Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD5 0 , and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD 50 sarinexposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD 50 soman-exposed guinea pigs. Positive dose-response relationships were observed in all low-level, 0.05 to 0.4 LD 50 , exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD 50 exposure. Of the five types of guinea pig

Objective Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α‐hydroxy‐5β pregnan‐20‐one), a GABA A receptor‐positive allosteric modulator, as an adjunct to benzodiazepine therapy against the chemical warfare nerve agent (CWNA) sarin (GB), using whole‐body exposure, an operationally relevant route of exposure to volatile GB. Methods Rats implanted with telemetry transmitters for the continuous measurement of cortical electroencephalographic (EEG) activity were exposed for 60 minutes to 3.0 LCt 50 of GB via whole‐body exposure. At the onset of toxic signs, rats were administered an intramuscular injection of atropine sulfate (2 mg/kg) and the oxime HI‐6 (93.6 mg/kg) to increase survival rate and, 30 minutes after seizure onset, treated subcutaneously with diazepam (10 mg/kg) and intravenously with pregnanolone (4 mg/kg) or vehicle. Animals were evaluated for GB‐induced status epilepticus (SE), spontaneous recurrent seizures (SRS), impairment in spatial memory acquisition, and brain pathology, and treatment groups were compared. Results Delayed dual therapy with pregnanolone and diazepam reduced time in SE in GB‐exposed rats compared to those treated with delayed diazepam monotherapy. The combination therapy of pregnanolone with diazepam also prevented impairment in the Morris water maze and reduced the neuronal loss and neuronal degeneration, evaluated at one and three months after exposure. Significance Neurosteroid administration as an adjunct to benzodiazepine therapy offers an effective means to treat benzodiazepine‐refractory SE, such as occurs following delayed treatment of GB exposure. This study is the first to present data on the efficacy of delayed pregnanolone and diazepam dual therapy in reducing seizure activity, performance deficits and brain pathology following an operationally relevant route of exposure to GB and supports the use of a neurosteroid as an adjunct to standard anticonvulsant therapy for the treatment of CWNA‐induced SE.

Intramuscular (i.m.) diazepam is included by the US military as an anticonvulsant in the standard therapeutic regimen for organophosphorus nerve agent intoxication. In this study we investigated the pharmacokinetics of diazepam after i.m. administration while monitoring pharmacodynamic (electroencephalogram, EEG) data in soman-exposed guinea pigs. Prior to experiments the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered pyridostigmine (0.026 mg x kg(-1) i.m.) 30 min prior to soman (56 microg x kg(-1), 2 x LD50; subcutaneously, s.c.), which was followed in 1 min by atropine sulfate (2 mg x kg(-1) i.m.) and pralidoxime chloride (25 mg x kg(-1) i.m.). All animals receiving this regimen developed seizure activity. Diazepam (10 mg x kg(-1) i.m.) was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure terminated or not terminated at 30 min after diazepam. Serial blood samples were obtained from each animal. Diazepam (10 mg x kg(-1) i.m.) terminated seizure activity in 52% of the animals within 30 min. The pharmacokinetics were characterized by a one-compartment model with first-order absorption and elimination. The maximum plasma concentrations (Cmax) were 991 and 839 ng x ml(-1) for seizure terminated and not terminated, respectively. Mean plasma concentrations of diazepam were significantly different (P < 0.05) for seizure terminated vs not terminated groups at 30 min. The plasma Cmax in seizure-terminated animals in this study is similar to the minimum range of plasma diazepam (200-800 ng x ml(-1)) reported to suppress seizure activity in humans. It has been reported in an earlier study that the minimum effective i.m. dose (0.1 mg x kg(-1)) required to prevent soman-induced convulsions in Rhesus monkeys produces a mean Cmax of 50 ng x ml(-1) for diazepam. The data from our current study suggest that a higher dose (and corresponding Cmax) is necessary to terminate ongoing seizure activity.

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