Adult rats were induced into status epilepticus (SE) using lithium chloride and pilocarpine. Ganaxolone (GNX) and diazepam (DZP), alone and in combination, were administered 15 minutes after SE onset. GNX (6 mg/kg) and sub-therapeutic DZP (5 mg/kg) together produced a complete block of SE, indicating a synergistic antiepileptic effect. Pharmacokinetic analysis found no interaction between the drugs. GNX effects on EEG were similar to propofol. The results suggest GNX enhances DZP's antiepileptic activity at GABA-A receptors and could improve treatment of refractory SE.

1. • Adult male Sprague-Dawley rats (10 weeks of age) were pre-implanted with cortical electrodes.
• Rats were given lithium chloride (127 mg/kg) followed by pilocarpine (50 mg/kg) to induce SE.
• SE was first identified by presence of large amplitude slow wave EEG activity and then behaviorally
using the Racine scale. Racine stage 3 seizures were used as the time-marker for SE onset and
subsequent treatment.
• GNX and diazepam (DZP) were administered
via IV bolus injection 15 min after detection
of SE onset. In combination studies, DZP was
administered just prior to GNX.
• Seizure magnitude and duration were
measured by EEG recordings. EEG power
(mV2/Hz) was calculated by fast Fourier
Transform, (FFT) in 1 Hz frequency bins from
1 to 96 Hz using ICELUS software. Frequency
ranges analyzed:
o Delta 0 – 5 Hz
o Theta 5 – 10 Hz
o Beta 10 – 30 Hz
o Gamma-1 30 – 50 Hz
o Gamma-2 50 – 70 Hz
o Gamma-3 70 – 96 Hz
• EEG power values for each animal were
normalized by subtracting the baseline value from all subsequent values.
• EEG power data were evaluated by 2-way ANOVA (treatment and time period) at each frequency
range using Prism Graphpad (version 6). Post-hoc analysis consisted of Holms-Sidak multiple
comparison tests, with significance set at P< 0.05.
• Ganaxolone (GNX) is the 3β-methylated analog of the naturally occurring neurosteroid
allopregnanolone that is being developed for the treatment of status epilepticus (SE).
• SE is a severe, life-threatening epileptic condition that is currently treated with benzodiazepines
to control SE followed by standard antiepileptic drugs such as phenytoin and then, if needed, by
administration of general anesthetics such as propofol.
• Patients with SE become progressively refractory to treatment over time. Moreover, more than
30% of SE patients are resistant to aggressive benzodiazepine administration.
• The rat lithium-pilocarpine model of SE is a clinically translatable model of this condition. Rats
subjected to experimental SE are refractory to benzodiazepine if treatment is initiated ≥ 15 min
after seizure onset; thus modeling a treatment resistant form of SE.
• GNX elicits its antiepileptic activity through positive allosteric modulation of both the synaptic
and the extrasynaptic GABAA receptors, unlike benzodiazepines which only modulate the
synaptic receptors.
• The current studies were designed to evaluate the combination antiepileptic effects of GNX with
diazepam in a benzodiazepine resistant SE paradigm.
Ganaxolone and Diazepam Administered IV Produce a Synergistic Antiepileptic
Effect in a Treatment Refractory Model of Status Epilepticus
Michael S. Saporito, PhD1, John A. Gruner, PhD2, Julia Tsai, PhD1, and Albena Patroneva, MD1.
1Marinus Pharmaceuticals, Inc, Radnor, PA, United States, 19087; 2 Melior Discovery, Inc. Exton, PA.
• We have previously demonstrated that GNX when administered alone 15 min after SE onset elicits a
complete block of SE at dose-levels of >12 mg/kg
• Diazepam is inactive up to dose-levels of 10 mg/kg when administered >15 min after SE onset
• When administered in combination 15 min after SE onset, DZP (5 mg/kg) and GNX (6 mg/kg) elicit a
complete block of pilocarpine induced seizures.
• The pharmacokinetics of GNX and diazepam were identical when administered alone or in
combination indicating that neither drug affected the pharmacokinetic disposition of the other.
• The data suggest that this synergistic enhancement of antiepileptic activity occurred at the level of
the GABAA receptor.
