593. doi: 10.1006/faat.1994.1065. [DOI] [PubMed] [Google Scholar]
  • 17.Janowsky DS. Central anticholinergics to treat nerve agent poisoning. Lancet. 2002;359:265–266. doi: 10.1016/S0140-6736(02)07434-2. [DOI] [PubMed] [Google Scholar]
  • 18.Lallement G, et al. Subchronic administration of various pretreatments of nerve agent poisoning. I. Protection of blood and central cholinesterases, innocuousness toward blood–brain barrier permeability. Drug Chem Toxicol. 2001;24:151–164. doi: 10.1081/dct-100102607. [DOI] [PubMed] [Google Scholar]
  • 19.Lallement G, Foquin A, Dorandeu F, Baubichon D, Carpentier P. Subchronic administration of various pretreatments of nerve agent poisoning. II. Compared efficacy against soman toxicity. Drug Chem Toxicol. 2001;24:165–180. doi: 10.1081/dct-100102608. [DOI] [PubMed] [Google Scholar]
  • 20.Dawson RM. Review of oximes available for treatment of nerve agent poisoning. J Appl Toxicol. 1994;14:317–331. doi: 10.1002/jat.2550140502. [DOI] [PubMed] [Google Scholar]
  • 21.Steppuhn KG, Turski L. Diazepam dependence prevented by glutamate antagonists. Proc Natl Acad Sci USA. 1993;90:6889–6893. doi: 10.1073/pnas.90.14.6889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kozikowski AP, et al. Delineating the pharmacophoric elements of huperzine A: Importance of the unsaturated three-carbon bridge to its AChE inhibitory activity. J Med Chem. 1991;34:3399–3402. doi: 10.1021/jm00116a010. [DOI] [PubMed] [Google Scholar]
  • 23.Ashani Y, Peggins JO, III, Doctor BP. Mechanism of inhibition of cholinesterases by huperzine A. Biochem Biophys Res Commun. 1992;184:719–726. doi: 10.1016/0006-291x(92)90649-6. [DOI] [PubMed] [Google Scholar]
  • 24.Tonduli LS, Testylier G, Masqueliez C, Lallement G, Monmaur P. Effects of huperzine used as pretreatment against soman-induced seizures. Neurotoxicology. 2001;22:29–37. doi: 10.1016/s0161-813x(00)00015-2. [DOI] [PubMed] [Google Scholar]
  • 25.Guidotti A, et al. GABAergic dysfunction in schizophrenia: New treatment strategies on the horizon. Psychopharmacology (Bere) 2005;180:191–205. doi: 10.1007/s00213-005-2212-8. [DOI] [PubMed] [Google Scholar]
  • 26.Giusti P, et al. Imidazenil: A new partial positive allosteric modulator of γ-aminobutyric acid (GABA) action at GABAA receptors. J Pharmacol Exp Ther. 1993;266:1018–1028. [PubMed] [Google Scholar]
  • 27.Auta J, Giusti P, Guidotti A, Costa E. Imidazenil, a partial positive allosteric modulator of GABAA receptors, exhibits low tolerance and dependence liabilities in the rat. J Pharmacol Exp Ther. 1994;270:1262–1269. [PubMed] [Google Scholar]
  • 28.Auta J, et al. Comparison of the effects of full and partial allosteric modulators of GABAA receptors on complex behavioral processes in monkeys. Behav Pharmacol. 1995;6:323–332. [PubMed] [Google Scholar]
  • 29.Auta J, Guidotti A, Costa E. Imidazenil prevention of alprazolam-induced acquisition deficit in patas monkeys is devoid of tolerance. Proc Natl Acad Sci USA. 2000;97:2314–2319. doi: 10.1073/pnas.97.5.2314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Costa E, Auta J, Guidotti A. Tolerance and dependence on ligands of the benzodiazepine sites expressed by GABAA receptors. In: Mohler H, editor. Handbook of Experimental Pharmacology. Vol 150. Berlin: Springer; 2001. pp. 227–250. [Google Scholar]
  • 31.Impagnatiello F, et al. Modifications of γ-aminobutyric acid A receptor subunit expression in rat neocortex during tolerance to diazepam. Mol Pharmacol. 1996;49:822–831. [PubMed] [Google Scholar]
  • 32.Rump S, et al. Effects of imidazenil, a new benzodiazepine receptor partial agonist, in the treatment of convulsions in organophosphate intoxications. Neurotox Res. 2000;2:17–22. doi: 10.1007/BF03033323. [DOI] [PubMed] [Google Scholar]
  • 33.Thompson DM, Auta J, Guidotti A, Costa E. Imidazenil, a new anxiolytic and anticonvulsant drug, attenuates a benzodiazepine-induced cognition deficit in monkeys. J Pharmacol Exp Ther. 1995;273:1307–1312. [PubMed] [Google Scholar]
  • 34.Qian BC, et al. Pharmacokinetics of tablet huperzine A in six volunteers. Acta Pharmacol Sin. 1995;16:396–398. [PubMed] [Google Scholar]
  • 35.Broomfield CA, et al. Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates. J Pharmacol Exp Ther. 1991;259:633–638. [PubMed] [Google Scholar]
  • 36.Gordon RK, et al. The NMDA receptor ion channel: A site for binding of huperzine A. J Appl Toxicol. 2001;21:S47–S51. doi: 10.1002/jat.805. [DOI] [PubMed] [Google Scholar]
  • 37.Costa E, Guidotti A. Benzodiazepines on trial: A research strategy for their rehabilitation. Trends Pharmacol Sci. 1996;17:192–200. doi: 10.1016/0165-6147(96)10015-8. [DOI] [PubMed] [Google Scholar]
  • 38.Rudolph U, et al. Benzodiazepine actions mediated by specific γ-aminobutyric acid (A) receptor subtypes. Nature. 1999;401:796–800. doi: 10.1038/44579. [DOI] [PubMed] [Google Scholar]
  • 39.Pibiri F, Nelson M, Guidotti A, Costa E, Pinna G. Decreased corticolimbic allopregnanolone expression during social isolation enhances contextual fear: A model relevant for posttraumatic stress disorder. Proc Natl Acad Sci USA. 2008;105:5567–5572. doi: 10.1073/pnas.0801853105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Blanchard RJ, Blanchard DC. Passive and active reactions to fear-eliciting stimuli. J Comp Physiol Psychol. 1969;68:129–135. doi: 10.1037/h0027676. [DOI] [PubMed] [Google Scholar]
  • 41.Pinna G, Galici R, Schneider HH, Stephens DN, Turski L. Alprazolam dependence prevented by substituting with the β-carboline abecarnil. Proc Natl Acad Sci USA. 1997;94:2719–2723. doi: 10.1073/pnas.94.6.2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephal Clin Neurophysiol. 1972;32:281–294. doi: 10.1016/0013-4694(72)90177-0. [DOI] [PubMed] [Google Scholar]

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