Efficacy of the GluK1/AMPA receptor antagonist LY293558 against seizures and neuropathology in a soman-exposure model without pretreatment and its pharmacokinetics after intramuscular administration

Abstract

Control of brain seizures after exposure to nerve agents is imperative for the prevention of brain damage and death. Animal models of nerve agent exposure make use of pretreatments, or medication administered within 1 minute after exposure, in order to prevent rapid death from peripheral toxic effects and respiratory failure, which then allows the testing of anticonvulsant compounds. However, in a real-case scenario of an unexpected attack with nerve agents, pretreatment would not be possible, and medical assistance may not be available immediately. To determine if control of seizures and survival are still possible without pretreatment or immediate pharmacologic intervention, we studied the anticonvulsant efficacy of the GluK1 (GluR5)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid (LY293558) in rats that did not receive any treatment until 20 minutes after exposure to the nerve agent soman. We injected LY293558 intramuscularly, as this would be the most likely route of administration to humans. LY293558 (15 mg/kg), injected along with atropine and the oxime HI-6 at 20 minutes after soman exposure, stopped seizures and increased survival rate from 64% to 100%. LY293558 also prevented neuronal loss in the amygdala and hippocampus, and reduced neurodegeneration in a number of brain regions studied 7 days after soman exposure. Analysis of the LY293558 pharmacokinetics after intramuscular administration showed that this compound readily crosses the blood-brain barrier. There was good correspondence between the time course of seizure suppression by LY293558 and the brain levels of the compound.

Fig. 1.

Time-course of behavioral seizure suppression by LY293558 (15 mg/kg) administered intramuscularly at 20 minutes after soman injection, along with atropine and HI-6. Data points are the group means ± S.E.M. (SOMAN, n = 18; SOMAN+LY293558, n = 18) of the maximum Racine scale scores, in blocks of 10 minutes. Vertical arrows indicate the time points of drug administration. ^#P < 0.05 in comparison with the SOMAN group (ANOVA, Bonferroni post-hoc test).

Fig. 2.

Administration of LY293558 (i.m.) stops soman-induced generalized seizures and reduces the total duration of status epilepticus (SE) in the 24-hour period after soman exposure. (A) Administration of 15 mg/kg LY293558 at 20 minutes after soman exposure completely blocked electrographic seizure activity within 40 minutes (bottom set of traces). (B) An example of seizures persisting in rats that did not receive LY293558. The numbers 1, 2, 3, and 4, in (A) and (B), refer to the electrodes/sites from where electrical activity was sampled, as shown diagrammatically in (C) (1, left frontal; 2, right frontal; 3, left parietal; 4, right parietal; 5, cerebellar reference electrode). (D) Left bar graph shows the duration of the initial SE (the SE that started within 10 minutes after soman exposure and was terminated by 15 mg/kg LY293558 in the SOMAN+LY293558 group, or spontaneously in the SOMAN group). Right bar graph shows the duration of SE throughout the 24-hour period after soman exposure. Sample size n = 5 for the SOMAN group, and n = 8 for the SOMAN+LY293558 group. **P < 0.01, ***P < 0.001 (unequal variance t test).

Fig. 3.

Administration of LY293558 (i.m.) protects against neuronal loss in the basolateral amygdala (BLA) and the CA1 hippocampal area, 7 days after soman-induced SE. (A) Panoramic photomicrographs of Nissl-stained sections of half hemispheres, outlining the amygdalar nucleus and the ventral hippocampal subfield where stereologic analysis was performed (red highlight). (B) Representative photomicrographs of Nissl-stained sections showing BLA and CA1 cells from a control rat that received saline in place of soman, a soman-exposed rat that did not receive anticonvulsant treatment, and a soman-exposed rat that received 15 mg/kg LY293558 at 20 minutes after soman exposure. Total magnification is 630× and scale bar is 50 µm. (C) Group data (mean ± S.E.M.; n = 18 for the SOMAN group and n = 18 for the SOMAN+LY293558 group) of stereologic estimation of the total number of Nissl-stained neurons in the BLA (left) and CA1 area (right), expressed as percent of the control. There was significant neuronal loss in the soman-exposed rats that did not receive anticovulsant treatment, whereas there was no neuronal loss in the soman-exposed rats that were treated with LY293558. *P < 0.05 in relation to the control group or the SOMAN+LY293558 group (ANOVA, LSD post hoc test).

Fig. 4.

Administration of LY293558 (i.m.) protects against neuronal degeneration, 7 days after soman-induced SE. (A and B) Panoramic photomicrographs of Nissl-stained sections showing the brain regions from where the Fluoro-Jade C (FJC) photomicrographs shown in (C) were taken. (C) Representative photomicrographs of FJC-stained sections from the brain regions where neuronal degeneration was evaluated, for the SOMAN and the SOMAN+LY293558-treated groups. Total magnification is 100× . Scale bar is 50 μm. (D) Bar graph showing the median neuropathology score and interquartile range (n = 18 for the SOMAN group and n = 18 for the SOMAN+LY293558 group) for the amygdala (Amy), piriform cortex (Pir), entorhinal cortex (Ent), the CA1 and CA3 subfields of the ventral hippocampus, hilus, and neocortex (neo-Ctx). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with the SOMAN group (Mann-Whitney U test).

Fig. 5.

Plasma (square) and brain (triangle) concentrations of LY293558 after i.m. injection of 15 mg/kg. For plasma samples: n = 6 at time points 0.25, 0.5 and 1 hour; n = 3 at time point 1.5 hours and later. For brain samples: n = 3 for all time points, except at 1 hour, where n = 6. Data points represent the mean and standard deviation.