Authors :
Presenting Author: Yam Nath Paudel, PhD – Virginia Commonwealth University
Robert Blair, PhD – Virginia Commonwealth University
Elisa Hawkins, BS – Virginia Commonwealth University
Jason Funderburk, MS – Virginia Commonwealth University
Melissa Morgan, BS – Virginia Commonwealth University
Matthew Halquist, PhD – Virginia Commonwealth University
Daniel Calvano, BS – Virginia Commonwealth University
Jennifer Koblinski, PhD – Virginia Commonwealth University
Hope Richard, MD, PhD – Virginia commonwealth University
Laxmikant Deshpande, PhD – Virginia Commonwealth University
Rationale:
Organophosphate (OP) compounds are chemical threat agents. Lethal OP exposure produces a hypercholinergic response that quickly leads to status epilepticus (SE). While treatment with standard-of-care therapy (SOC: atropine, 2-PAM, midazolam) reduces acute OP-SE mortality, chronic morbidities, including seizures and memory deficits, can still occur. Neuronal injury and neuroinflammatory pathways have been targeted to improve outcomes in OP-SE in rat models. Verapamil (VPM) is a calcium channel blocker for blood pressure management. VPM is reported to be neuroprotective and anti-inflammatory in experimental models of CNS injuries. Here, we investigated the safety, toxicity, pharmacokinetic (PK) profile neuroprotective effect of i.m. VPM therapy in a rat model of OP diisopropyl fluorophosphate (DFP) induced SE.
Methods:
Rats were injected with DFP (4 mg/kg, s.c). One minute later, atropine (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m) were administered, followed by midazolam (1.78 mg/kg, i.m.) at 1-h post SE onset. At this point, DFP rats received VPM (10 mg/kg, i.m.) b.i.d. or saline (SAL) for 3 days. On the fourth day, rats were perfused, and brains and quadricep muscles were dissected and sectioned for histology. Neuronal injury was assessed using fluoro-jade C (FJC), while hematoxylin & eosin (H&E) were used to evaluate inflammation at the i.m. injection site. For PK studies, no-SE control and DFP-SE rats were treated once with VPM (p.o. or i.m.), and blood and brain levels were analyzed over 24 hours using LC-MS/MS.
Results:
DFP exposure produced a rapid onset of SE. VPM+SOC produced no significant increase in mortality compared to the SAL+SOC group. H&E stained quadricep sections from DFP-VPM were scored on a four-point pathology scale and showed inflammation scores that were not significantly different from DFP-SAL (2.0 ± 0.7 vs. 1.38 ± 0.7, p= 0.1, n= 8, t-test). Non-compartmental PK analyses revealed that AUC for i.m. VPM was at least 60% compared to p.o. VPM while values for i.m. Tmax, T1/2, and Cmax were between 20-500% compared to p.o. VPM. Peak brain levels of VPM up to 1000 ng/g were noted within 30-min following DFP-SE (n= 3 rats/group/time point). Finally, extensive FJC+ staining indicative of dying neurons was seen in multiple brain regions from DFP-SAL rats, which was markedly reduced in DFP-VPM rats. Robust neuroprotection was seen in CA1 (42%), CA3 (48%), DG (60%), thalamus (61%), and parietal cortex (55%) regions (n =7, two-way ANOVA, p< 0.05).