• GNX administered alone in a dose-escalation fashion elicits EEG changes similar to that of propofol.
• These data have clinical implications in the treatment of SE when GNX is to be administered to
patients who are or have been treated with benzodiazepines.
Skull Image
From Watson,
“The Rat”
anchor
screw
(sinus)
Frontal cortex
electrodes
3mm ant.
to Bregma
2.5mm lateral
Hippocampal
Electrodes
4mm post.
to Bregma
3mm lateral
Skull
sutures
anchor
screw
(vertex)
EEG
Ch1
Ch2
Neck
Muscles
InterauralLine
Lambda
Bregma
Bregma
Figure 1. Electrode Placement
Figure 2. Status epilepticus was induced by administration of lithium and pilocarpine. DZP (5 mg/kg; IV) or GNX were
administered 15 minutes after SE onset. For combinations, DZP and GNX were administered sequentially. Seizures were
measured by EEG response. Data are expressed as a EEG power (Log mV2/Hz). Data represent the 10-30 Hz frequency
range. Data were analyzed by two-way ANOVA followed by a post-hoc Dunnett’s test. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001 from vehicle control.
Figure 2. Ganaxolone Combined with Diazepam Elicits a Synergistic Antiepileptic Effect in
Status Epilepticus
0 2 4 6
-1
0
1
2
Time Relative to SE Onset (hrs)
EEGPower
(LogmV2/Hz)
DZP (5 mg/kg)
Vehicle
Treatment
0 2 4 6
-1
0
1
2
Time Relative to SE Onset (hrs)
EEGPower
(LogmV2/Hz)
Vehicle
DZP (5 mg/kg) + GNX (3 mg/kg)
DZP (5 mg/kg) + GNX (6 mg/kg)
Treatment
**
*
**** ****
**
***
0 2 4 6
-1
0
1
2
Time Relative to SE Onset (hrs)
EEGPower
(LogmV2/Hz)
GNX (3 mg/kg)
GNX (6 mg/kg)
Vehicle
Treatment
SE-onset
Vehicle
Pilocarpine Treatment
Diazepam (5 mg/kg)
Ganaxolone (6 mg/kg)
Diazepam (5mg/kg)
+ Ganaxolone (6 mg/kg) SSSSSE-onset
EEG Traces SE-onset and Treatment
Figure 3. Representative EEG
traces of vehicle, diazepam
alone (5 mg/kg), GNX alone
(6 mg/kg) or diazepam and
ganaxolone in combination
Figure 4. Diazepam (5 mg/kg) and GNX (6 mg/kg) pharmacokinetics (IV) were measured alone and in combination.
For combination, GNX was administered immediately after DZP. Plasma levels were measured by LC/MS/MS.
Predose
3 mg/kg
9 mg/kg
15 mg/kg
24 mg/kg
30 mg/kg
Propofol
6 sec
Ganaxolone
Figure 5. In order to evaluate
the effects of GNX on normal
awake rats, the drug was
administered at 3 mg/kg
(bolus; IV) every 3 minutes.
EEG signal was measured
continuously for 6 hours.
EEG traces represent 6
seconds beginning 2 min
after the injection. EEG
changes are consistent with
anesthetic effects and are
first detected after the 3rd
administration.
Figure 3. Diazepam and Ganaxolone Produce a Synergistic Antiepileptic Response
Figure 4. Ganaxolone and Diazepam Pharmacokinetics Were Not Altered by Co-administration
Figure 5. Ganaxolone Shows Similar Effects to Propofol on EEG
OVERVIEW
SUMMARY AND CONCLUSIONS
0 20 40 60 80 100
0
500
1000
1500
2000
2500
Diazepam Pharmacokinetics
Time (min)
Diazepam(ng/mL)
DZP (5 mg/kg)
GNX (6 mg/kg) + DZP (5 mg/kg)
0 20 40 60 80 100
0
1000
2000
3000
4000
Ganaxolone Pharmacokinetics
Time (min)
GNXLevels(ng/ml)
GNX (6 mg/kg)
GNX (6 mg/kg) + DZP (5 mg/kg)
METHODS