Abstracts

Rationale: Organophosphate (OP) compounds are chemical threat agents. OP exposure produces a hypercholinergic response that quickly leads to status epilepticus (SE). SE particularly targets the heart and the brain and despite existing therapies it is still associated with significant mortality and morbidity. In this study, we investigated effect of an intramuscular (i.m.) adjunct therapy of atenolol (AT) and levetiracetam (LV) on paraoxon (POX) induced SE. We also investigated the safety profile of this i.m. combination therapy and also compared bioavailability of these agents when administered both i.m. and orally. Methods: Male Sprague-Dawley rats (250-300g) were injected with POX (2 mg/kg, s.c). One minute later, rats were injected with atropine sulfate (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m). Rats quickly developed SE, which was then stopped with midazolam (2 mg/kg, i.m.) at 1-h post SE onset. Rats were than treated with AT (5 mg/kg, i.m.) and LV (50 mg/kg, i.m.) b.i.d. for 7-days. Mortality was assessed at 30-days after the end of adjunct therapy. At 1-day, 2-months, and 4-months after the end of adjunct therapy, rats were sacrificed and quadricep muscles were dissected and analyzed for muscle pathology. In separate experiments, control rats were treated once with AT and LV orally or i.m. and blood was drawn at various time points ranging from 0-min to 48-h. Blood plasma was analyzed for AT and LV levels using LC-MS/MS and pharmacokinetic parameters were compared between these two routes of administration using non-compartment analyses. Results: POX exposure produced rapid onset of SE that was treated effectively with standard atropine, 2-PAM and midazolam therapy. Percent mortality assessed at 30-days post POX SE was 33 ± 6%. A 7-day AT+ LV therapy produced a 66 ± 6% reduction in SE mortality. Muscle safety following repeated 7-day AT+ LV injections was assessed in sections of quadriceps using a standard 4-point inflammation scale. AT+ LV therapy exhibited pathology inflammation scores that were not significantly different from saline treated controls when examined acutely at 1-day and chronically at 2 and 4 months after injections. Pharmacokinetic analyses revealed that i.m. to oral AUC was at least 60% and other parameters Tmax, T_1/2, and Cmax were between 20 to 500% for oral compared to i.m. administration for AT and LV.    Conclusions: These studies show that a 7-day treatment regimen with AT and LV significantly reduced mortality following POX SE. In addition, our studies have provided the first direct evidence that the i.m. route of administration is an effective method to deliver AT and LV following SE. The data from this study demonstrate that the i.m. route can achieve stable therapeutic blood levels and kinetics in comparison to oral administration. The results also establish the safety of repeated i.m. AT+ LV therapy. The optimal dosing regimen of 7 day AT + LV did not cause significant muscle pathology acutely at 1 day after injection or chronically at 2 and 4 months after injection. Our data provides evidence of safety and efficacy of i.m. AT+LV therapy for reducing mortality following OP-induced 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. U01NS105058-01 to R.J.D.