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

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 GABA(A) 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 approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/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 ED(50) of IMI to block DFP-induced mortality is approximately 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.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

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.

Fig. 5.

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.

Fig. 6.

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.)

Fig. 7.

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.