Abstracts

Rationale: Hyperpolarizing GABAAR currents are critically dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter (KCC2). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to benzodiazepine (BDZ-RSE). Here we aim to test the efficacy of a KCC2 activator, OV350, in a rat model of BDZ-RSE induced by the nerve agent, soman. OV350 was evaluated as an adjunctive therapy to standard medical countermeasures (SMCs) atropine, 2-PAM, and midazolam.

Methods: Methods were based on a model of nerve agent-induced BDZ-RSE that has been previously published [1]. Adult Sprague Dawley rats (male, n = 61) were surgically implanted with jugular vein catheters and electroencephalogram (EEG) electrodes approximately one week prior to soman exposure. On exposure day, approximately one hour of baseline EEG was recorded for each rat. To improve survival in this model, HI-6 (125 mg/kg) and atropine methyl nitrate (2 mg/kg) were administered 30 minutes before and 1 minute after, respectively, soman exposure (126 µg/kg, SC). At 20 minutes after the electrographic onset of SE (~ 5 min after soman treatment), the SMCs atropine sulfate (0.45 mg/kg), pralidoxime chloride (2-PAM; 25 mg/kg), and midazolam (1.8 mg/kg) were administered by intramuscular injection concurrently with the test compound or corresponding vehicle administered intravenously via the pre-implanted jugular vein catheter. Three doses of OV350 (50, 100 or 150 mg/kg) and one vehicle control group were tested. EEG was continuously monitored for at least 4 hours after administration of SMCs plus test compound/vehicle, after which rats were euthanized via transcardial perfusion. Brains were extracted, sectioned, and stained with H&E and a board-certified pathologist scored brain regions that are particularly vulnerable to damage following nerve agent-induced SE. EEG recordings were reviewed post-hoc for seizure onset and latency to termination (LTT). Termination was defined as a reduction of EEG amplitude to less than twice that of baseline coupled with cessation of rhythmic spiking activity lasting for at least 5 minutes.

1. Jackson, C., et al., J Pharmacol Toxicol Methods, 2019. 97: p. 1-12.

Results: Of the 61 animals enrolled, 47 survived the soman exposure long enough to receive the SMCs with OV350 or vehicle treatment. Only 7% (1 of 14) in the vehicle group demonstrated seizure termination, whereas 36% (5 of 14 animals), 42% (5 of 12 animals) or 14% (1 of 7) with 50, 100 or 150 mg/kg OV350, respectively, were protected. The mean LTT for OV350 at 50 mg/kg was 54 ± 51 min and 26 ± 30 min for 100 mg/kg. Survival improved with low-dose OV350 with 71% of the animals surviving to the 4 hours timepoint compared to only 50% with vehicle, 58% with 100 mg/kg or 14% with 150 mg/kg OV350.

Conclusions: These data suggest that the KCC2 activator, OV350, can act to terminate seizures, improve survival, and protect from the associated brain tissue damage following soman exposure plus SMCs.

Funding: Studies funded by Ovid Therapeutics