USAMRICD Research, Education, and Training for Medical Chemical Defense 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)
Transcript
USAMRICDResearch, Education, and Training for Medical Chemical Defense
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 DefenseAnalytical 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)
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Background
• Exposure to high doses of soman (GD) can induce prolonged seizures and subsequent excitoxicityresulting in profound neuropathology
• Current therapies (oximes, anticholinergics, and benzodiazepines) promote survival but are not fully protective
• Neuroactive steroids are effective neuroprotectiveagents in a variety of animal models and are potential adjunct therapeutics
USAMRICDResearch, Education, and Training for Medical Chemical Defense
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Neuroactive Steroids
• Modulate excitability through interaction with membrane receptors and ion channels
• Not active at intracellular steroid receptors
• Potentiate GABA-evoked Cl-current
• Directly activate GABAAchannels in high concentrations
• Potentiate the effects of benzodiazepines and barbiturates Belelli and Lambert (2003) Nature Rev Neurosci
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Therapeutic Potential
• ALLO and PREG are effective against PTZ-, bicuculline- and picrotoxin-induced seizures
– Belelli et al. (1989), Högskide 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)
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Rat Model
• Male Sprague-Dawleyrats were surgically implanted with the F40-EET transmitter (Data Sciences International)
• EEG activity was continuously monitored throughout the duration of the experiment
www.datasci.com
USAMRICDResearch, Education, and Training for Medical Chemical Defense
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
Seizure Onset
30
-30
01
HI-6Atropinesulfate
GD 1.0xLD50
Diazepam
≈8-10
Exp. 2PREG
Exp. 1PREG
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Soman: Pregnanolone
GroupSaline GD GD/Preg(pre) GD/Preg(post)
Seiz
ure
Dur
atio
n (m
inut
es)
0
50
100
150
200 ***
*
• 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
• 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 Body Weights
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Soman: Pregnanolone
Saline GD GD/Preg(pre) GD/Preg(post)
Neu
ropa
thol
ogy
Scor
e
0
25
50
75 ***
• 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
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Rat Model-ALLO
-30
01
HI-6Atropinesulfate
GD 1.0xLD50
Diazepam
Seizure Onset
≈8-10
30
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
USAMRICDResearch, Education, and Training for Medical Chemical Defense
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
USAMRICDResearch, Education, and Training for Medical Chemical Defense
10 post exposureNo GD + Vehicle
GD + Vehicle
GD + ALLO
Baseline 10 min Post-Exposure 1 hr Post-Diazepam
USAMRICDResearch, Education, and Training for Medical Chemical Defense
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
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Conclusions
• Standard treatment failed to protect rats from the neuropathology resulting from exposure to 1.0xLD50GD
• Pretreatment with PREG reduced the number of rats displaying GD-induced seizures and seizure-associated neuropathology
• Post-exposure treatment with PREG reduced seizure duration and neuropathology
• Pretreatment with ALLO prevented GD-induced seizures and subsequent neuropathology completely.
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Future Directions
ALLO and PREG• Sedation/motor toxicity
– Potentiate effects of BDZs• Low bioavailabilty• Metabolism (3α-OH)
– Rapid sulfate or glucoronideconjugation
– Oxidation to ketone
Ganaxolone• 3β-methylated synthetic
analog of ALLO• Methyl group partially protects
3α-OH from metabolism• Orally available• Currently in Phase II clinical
trials for treatment of epilepsy
USAMRICDResearch, Education, and Training for Medical Chemical Defense
Acknowledgements• Lumley lab members:
– Ariel Capilli Stephen Estes Rose KajihKristen Kamberger Nathan Kelley Rachel KnoppJosh Kraft Wuya Lumeh Dr. Govinni MohanAlison Nelson Mike Pham Kristy ReddingCaroline Schultz Mark Schultz Julia SchwartzMike Stone Theresa Ward
• 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.
