Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents

Abstract

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer's disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.

Fig. 1.

Pretreatment with galantamine prevents the acute toxicity of lethal doses of OPs: Comparison with pyridostigmine and huperzine. In all experiments, guinea pigs received an i.m. injection of selected doses of galantamine, pyridostigmine, or huperzine followed 30 min later by a single s.c. injection of 1.5× LD₅₀ (42 μg/kg) or 2.0× LD₅₀ (56 μg/kg) soman, 1.5× LD₅₀ sarin (56 μg/kg), or the indicated doses of paraoxon. At 1 min after the OP challenge, all animals received atropine (1–10 mg/kg, i.m.). (A–C) Dose–response relationships for galantamine or atropine to maintain 24-h survival of animals challenged with nerve agents. (D) Dose–response relationship for paraoxon-induced decrease in 24-h survival of atropine-treated guinea pigs that were pretreated with saline or galantamine. (E) Effects of increasing doses of pyridostigmine or huperzine in maintaining 24-h survival of soman-challenged, atropine-treated animals. Each group had 8–12 animals. Percent survival represents the percent of animals that were kept alive because they presented no life-threatening symptoms.

Fig. 2.

Long-term effectiveness and acute toxicity of different antidotal therapies against OP poisoning. (A) Seven-day survival of guinea pigs treated with galantamine at 30 min before and atropine at 1 min after their challenge with 1.5× LD₅₀ soman. Each group had 8–12 animals. (B) Seven-day follow-up of the weight of animals subjected to different treatments. Weights are expressed as percent of the weights measured 1 h before a treatment. Control groups consist of animals that received a single i.m. injection of atropine, galantamine, huperzine, or saline. The soman/atropine groups consist of animals treated with galantamine or huperzine at 30 min before and atropine at 1 min after soman (n = 5–8 animals per treatment). (C) Graphs of the average total distance traveled and stereotypy of guinea pigs in an open-field arena at the indicated times after they received an i.m. injection of saline, galantamine, or huperzine (n = 6 animals per treatment). In B and C, results are presented as the mean ± SEM. Asterisks indicate that results from huperzine- and saline-treated animals are significantly different at P < 0.05 (ANOVA followed by Dunnett’s post hoc test).

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 mg/kg galantamine at 1, 2, 3, 4, or 5 h before the s.c. injection of 1.5× LD₅₀ soman that was followed 1 min later by an i.m. injection of 10 mg/kg 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 1.5× LD₅₀ soman or 2–3 mg/kg paraoxon, respectively. Each group had 8–10 animals.

Fig. 4.

Soman-induced neurodegeneration is not present in the hippocampus, pyriform cortex, and amygdala of guinea pigs pre- or posttreated with galantamine. (A and B) Representative photomicrographs of the hippocampal CA1 field, the pyriform cortex, and the amygadala of guinea pigs that were euthanized 24 h after an i.m. injection of saline (A) or 8 mg/kg galantamine (B). No FJ-B-positive neurons were seen in the brains of these animals. (C) Large numbers of FJ-B-positive neurons were seen in all three index areas of the brain of a guinea pig that survived for 24 h after the challenge with 1.5× LD₅₀ soman. (D and E) FJ-B-positive neurons were rarely seen in brain sections of animals that received galantamine (8 mg/kg, i.m.) at 30 min before (D) or 5 min after (E) soman. In C–E, all animals received atropine (10 mg/kg, i.m.) at 1 min after the OP, and they were euthanized at 24 h after the OP challenge. Photomicrographs are representative of results obtained from each group, which had five animals.

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 = 4–6 animals per time point) with galantamine (8 mg/kg, i.m.) is plotted on a logarithmic scale against time. (C) AChE activity measured in samples from saline-treated animals was taken as 1 and used to normalize the enzyme activity measured in samples obtained at various times after treatment of animals with galantamine (8 mg/kg, i.m.). Normalized inhibition (1 − normalized activity) is plotted against the time at which samples were obtained. Asterisks indicate that results from galantamine- and saline-treated animals are significantly different at P < 0.001 (***) or < 0.01 (**) (ANOVA followed by Dunnett’s post hoc test). In A–C, the first point corresponds to results obtained at 5 min after the treatment. (D) Increasing concentrations of galantamine were added in vitro to brain homogenates and blood samples obtained from naive animals. AChE activity in untreated samples was taken as 1, and it was used to normalize activity measured in galantamine-treated samples. The graph of normalized AChE activity vs. galantamine concentrations was fitted with the Hill equation. Results are presented as the mean ± SEM (n = 4–6 animals per galantamine concentration).