Pretreatment with the neurosteroid allopregnanolone prevents soman-induced seizures and brain pathology in rats.

Mark C. Moffett, Julia E. Schwartz, Michael F. Stone, Deanna Maida, Mark K. Schultz, Lucille A. Lumley
U.S. Army Medical Research Institute of Chemical Defense Analytical Toxicology Division Neurobehavioral Toxicology Branch

This research was supported by the Defense Threat Reduction Agency-Joint Science and Technology Office, Medical S&T Division (1.E0028_08_RC_C; Dr. Lucille Lumley, PI)
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Background
Exposure to high doses of soman (GD) can induce prolonged seizures and subsequent excitoxicity resulting in profound neuropathology Current therapies (oximes, anticholinergics, and benzodiazepines) promote survival but are not fully protective Neuroactive steroids are effective neuroprotective agents in a variety of animal models and are potential adjunct therapeutics

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Cholesterol

P450scc

Pregnenolone
3HSD-1 3HSD-2

P450c17

17-OH-Preg

P450c17

DHEA

Progesterone
5-reductase 5-reductase

5-DHP
3-HSD

5-DHP
3-HSD

35-THP

35-THP

(Allopregnanolone) (Pregnanolone)

DHEA, dehydroepiandrosterone HSD, hydroxysteroid dehydrogenase THP, tetrahydroprogesterone

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Neuroactive Steroids
Modulate excitability through interaction with membrane receptors and ion channels Not active at intracellular steroid receptors Potentiate GABA-evoked Clcurrent Directly activate GABAA channels in high concentrations Potentiate the effects of benzodiazepines and barbiturates

Belelli and Lambert (2003) Nature Rev Neurosci

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Therapeutic Potential
ALLO and PREG are effective against PTZ-, bicuculline- and picrotoxin-induced seizures
Belelli et al. (1989), Hgskide et al. (1988), Kokate et al. (1994)

PREG attenuated NMDA-induced convulsions and both ALLO and PREG prevented NMDA-induced lethality
Gasior et al. (1997)

Protective index (TD50/ED50) is comparable to clinically used antiepileptic drugs No tolerance to the anticonvulsant effects of PREG in mice
Kokate et al. (1998)

Inactive doses of ALLO and PREG potentiated the anticonvulsant effects of diazepam
Gasior et al. (1997)

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Rat Model
Male Sprague-Dawley rats were surgically implanted with the F40EET transmitter (Data Sciences International) EEG activity was continuously monitored throughout the duration of the experiment
www.datasci.com

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Rat Model-PREG
30 min pretreatment with the oxime HI-6 (125 mg/kg, ip) Experiment 1: PREG (4.0 mg/kg, iv) or vehicle (30% HPCD, 1 ml/kg, iv) immediately prior to GD 1.0xLD50 GD or saline (0.5 ml/kg, sc) followed 1 min later with atropine sulfate (2.0 mg/kg, im) Diazepam (10 mg/kg, sc) was administered 30 min post-seizure onset Experiment 2: PREG (4.0 mg/kg, iv) or vehicle (30% HPCD, 4 ml/kg, iv) administered 30 min post-seizure onset
30
HI-6
Exp. 1 PREG Atropine sulfate

Diazepam

0 -30
GD 1.0xLD50

8-10 1
Seizure Onset
Exp. 2 PREG

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Seizure Duration
Soman: Pregnanolone
200

Body Weights
Rats treated with the standard therapy had a significant drop in body weight following GD exposure PREG treated rats showed a lesser drop in body weight compared to GD-exposed controls

***

Seizure Duration (minutes)

150

100

50

0 Saline GD GD/Preg(pre) GD/Preg(post)

Group

GD 50% (2/4) mortality within 48 hrs 0% mortality in PREG (pre and post) treated rats Average duration of the initial seizure was significantly reduced in post-PREG rats

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Soman: Pregnanolone
75

***

Neuropathology Score

50

25

0 Saline GD GD/Preg(pre) GD/Preg(post)

GD-exposed rats displayed sever fiber degeneration in the piriform cortex, thalamus and cingulum PREG administered prior to GD exposure or 30 min post-seizure onset prevented brain pathology

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Rat Model-ALLO
30 min pretreatment with the oxime HI-6 (125 mg/kg, ip) Allopregnanolone (4.0 mg/kg, iv) or vehicle (30% HPCD, 1 ml/kg, iv) immediately prior to GD 1.0xLD50 GD or saline (0.5 ml/kg, sc) followed 1 min later with atropine sulfate (2.0 mg/kg, im) Diazepam (10 mg/kg, sc) was administered 30 min post-exposure

HI-6

ALLO

Atropine sulfate

Diazepam

0 -30
GD 1.0xLD50

8-10 1
Seizure Onset

30

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ALLO

60% (3/5) mortality in vehicle + GD rats 14% (1/7) mortality in ALLO pretreated rats Pretreatment with ALLO prevented GD-induced drop in body weight

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Baseline

10 min Post-Exposure

1 hr Post-Diazepam

No GD + Vehicle

10 post exposure

GD + Vehicle

GD + ALLO

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ALLO

Neuronal tract degeneration was evident in vehicle treated GD-exposed rats in the piriform cortex, thalamus and medial amygdaloid nucleus No degeneration is present in the ALLO pretreated rats

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Conclusions
Standard treatment failed to protect rats from the neuropathology resulting from exposure to 1.0xLD50 GD Pretreatment with PREG reduced the number of rats displaying GD-induced seizures and seizureassociated neuropathology Post-exposure treatment with PREG reduced seizure duration and neuropathology Pretreatment with ALLO prevented GD-induced seizures and subsequent neuropathology completely.

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Future Directions
ALLO and PREG
Sedation/motor toxicity
Potentiate effects of BDZs

Ganaxolone
3-methylated synthetic analog of ALLO Methyl group partially protects 3-OH from metabolism Orally available Currently in Phase II clinical trials for treatment of epilepsy

Low bioavailabilty Metabolism (3-OH)

Rapid sulfate or glucoronide conjugation Oxidation to ketone

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Acknowledgements
Lumley lab members:
Ariel Capilli Kristen Kamberger Josh Kraft Alison Nelson Caroline Schultz Mike Stone Stephen Estes Nathan Kelley Wuya Lumeh Mike Pham Mark Schultz Theresa Ward Rose Kajih Rachel Knopp Dr. Govinni Mohan Kristy Redding Julia Schwartz

The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Department of Defense The experimental protocol was approved by the Animal Care and Use Committee at the USMRICD and all procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and the Animal Welfare Act of 1966, as amended.

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