Abstracts

Rationale: Organophosphate (OP) compounds are chemical threat agents and include commonly used pesticides and nerve gas agents. Accidental or intentional OP exposure produces a rapid onset status epilepticus (SE) that particularly damages the heart and the brain. There is an unmet need for therapies that could be administered after OP exposures that significantly reduce the OP-SE mortality. In this study, we investigated whether delayed treatment with intramuscular (im) adjunct combination therapy of atenolol (AT) and levetiracetam (LV) would protect the heart and the brain from SE vulnerability and extend significant protection from OP mortality. We used diisopropyl fluorophosphate (DFP), paraoxon (POX), and sarin (GB) as OP agents. Methods: Male Sprague-Dawley rats (280-300g) were injected with DFP or POX (4mg/kg, sc). One minute later, rats received atropine (0.5mg/kg, im) and 2-PAM (25mg/kg, im). Rats quickly developed SE which was stopped with midazolam (1.8mg/kg, im) at 1-h post SE onset. For sarin studies, GB (132µg/kg, sc) was followed with atropine (2 mg/kg, im) and 2-PAM (25mg/kg, im). At 1-h after SE onset, GB rats were treated with midazolam (5mg/kg, im). Rats were then treated with AT (5mg/kg, im) and LV (50mg/kg, im) b.i.d. for 7-days. Mortality was assessed at 30-d after the end of the adjunct therapies. Pharmacokinetic properties of i.m. administered AT and LV were assessed in whole blood using liquid chromatography tandem mass spectroscopy. Muscle pathology was conducted to assess safety of im AT and LV theray. Results: DFP exposure produced a rapid onset SE that was treated effectively with atropine, 2-PAM and midazolam therapy. Percent mortality assessed 30-d post DFP SE was 52.2 +- 5.6 and with the 7-d treatment with AT + LV the mortality was reduced to 31.5 +- 2.4. (n= 24), representing a significant 40% reduction in mortality due to DFP SE. These numbers are in agreement with protection observed against POX SE mortality. The percent mortality following sarin SE was 20% and with the 7-d treatment with AT + LV, the mortality was reduced to 6.6% (n=15), representing a 67% reduction in mortality following sarin-induced SE. Pharmacokinetic analyses revealed comparable Tmax, t1/2, and Cmax, values for oral and i.m. administered AT and LV (n= 4 at each time-point). Muscle pathology at injection site was assessed using standard four-point inflammation scale and showed no significant pathological changes acutely 1 day after injection or chronically at 2 and 4 months after injection. At the sub-chronic and chronic time-points, inflammation score was below 1.0 (n= 6). Conclusions: Our data provide evidence that similar to lethal POX exposures, mortality following DFP or GB exposures could be significantly reduced by im treatment with AT and LV, even when the intervention is delayed by 1-h. The im route produced stable therapeutic blood levels and favorable kinetics for AT and LV and insignificant muscle pathology established safety of repeated AT+LV therapy. These studies also show that im route is an effective, faster and safe method to deliver AT and LV during SE. Funding: This work was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke Grant No. (U01-NS105058)